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Quantity One® - Bio-Rad

1. a ee GEL eal ER v MU T Fig E 2 GS 800 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters These are numbered 1 40 top to bottom and 1 30 left to right if the light source is reflective and 1 40 top to bottom and 1 29 left to right if the light source is transmissive To hide the gridlines select the Hide Grid checkbox under Options Below the main scanning window is the calibration strip window Every time the densitometer calibrates an image of the calibration strip will appear in this window Quantity One User Guide The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are five basic steps to scanning an image using the GS 800 l 2 E 2 Select the application Select the scan area Select the resolution of the scan Calibrate the instrument This is automatic after you enter the step tablet values before you first scan after installation Acquire the image Step l Select Application To set the parameters for your particular scan you can 1 2 Select from a list of possible applications or Choose your own filter and light source settings Selecting from the List of Applications When you select from the list of applications the software automatically sets the appropriate filter s and other parameters for that partic
2. sees H 4 Selecting resolution o tete Brei esa cen Hag Hee Med H 13 Selecting scan area esce p eee eee etre eret Pertinet H 12 Simulation MOE ve eicit ite ti pe ho epe e nn H 2 Phylogenetic Trees Clustering methods cote irte tee teen eem titers 12 15 Display options cee eis Ae Bep etes 12 18 Displaying MU 12 14 Preferences DISPLAY eC 2 17 Pile pad eerta ate AE pt Os 2 17 General mt pee eto pn ende E eed Sa ds 2 15 Relative Front Calculation ssseseeeeeeeeenerenen rennen 2 20 Relative Percent Calculation sese 2 20 Toolbars oubli aie Re eek Bes Beate Soha gems 2 19 Lose pU da tt ie a 13 1 Printing Image Report ita ria exten ee ie 13 3 Vadeo Pini oen a A segna 13 4 Propagate Badd Etude t oit shes d eoe oe e 6 21 QUAN AT E TR 1 14 Calibrated e iras do did a a dl EE 5 15 CORLOUE Letter e iet eri e ede aee e ecd Seda 5 15 Normalized mi A Rete 5 15 JEN ET C n 5 15 5 17 Trace a aede Eee RENE BE SIE 5 3 5 15 6 26 Index 11 Quantity One User Guide Quantity Standards Applying to bands eae eee drid 6 30 Calibration curve iti es ino mte ep RORIS 6 28 Checking imported values sese eee 6 31 Creatine ste np POESIE POI nien ER POS 6 24 Definition ona RAT eii ti tpe reete 6 24 Dilution series i iiiter tme pepper ted ts 6 30 Entering standards sese eene nennen eene tne 6 27 ImpOrUfip iier a ri
3. sese 5 13 Doublets det Ctig tete o I eR PERDRE 5 8 Drawing des o ds 5 24 Gaussian modeling fc feb e eot te teen eerie e deest rete ied 5 18 Identification and quantitation seen 5 2 Identifying individually cue iR ette ote S Rie eus 5 10 Irregular sh pes 4 asi eee ed bes 5 21 Normalized for quantity cuco t eec ete ehe 6 22 NUDE eue e e ERES 5 14 Plotin traces E EA 5 12 Shadow bands rejecting esses nennen nennen 5 6 Bio Rad Technical Service ie eret e een eie ed 1 14 C Calibrated quantity ermita 5 15 Categories and attributes essere nennen eren entren J 1 Centerns an Ma e sese eee pete ipee tib cen eot pe pe RU Ree S 3 4 Chemi Doc Acquiring the ape ed epe rs C 5 D 6 Adjusting the display inserire ieee ee e eree nennen nnne C 11 Annotating AMAL CS uoce dp mie eed e edite ee bee A 10 C 12 Auto EXPOS i ee EAA A Ree C 6 D 6 AULOEXPOSUTS Settings ne d tete aet pecie miis C 15 D 14 Backup images oo eee sese C 16 D 14 Index 2 Chemi mode 2th eee Adri C 5 DAC Settings cte Euboea dete te opened uds C 14 Exposure Status bar eee peteret RUP RS C 13 D 9 Exposure time eee RS Ree e RS REIR I eee A 7 C 7 D 7 Errezil C 8 Image mode 3 6220 ote ie eter dO etie C 5 Imaging rea tire etre reU e RERO C 15 D 12 Invert display eee eiue ted rte dee es C 11 Live ACQUIIE nicas C 8 D 8 Live image display antes eee ah ee Aarti ee C 4 Manual EXP
4. Enable 21 CFR Part 11 mode ogram times out if inactive minutes S bam on startup iX Automatically create backup for new images Enable Secure Mode to prevent file changes Y OK X Cancel 2 Help Fig K 1 Security Preferences The following options are available on the Security tab of the Preferences dialog box Enable 21 CFR Part 11 mode If this box is unchecked all other options are inactive To activate the other options and CFR mode check this box Program times out if inactive When this box is checked you must enter your password to resume using Quantity One The minutes field indicates the amount of time that must elapse before the application times out Require login on startup If this box is checked you must enter your password when when Quantity One starts This password is the same password as the one used for logging on to Windows This is an added security measure to prevent unauthorized users from starting Quantity One on a machine where they are not logged in Automatically create backup for new images This option when selected creates a backup file when you save a newly acquired or imported image Locate the backup file in the same folder as the original The backup file is identified by backup of preceding the filename of the original Enable Secure Mode This option is not associated with CFR For further information on Secure Mode See Section 2 5 Preferences
5. Q Live Focus IRIS ZOOM FOCUS Open 4 Tele 4 Near Close Y wide Y Far Step Il Image Mode UY White C Chemi c Step IIl Acquire Image Auto Expose O Freeze Step IV Select Output E Video Print Save Dptions 2 Help Fig C 11 Simple Acquisition Window To change the acquisition window to Simple Acquisition Mode check the box marked Simple Acquisition Mode The change will take effect the next time you open the acquistion window C 17 Quantity One User Guide C 18 Appendix D ChemiDoc XRS Fig D 1 ChemiDoc XRS Before you can begin acquiring images the ChemiDoc XRS system must be properly installed and connected with the host computer See the ChemiDoc XRS hardware manual for installation startup and operating instructions D 1 Quantity One User Guide To use the ChemiDoc XRS the instrument must be connected to a fireWire port on your computer If your computer does not have a fireWire connection you will need to install the Bio Rad supplied fireWire card prior to connecting the instrument Note Itis recommended you install the software before connecting the instrument Make sure that your ChemiDoc XRS camera is turned on If the camera is not turned on the ChemiDoc XRS acquisition window will not open Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acq
6. Match Unmatch Outlier Normalize Matched Band Set Show Band Types Shift F5 Show Band Models Ctrl F5 Match Graphs Fig 6 11 Matching tools Select Match from the menu or toolbar and click on a representative experimental lane in the gel image This may be a lane that contains most or all of the bands that you are interested in and or a lane in which the bands are particularly well resolved Each band in this lane will be designated as a different band type Note If you have defined standards in the gel a pop up box will warn you that the regression model for calculating band values will be restricted to point to point semi log If you selected a different regression model when defining standards it will be changed The first time you click on an experimental lane with the Match command a pop up box will prompt you to specify the matching tolerance 6 14 Chapter 6 Standards and Band Matching Quantity One x A Do the whole lane Select yes to make a new band type for each band in this lane Tolerance 3 00 No Cancel Help Fig 6 12 Query box Apply matching to the whole lane Tolerance is the minimum spacing that the matching model expects to find between unique bands It is expressed as a percent of lane height You can enter a value between 0 2 and 10 percent If the bands are very close together enter a tolerance of 2 5 percent or less
7. 3 5 3D Viewer The 3D Viewer allows you to see a three dimensional rendering of a portion of your image This is important for such instances as determining whether a selected band is actually two or more separate bands To see a 3D rendering of a portion of your image select 3D Viewer from the View menu Your curser turns into a crosshair Click and drag your curser over the image area you would like to view creating a box Note viewing a large area of your image may reduce performance Toreposition the box position your cursor at the center of the box The cursor appearance will change to a multidirectional arrow symbol You can then drag the box to a new position e Toresize the box position your cursor on a box corner The cursor appearance will change to a bi directional arrow You can then drag that corner in or out resizing the box e To redraw the box position your cursor outside the box and click once The box disappears and you can then draw a new box To view the selected area position your cursor inside the box slightly off center The cursor appearance will change to an arrow Click once to open the 3D Viewer Note If you are having problems using the 3D Viewer install the latest drivers for your graphics card If after updating your drivers you are still having problems go to the Display tab of the Preferences dialog box and select Disable hardware Chapter 3 Viewing and Editing Images acceleration f
8. After you select a tolerance click on Yes to automatically match all bands across the lanes Click on No to match only the specific band you clicked on When you click on Yes the bands in the lane you selected will change to green indicating that they are known band types that have been identified by you A band type number will appear next to each band The automatic matching mechanism will attempt to match the bands in the other lanes to the known band types Matched bands are labeled in red with the number of the band type appearing next to each band These matched bands are connected by modeling lines Yellow bands are bands that the software cannot accurately match The matching algorithm is deliberately conservative to avoid incorrect labeling so a number of yellow bands may appear on the image Your next step will be to identify the yellow bands as either new band types or existing band types that could not be automatched To summarize e Green bands are known band types Quantity One User Guide Red bands are bands that have been automatically matched with the known band types Yellow bands are bands that have not been matched and are unclassified Displaying Band Types and Modeling Lines To display the band type numbers on the image select Show Band Types from the Match menu or toolbar Band type modeling lines reveal the path along the gel image that the software uses to match bands of the same type in d
9. Make Backup Copy Hide Grid P x Warn On Saturated Pixels Q Info Help RE S T U 10 11 12 13 14 15 16 Fig F 2 Personal FX acquisition window The default scanning window is marked by grid lines that divide the area into quadrants There is also an outer box and inner box marked by thicker lines The quadrants are numbered 1 16 left to right and lettered A U top to bottom If you prefer a scanning window measured in centimeters deselect the Quadrant Mode checkbox in the control panel by clicking on it To hide the gridlines click on the Hide Grid checkbox under Options The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are three basic steps to scanning an image using the Personal FX 1 Select the scan area Quantity One User Guide 2 Select the resolution 3 Acquire the image F 2 Step Select Scan Area To select a scan area drag your mouse within the scanning window In the scanning window your cursor appearance will change to a cross The border of the scan area you are selecting is marked by a frame Drag cursor to ae scan ea N 4 Fig F 3 Selecting a scan area If you are in quadrant mode note that the frame locks onto the next quadrant as you drag When you release the mouse button the border changes t
10. Advanced Crop Commands To ensure that your scans are exactly the same size and shape you can use the tools on the Image Advanced Crop submenu to save the crop box from one scan and apply it to others These tools also allow you to crop a gel of the same scan To define and save a crop box and apply it to another scan 1 Select Define Crop Area from the submenu and drag a crop box on an image Position the box as described in Cropping Images Select Place Crosshair from the submenu and click a landmark inside the box that is present in all the gels you want to crop This could be a spot or some other image detail The crosshair will make it easier to position the box in the other images so that it encloses the same area Select Save Crop Settings from the submenu enter a name for the current crop settings in the pop up box and click Apply Complete the crop action in the current image by positioning your cursor inside the box slightly off center and clicking to perform the crop as described in Cropping Images Open or select the next image you want to crop select Load Crop Settings from the submenu and select the name of the settings you saved The crop box and crosshair will appear on the image Reposition the crop box so that the crosshair is correctly aligned with the appropriate image object then complete the crop as described in step 4 above To delete any crop settings you have saved select Delete Crop Settings fr
11. Color Palette x E Quantity One xj Colormap Name coomass Select Color Preferences Load Delete BEIGE Color Group LightN Overlay Colors Coomass EtBr False Gray Standard Psuedo Silver Spectrum Sybr Green Sypro Ruby A user defined set of colors _ _ User defined deep purple Cancel Fig 3 12 Selecting a Colormap From the list displayed click on the set of colors you want to apply To create your own colormap adjust the colors within the color groups as described above and type in a new colormap name Click on OK to apply the changes To remove a colormap click on the Delete button Select the colormap to be deleted from the displayed list A pop up box will ask you to confirm the deletion To return to the Standard colormap click on the Reset button All colors will reset to their default values 3 8 Transform Use the Transform dialog to adjust the image brightness and contrast and optimize the image display These controls affect the image display only and will not change the underlying data With an image open select Transform from the mage menu or main toolbar 3 17 Quantity One User Guide c eb de 4 S gs EJ D ieeem Lane Band Match Yolu Transform Crop Fig 3 13 Transform command The Transform dialog contains a Preview Window a Frequency Distribution histogram a Transform Plot and three ma
12. Each allows you to choose between aligning the tree to the right or proportionally aligning it Right alignment uses a fixed branch length for displaying the distance between nodes proportional alignment differs for Neighbor Joining and other tree modes With the Neighbor Joining method proportional alignment shows branch length sizes proportional to the min max values set in the Options dialog Other methods plot the nodes at locations determined by their similarity values The Fit Tree in Window option scales the tree so that the entire tree will fit onto a single printed page With this turned off the entire tree will appear with the correct distances between nodes preserved and the printed tree can be tiled across multiple sheets of paper The Neighbor Joining Options dialog click on the Designate New Root button to designate a new root node the node at the very top of the tree enabling you to look at the relationships between lanes from a different perspective This will highlight all the nodes in the tree Click on the node that you want to serve as the new root and the 12 18 Chapter 12 Reports dendrogram will be recalculated To return to the original tree click on Restore Original Tree The Min and Max sliders are active only for proportionally aligned Neighbor Joining trees Use them to adjust the minimum and maximum distances between nodes on branches to highlight specific regions of data Distances below the Min
13. Edit Auto Rename Label OK Cancel 2 Help Fig 7 6 Volume Properties dialog box 7 5 Quantity One User Guide At the top of the Volume Properties dialog box is the current label for the selected volume Volume Type lists the types of volumes Unknown Standard and Background The type of the current volume is indicated by the selected radio button Choose a new type by clicking a different radio button If you choose Standard as the new volume type you must enter a concentration greater than zero in the Concentration field Note If you change a volume s type e g change an unknown to a standard any subsequent volumes of the original type will be renumbered For example if you create volumes U1 and U2 and then designate U1 as a background volume U2 will be renumbered U1 7 2 a Editing Volume Labels In the Volume Properties dialog box you can choose to give the selected volume a User label as well as modify the style of the Auto labels Editing User Labels To create or edit a User label for the selected volume click Edit User Label to open the Text Overlay Properties dialog box In the Text Overlay Properties dialog box you can change the font size color orientation and background of the selected text The text field displays the current volume label To change the text of the label type a new label in the text field Note A change to the text of the label will not cause a renumbering of unedi
14. Fig 8 13 Creating a custom filter When you have finished selecting the filters click OK Any applications that cannot use the new filter configuration will be unavailable in Step I Select Application If any selected applications are no longer usable the channel reverts to the default application selection Note Depending on the filter types you select some or all existing custom applications may become unavailable H 17 Quantity One User Guide H 18 Appendix VersaDoc Fig l 1 VersaDoc Before you can acquire images using the VersaDoc or VersaDoc MP Imaging System you need to install the Roper Scientific interface adapter and its associated device drivers in your computer The driver installation procedures for Windows operating systems inlcude pre installation and use of the Windows Hardware Wizards Note Important Please read and follow the driver installation procedures contained in the README documents before you install the VersaDoc camera interface adapter You can find the README documents in the VersaDoc Drivers folders on The Discovery Series TDS CD Quantity One User Guide The CD also contains Roper Scientific camera interface driver installation instructions for Macintosh computers See the VersaDoc hardware manual for further installation startup and operating instructions for the VersaDoc Imaging System The VersaDoc Imaging System contains white and UV flourescent bulbs whereas the
15. Quantity One User Guide E wa00196 1D Scan New Al ES o Anchor point o Lane in red Border and anchor lines in white mee 3 6 no Es til o HEHH EEH H o o QDD in F m Fig 4 2 Features of a lane frame 4 1 a Lane Frames The fastest way to define all the lanes on a gel is to create a lane frame using the Auto Frame Lanes command If this command doesn t work well on your images you can create and place a lane frame manually Auto Frame Lanes Auto Frame Lanes works best on gels with large numbers of clearly defined lanes and bands Also the lanes should be reasonably vertical and contain approximately the same amount of sample Note Select Auto Frame Lanes from the Lane menu or toolbar The program will automatically detect the lanes and place a frame over them 4 2 Chapter 4 Lanes The lane frame contains individual lane lines numbered sequentially from left to right The border and anchor lines of the frame are marked with dashed white lines the lanes are solid red lines and each anchor point interior and corner is marked with a circle The top and bottom of the frame are parallel with the top and bottom of the image However the interior anchor points and lines will bend the frame to follow the actual lanes in the gel compensating for any curving or distortion in the gel If Auto Frame Lanes detects too few or too many lanes you can add o
16. The default sensitivity setting is 10 00 If the gel has faint bands e g O D 0 05 counts 2 000 you may want to increase this value to 20 00 Chapter 5 Bands Lane Width The Lane Width setting determines the width along the lane lines that are sampled for band detection and quantitation When a band is detected an average intensity for each horizontal row of pixels within the band brackets is calculated The lane width determines the number of pixels in each row Study the band lines in the image while you adjust the width Select a width that is slightly wider than the bands in the gel Lane Width 4 921mm al Adjust the lane width until the band lines are slightly wider than the bands in the image Fig 5 4 Adjusting the lane width You can also change the widths of individual lanes using the Lane Width command see section 4 1 d Lane Width Minimum Density When Normalize is turned off the Min Density defines the lowest signal intensity that will be counted as a band Before selecting a Min Density value use the Plot Lane command on the Lane menu to plot a trace of a lane that includes some faint bands Then enter a value that is lower than the intensity of the peak of a faint band but is still above the background If faint bands are still undetected after adjusting this parameter you may want to increase the Sensitivity setting Quantity One User Guide If Normalize is turned on Min De
17. Always Auto Scale Select this checkbox to automatically Auto Scale every new image that you open The software will examine the data range in every image and optimize it accordingly This setting disables the other image optimization controls in the Transform dialog Reset To return to an unmodified view of the image click on Reset 3 9 Resizing and Reorienting Images The mage menu and toolbar contain commands for changing the size and orientation of images 3 23 Quantity One User Guide Image Lane Band Match Volume Transform Ctrl B Image Tools me hh D Crop Advanced Crop gt Vertical Flip Horizontal Flip Rotate d Rotate 90 Left Rotate 90 Right Subtract Background Rotate 180 Filter Wizard Filter List Custom Rotation Invert Data Fig 3 16 Resizing and reorienting tools Note Many of these commands will permanently change the image You will be prompted to confirm any permanent changes 3 9 a Cropping Images Use the Crop tool to eliminate unwanted parts of an image such as border space around the gel You can also use this command to reduce the file size of an image Select Crop from the mage menu or toolbar The cursor will change to a Crop symbol Define the region to be cropped by dragging the cursor across the image creating a box Everything outside the box will be deleted The dimensions of the crop area in millimete
18. SI LP 8 3 White and blue colonies eeeesseeeeeeneeeen nennen enne 8 7 A i T iren nem UP PPIeg 3 14 Compare Lane Images uti ie ettet E beret etes 12 11 Compare Lanes rro nae ern i eee deep 4 13 Comparison reports eti ice ei ette obe aet diet awe intei eee 12 9 Computer requirements MacidtOSD herede 1 6 nom DE 1 5 Context menu sioe eas tede team RERO EAE eie 2 5 Contour area M 5 15 Contour quantity cimas 5 15 Contours See Bands Contouring Contrast See Transform Crop Saving crop settinpgs suisse RUD RR TI EUIS ERR DRE dS 3 26 epp EE 3 24 Custom scaena ieu bie REB D 4 Custom Rotation cre e Fete Ue ed de ele edipi de ee 3 27 Default Automation RR Gem ree ote i e reip re ERU E P ERE LEUR 3 45 Density in REPO iets hes heen he et ee i ie IDEA 5 23 Density TOONS 4er Fe bte t e Oe ei eo eie Fees 3 5 Density at Cursor teret dee e etu 3 5 Density 3n BOX a eerte Uo e ERU UHR PERRO SERERE DERE HE ERN 3 5 Plot Cross section sireni e E S nennen nenne treten rennen eren 3 6 Plot Density Distribution eet rho t ee eter eie edens 3 5 Plot Vertical Trace 13 oerte a 3 6 Detect Bands See Bands Detecting automatically DEVICES sie ae ei A Rt eoi tdm PIT 2 21 Dice Coefficient sentada o ed be e eoi 12 10 Differential Display ere e een Ee era EC ais 11 1 DOS hle names Parsi ep ied eee dextre tette OR URSUS 2 16 Downloading from Internet Index 4 WIN O WS sectorial pittore ies 1 13
19. Saving the Image After the scan is complete a message will appear asking you if you want to keep the scan If you select Yes a separate window will pop up containing the new image You can then save and analyze the image using the standard menu and toolbar functions F 6 Appendix F Personal FX F 5 Options Auto Save After Scan To automatically save any scan you create click on the Auto Save After Scan checkbox Note In PDQuest this option is preselected and cannot be turned off All images must be automatically saved when acquired With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be promted to give it a new name This protects the backup file and preserves it from any changes Highlight Saturated Pixels When this box is checked any saturated pixels in the image wi
20. r Step Position Gel Q Live Focus IRIS Z00M FOCUS Open 4 Tele 4 Near Y Close wide Fa C Show Alignment Grid Fig A 3 Newer Gel Doc EQs feature camera control buttons in the acquisition window You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging Area dimensions under Options see section A 9 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are A 4 Appendix A Gel Doc focusing on you can place a ruler in the Gel Doc EQ cabinet so that it is visible in the image A 3 Step Il Select Image Mode The Image Mode option buttons allow you to set the type and scale of your data UV Select this mode for fluorescent samples With this mode selected the data will be measured in linear intensity units White Light Select this mode for reflective and transmissive samples With this mode selected the data will be measured in uncalibrated optical density uOD units A 4 Step Ill Acquire Image For many white light applications you can skip this step and save and print images directly from Live Focus mode For UV light or faint samples you can take an automatic exposure based on the number of saturated pixels in the image or you can enter a specific exposure time Note Exposure refers to the integration of the image on t
21. see 3 29 3 11 Filtering Images 2 rete eee eire pens 3 33 3 12 Invert Data eee e REN COE ERE RE etes 3 39 3 13 Text Overlays not Reto ed RU RERO 3 39 3 14 Erasing All Analysis from an Image eee 3 42 3 15 Sortiand Recalculate icc 2 need e n Pene 3 42 3 16 Automation Manager eese eene cnn non arco non nre ene 3 42 4 Canes iii eii 4 1 41 Defining AMES ERR REFUSER S USE 4 1 4 2 Lane Based Background Subtraction eee 4 0 4 3 COMP Lanes pepper ege e REOR ien 4 13 4 4 Lane based Arrays ento mide eR e ENS ees 4 16 LM M 5 1 5 1 How Bands Are Identified and Quantified esses 5 2 5 2 Band Detecta 5 3 5 3 Identifying and Editing Individual Bands sess 5 10 5 4 Plotting Traces of Bands eoe oer 5 12 5 3 Band Attributes muscle 5 13 9 6 B nd Information cerei eid epe teo to eec lere ith 5 16 5 7 Gauss Modeling Bands eese eee eene 5 18 5 8 Irregularly Shaped Bands in Lanes eee 5 21 6 Standards and Band Matching 6 1 6 1 Stain dards chart ree delete e eei Enero ete beige de s 6 1 6 2 Band Matching e pee ette tentent aene tere teer prie 6 13 6 3 Quantity Standards niece dee dediti delo ae 6 24 7 10 11 Contents Volume TOONS iniciada 7 1 TA Creating a Volume etre ee dead anaes eee 7 1 7 25 Volume Lab
22. 99 5 Data range 0 0 1 00 0D Memory size 400 29 Kb History OK Cancel Print Fig 2 9 Image Info box History lists the changes made to the image including the date If you have Security Mode active the name of the user who made the change is also listed See Section 2 5 Preferences for information on Security Mode To print the file info click on the Print button in the dialog box Changing the Image Dimensions You can change the dimensions of certain images using the Image Info dialog box This feature is only available for images captured by a camera or imported TIFF images in which the dimensions are not already specified 2 11 Quantity One User Guide For these types of images the Image Info dialog box will include fields for changing the image dimensions Colony Counting 2 1D Scan x Cc WINNT File Profiles Info swarric Desktop Colony Counting vZ lsc Description Scan date 09 Jun 1999 12 30 Scanner gt Scan area mm X 167 3 Y 163 3 Scan pixels 4 40 3 y b Pixel size um lt 403 2 Y 403 2 Data range 0 0 255 00 Int Fields for changing the image dimensions Memory size 166 62 Kb History Cancel Print Fig 2 10 Image Info dialog box with fields for changing the image dimensions Enter the new image dimensions in millimeters in the appropriate fields Note that the pixel size in the image in micrometers will ch
23. A 16 Appendix B Gel Doc XR Fig B 1 Gel Doc XR Before you can begin acquiring images the Gel Doc XR system must be properly installed and connected with the host computer See the Gel Doc XR hardware manual for installation startup and operating instructions B 1 Quantity One User Guide To use the Gel Doc XR be sure the system is properly installed Installation instructions can be founds in the Gel Doc XR Hardware Installation Manual Make sure that your Gel Doc XR system is powered on If not the Gel Doc XR acquisition window will not open and an error will be displayed stating the Gel Doc XR camera could not be found Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated B 1 Gel Doc XR Acquisition Window To acquire images using the Gel Doc XR go to the File menu and select Gel Doc XR The acquisition window for the instrument will open displaying a contr
24. Ed keere e ii iaa ido 7 6 E e o o 2 15 Expor e TP TES arriban tina 13 5 EX Quest esichin ae iin bate hi sie AAI SAI WAS LS edis 10 1 Cahlbration atid SETUP rei eee een ee tee ien totes 10 2 Lane Volume Excision eese ener 10 18 Manual EXCISION creed eei demit ER EE eR 10 6 File locations in Windows sees eene nemen treten ene 1 8 Falter Wizard cc Meet ta 3 34 Filtering image NOISE iecit e epe anti rtt cases ERE E EES 3 33 Flipping amages entrer tope eR HU DEO HR cis 3 26 Fluor S MAX Multilmager File size iol 1M ages eene ipei ete ater eei peer I 19 Fluor S Multilmager File size of 1m ges outre eemeiieg epe DU o fe eire D 15 Saturated pixels highlighting essere D 15 FX see Molecular Imager FX Gaussian modeling of bands o oooccnocnonccoconononnnoncnononancnon cnn nonccono ro nono nennen rennen 5 18 Gaussian Peak Density siise ennnen eee aaia nennen rennen 5 15 Gaussian Trace Quantity 4 eot IRI Oe REOR Rien 5 15 Gel Doc Acquiring the Tage s oreet idet eie nct tte ete e A 5 Index 5 Quantity One User Guide A Uto eXpOSe inne eoe rete aie rete E eR A 5 Auto exposure settings oooocononconcoocconccnncononnnonnnonn conc nn nennen enne A 13 B 13 Backup Uma ges eonim guter eee A 13 B 13 DAC Settings ette aet eei A 12 Display settings 3er rete trt E edere A 8 Exposure Status bar eene ete niente nene A 11 B 11 IEI A eme E DH EU Ps A 7
25. Load or Delete field and select from the list of saved settings Then click on Load or Delete To display the report click on the Report button The report will be displayed in a standard report window see section 12 1 Report Window 12 3 Band Types Report The Band Types Report shows the presence or absence of specific bands in specific lanes of a gel The report window displays a schematic representation of all the bands and lanes in the gel with the presence or absence of bands shown as specified in the options dialog 12 6 Chapter 12 Reports Band Type Report For Proteins 1SC Band Type 1 Band Type 2 Band Type 3 Band Type 4 Band Type 5 Band Type 6 Band Type 7 Band Type 8 Band Type 9 Band Type 10 Band Type 11 Band Type 12 Band Type 13 Band Type 14 Band Type 15 Matching Tolerance 3 0096 GO a 4 ge x Page J ose LIL ajaj Page Setup Print Preview Fig 12 6 Band Type Report Note This report requires band matching see section 6 2 Band Matching Select the report from the Reports menu and select the display options in the Band Types Report Options dialog To save the report options click on the Settings button in the options dialog and enter a name for the settings in the field To load or delete previously saved settings click on the button next to the Settings to Load or Delete field and select from the list of saved settings Then click on Load or Delete
26. SIM edd E 3 10 A Annotations See Text Overlays Arrays L ane b sed ue iet ca eei eti eet aes 4 16 Volume inuenio iii dc 7 12 ATOW KO Social elias EE 3 3 Automation Manager 3 enint eet eite her rie e bee iras 3 42 Average density ies sess etae coe o e et A eee eei tei eig 5 15 B Background subtraction Entire IMA e te re ted e nee p esi E SU bc 3 29 Lane based rn 5 sanam p pO aeree eoe ned 4 9 Volume based ivi ee dete Fee eee ntes 7 10 Band data displaying sese rennen rennen 5 13 Band Set form Listofbandtypes ini 6 18 TOM DAM d 6 19 hEuRE MMC PEE 6 17 Band types Defining o end 6 14 ISO li it iio 6 18 Index 1 Quantity One User Guide Band Ty pes report oec eo Ite petet Pepper Reed 12 6 Bands AUS tecto Dem Re eem pU DUI e 5 12 Attributes displaying sessseeeeeseeeeen nennen nennen ener 5 13 Band information displaying ooooonncnnnnoninoconnconoconcnnnnnnonn conc non nrancnn con nc ronca corno 5 16 Brackets or lines preference oooooooconocinocconconnnnnconccnnconnnnnonn non nono rnonn crono nn ncn nono 2 18 E Dl RU 5 22 Dbel ting eet eaves oan ar RG eagles Sen a ds 5 12 Detectng a tomatcally eiit E ge neigen 5 3 Detection parameters iseset ieie eee re EEEE e nennen netten 5 5 Detection parameters saving and loading sse 5 9 Displaying as brackets lines
27. Select scan area After you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later H 4 Step Ill Select Resolution The PharosFX acquisition window allows you to scan at 50 100 200 or 800 micrometers These resolutions are listed as option buttons in the control panel Step Ill Select Resolution 50 micrometer e 100 micrometer 200 micrometer 800 micrometer Fig H 11 Resolution option buttons The resolution you select should be based on the size of the objects e g bands spots you are interested in For example e 50 micrometer resolution should be reserved for images requiring the highest H 13 Quantity One User Guide level of detail e g high density in situ samples 1 536 well microplates high density arrays samples with very closely spaced features such as bands on a 1D gel Files scanned at 50 micrometers can be very large 100 MB e 100 micrometer resolution is useful for typical gels and arrays e 200 micrometer resolution is useful for gels with large bands and dot blots e 800 micrometer resolution should be reserved for very large objects such as CAT assays File Size of Images Image File Size below Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for th
28. The Calibration summary describes all changes made during calibration PDQuest generates a log that will be updated each time the spot cutter is calibrated To view the log click View Log Note If this is the first time calibrating the spot cutter the log is unavailable Click Finish to close the wizard You are now ready to cut spots using the EXQuest Spot Cutter 10 5 Quantity One User Guide 10 2 Manual Excision Manual Excision is used for basic spot cutting operations If you want to excise entire lanes or create volumes to excise use Lane Volume Excision Before using this tool make sure the spot cutter is connected is switched on and has warmed up then select Manual Excision from the Excision menu The software will connect with the spot cutter platform and camera and the Manual Excision dialog box will open Manual Excision Tool connected to EXQuest with Gel Head simulated Acquire Image White Trans Acquire image ER Specify Options Cut Plate Hydration Wash Make multiple cuts when possible Maximum cuts object Cutting tip size 1 0 mm 51 5 mm Material Free gel 4 I TT TIT Specify Cuts T Add move cut X Snap to peak Run Info Cuts 0 Wells 0 iu Plates Min Well Volume 000 Show xj Well jPosition mm al ea RY a al Xj Zoom on cut Y Prime pump E Save Import Ea Close g Help Fig 10 4 Manual Excision
29. When looking at a lane trace these bands appear as flat or gently sloping abutments to darker better defined bands i e there is no dip on the trace between the two bands Increasing the Shoulder Sensitivity will result in more shoulders being detected as bands Changing this setting to zero will result in no shoulders being recognized as separate bands If band detection calls a doublet a single band check the lane trace to see if there is a dip between the peaks of the two bands If there is no dip increasing the Shoulder Sensitivity value will help resolve the two bands Chapter 5 Bands Size Scale The Size Scale field helps distinguish between trends in signal intensity and random intensity fluctuations It is the number of pixels in a vertical column that are taken together to determine whether a band is present The Size Scale parameter is similar to the Noise Filter in that it uses the size of objects in the image to determine the nature of those objects The default Size Scale setting of 5 pixels is optimal for most gel images It can be set to any whole number greater than or equal to 3 If a gel image has high levels of background noise a larger Size Scale is appropriate At low noise levels a smaller value is preferable You can also increase the Size Scale if the gel only has a small number of thick bands scanned at high resolution 5 2 6 Parameter Sets You can save detection parameters for use on similar images
30. and you will not be able to start a cut run 10 25 Quantity One User Guide 10 3 d Performing the Cut Run After you have identified all the cuts you want to make and selected the appropriate options you are ready to begin the cut run Close the door of the spot cutter before proceeding Click the Begin Resume button Plate Selection The Plate selection dialog box lists all the plates available for the cut run The table shows the plate name barcode ID plate size 96 well 384 well 96 tube wells available on the plate and used wells If this is a new cut run the number of plates listed is determined by the number of wells required for the cut run li Plates for Cut Run Wells needed 4 Wells available 96 Use Name Barcode ld Size Order Avail Used Edit EPllezavevos 96 wels 41 A2 96 0 EEJAdd plate X Cancel 2 Help Fig 10 24 Select plates In the Plate selection dialog box you can change the name of a plate and enter a barcode ID directly in the table The barcode helps to properly place the plates in the spot cutter 10 26 Excision To add a plate click Add a plate To remove a plate from the list click Delete If the Use checkbox is cleared the plate will be skipped in the cut run even if it has wells available To skip specific wells in a plate to reserve them for such things as standards click the icon under Edit in the table This opens a dia
31. button Select the category to be applied from the available categories on the list or select lt none gt Click on the Attribute button to specify an attribute Click OK to apply your selection to the Standards box J 2 Gel Layout In Diversity Database you can use the Gel Layout dialog box to compare samples across multiple gel images In Quantity One you can use it to enter general information about your image Appendix J Other Features To open the Gel Layout form select Gel Layout from the Edit menu Proteins Layout x Name Proteins Description 123 rat organs lt category lt category Date Run 03 Sep 1996 Lanes 15 Width fz O3mm Background Subtract On Off Lane Disk Size eo Lane Name CPM Loaded category Sli Protein ney z gt 1L NENNEN EEE 1o NENNEN B ie BEEN Els nm es Ele iung soz Ele fee n Close Help Fig J 3 Gel Layout dialog box The following information about your gel can be specified at the top of the Gel Layout form e Category and attribute information see above for the whole gel A brief description of the gel e Gel run date Number of lanes e Lane width Quantity One User Guide Lane based background subtraction and disk size Each lane in your gel has its own line on the Gel Layout form The following information can be specified for a lane The
32. e Texture gives the image texture Use the Scale function to scale the image This is useful for viewing shallow spots in the 3D Viewer If you lose the image because you moved it too far past the window border or rotated it and disoriented the view click Reset View to return the image to the original view Note Reset view does not change the scale factor To reset the scale factor close the 3D Viewer and click the box again to re open the 3D Viewer with the original scale factor 3 12 Chapter 3 Viewing and Editing Images 3 6 Image Stack Tool Use the Image Stack Tool to scroll through a series of related gel images You can easily compare bands that appear disappear or change size in different gels run under different conditions Note The images should be close to the same size with bands in the same relative positions You can use the Crop tool to resize images With all the images open select Image Stack Tool from the View menu The Image Stack Tool window will open Image Stack Tool L OL x Select gels to display Image l Raw l D Ima mbInage 2 Raw l D Ima Image 3 Raw l D Ima Image 4 Raw l D Ima EIN gt r Playback gt 4 Step t Auto slow I fast rImage order 4 Move displayed image w Done 2 Help Fig 3 8 Image Stack Tool Quantity One User Guide In the mage Stack Tool window all open
33. fo s3 nu 17 2 52 e t Bottom o o 7 oss ks 18 z es 3 3 Ela Bs rStep Ill Select Resoluti Attached to the outside of your GS 800 you will find a copy of the manufacturer s printout of the diffuse density values for each segment of the transmissive step tablet as well as a serial number for the tablet These exact density values must be entered into the software to associate a correct density value with each step on the step tablet Note Scanning in transmissive mode with incorrect step tablet values can cause significant errors in the reported densities of your scans First type the serial number for the tablet into the Tablet Serial Number field The Quantity Offset field does not apply in the GS 800 in transmissive mode This value should remain at zero Next enter the step tablet values into the appropriate fields under the Diffuse column After the step tablet is scanned the software will associate each density value with its corresponding segment on the step tablet The density values do not need to be reentered each time you calibrate When you are finished entering the transmissive step tablet values click on OK E 11 Quantity One User Guide Reflective Step Tablet For the reflective step tablet it is recommended that you use the default target values in the software Although you can edit these values based on your own measurements and testing the default values have been preset based on careful testi
34. promted to give it a new name This protects the backup file and preserves it from any changes l 6 c Imaging Area Size The imaging area is the area of the sample in centimeters that is captured by the camera and displayed in the scan window To specify the size of this area enter a dimension in the appropriate field under Imaging Area in the Options dialog box Click on the Options button in the acquisition window to open this dialog When you change one imaging area dimension the other will change to maintain the aspect ratio of the camera lens The imaging area will change depending on your zoom factor For example if you have zoomed in on a area that is 4 5 x 3 5 cm then you would enter 4 5 for the width 3 5 for the height would be calculated automatically 1 17 Quantity One User Guide Note Your imaging area settings must be correct if you want to do 1 1 printing They must also be correct if you want to compare the quantities of objects e g using the Volume Tools in different images The imaging area dimensions also determine the size of the pixels in your image i e resolution A smaller imaging area will result in a higher resolution 1 6 d Auto scale Transform Auto scale Transform after Acquisition allows the user the option of having the image automatically perform the Auto Scale transform function upon completion of image acquisition This eliminates the need to transform an image or re scan an image when
35. the acquisition time was too short or the iris not opened enough To enable the auto scale transform function check the box labeled Auto Scale Transform after Acquisition in the Options dialog l Other Features Display Options x Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig l 10 Other VersaDoc acquisition window features Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Transform command 1 18 Appendix I VersaDoc File Size of Images Image File Size shows the size of the image file you are about to create This size is determined by the resolution of the camera and any binning you perform when capturing the image If you do not have enough computer memory for the specified file size an error message will appear when you attempt to acquire an image Macintosh users can increase the application memory partition See your Macintosh computer documentation for guidance 1 19 Quantity One User Guide 1 20 Appendix J Other Features The following features are available in Quantity One but have more utility in its more powerful companion application Diversity Database You can examine your gel images in Quantity One and then database them using Diversity Database J 1 Categories and Attri
36. 1 8 Zoom BOK s deett D im P Uter ue ete E 3 2 Zoom commands imitating in other windows eeeeeeee 2 18 Zoom In Zoom QUE i aet ea tete te eI iR ge pere te herr tire 3 3 Zoom tools Changing behavior i eei benepuetei petere ree 2 17 Index 16 The Discovery Series Software Software License Agreement READ THIS THIS IS A LEGAL AGREEMENT BETWEEN YOU THE CUSTOMER AND BIO RAD LABORATORIES INC BIO RAD CONCERNING THE ENCLOSED COMPUTER PROGRAM THE PROGRAM YOU SHOULD REVIEW THE FOLLOWING TERMS AND CONDITIONS OF THIS AGREEMENT CAREFULLY BEFORE OPENING THIS PACKAGE BY OPENING THIS PACKAGE YOU INDICATE YOUR ACCEPTANCE OF SUCH TERMS AND CONDITIONS IF YOU DO NOT AGREE WITH ANY OF THEM YOU SHOULD PROMPTLY RETURN THE PACKAGE UNOPENED YOUR MONEY WILL BE REFUNDED LICENSE Pursuant to the license granted to you by this Agreement you may a use the Program on a single computer b copy the Program into any computer readable or printed form for back up or modification purposes in support of your use of the Program on the single computer certain programs however may include mechanisms to limit or inhibit copying they are marked copy protected c modify the Program and or merge it into another program for your use on the single computer and d transfer the Program and license to another party if the other party agrees to accept the terms and conditions of this Agreement Any portion of the Program merged
37. 14 Security Preferences The following options are available on the Security tab of the Preferences dialog box Enable 21 CFR Part 11 mode If this box is unchecked all other options are inactive To activate the other options and CFR mode check this box Program times out if inactive When this box is checked the user must enter their password to resume using Quantity One The minutes field indicates the amount of time that must elapse before the application times out Require login on startup Check this box to require the user to enter the user s password when the application opens This password is the same password as the one used for logging on to Windows This is an added security measure to prevent unauthorized users from starting Quantity One on a machine where they are not logged in Automatically create backup for new images This option when selected creates a backup file when you save a newly acquired or imported image Locate the backup file in the same folder as the original See Appendix K 21 CFR Part 11 for further information 2 22 Chapter 2 General Information Secure Mode Secure mode is not associated with CFR However you can enable Secure mode when CFR is enabled Secure mode prevents any user from making changes to raw image data on the local machine If a user opens any image on a machine where Secure mode is enabled any commands that change the data such as Subtract background Filter Wizard
38. Batch mode New batch pen batch Save Count Load Nest Batch File batchool xls Count Name Colony Counting lsc 1999 05 17 l hr O Count Comment Fig 8 8 Batch Mode controls Creating Opening a Batch File To create a new batch file click on the New Batch button This will open a dialog in which you can specify the name and location of the spreadsheet you want to create Write counts to file I2 x Save in 310 e E 2 backup File name batch001 Save as type Spreadsheet File xls Cancel Fig 8 9 Creating a batch file When you click on Save the new batch file name will be displayed in the Colony Counting dialog Chapter 8 Colony Counting To open an existing batch file click on the Open Batch button This will open a similar dialog Select the Excel file you want to open from the appropriate directory Naming Saving a Count Enter a name for the count you want to save in the Count Name field or use the default name the file name plus a time stamp Enter any comments in the Count Comment field This data will be included in the spreadsheet To save the currently displayed count to the batch file click on the Save Count button The number of colonies as well as associated count settings will be added to the spreadsheet After you have saved the current count the Save Count button will become deactivated If you adjust the count in any way the button will become acti
39. CFR Part 11 System Security features of Quantity One Quantity One utilizes these security features in concert with the operating system to assist the laboratory research institution etc in becoming CFR compliant These features include audit trail electronic signature and secure user login Note Bio Rad makes no claim that Quantity One is CFR compliant in and of itself nor does it guarantee compliance for the user The organization must establish policies and standard operating procedures that work in conjunction with the tools provided by Bio Rad to ensure compliance with 21 CFR Part 11 Audit Trail Section 11 10 e of 21 CFR Part 11 requires the use of secure computer generated time stamped audit trails to independently record the date and time of activities within the system In System Security mode Quantity One automatically records any events that affect data and analysis in the Audit Report For instance such things as K 1 Quantity One User Guide cropping and band detection are recorded in the Audit Report while transform will not because it does not affect the data Although there are software controls to detect modification of data protection of the data from deletion must happen at the level of the operating system As a result data must be stored only on an NTFS partition Access to directories where data is stored should be granted only to those who need to access that data Ideally access for a single user wi
40. Cancel Fig 4 18 Setting the array cell height When you click on OK the cell brackets will adjust to the specified height If you aren t sure of the exact height you can experiment with different values Select Array Cell Width and enter the width in millimeters of all the cells in the array E Quantity One x A Enter default sample width 200 73 mu Cancel Fig 4 19 Setting the array cell width When you click on OK the cell brackets will adjust to the specified width If you aren t sure of the exact width you can experiment with different values Analyzing Array Data When the brackets fully enclose each cell in the array you are ready to analyze the data You can display various measures of cell quantity on the image using the Band Attributes command on the Band menu With the Band Attributes dialog open select from Peak Density Average Density Trace Quantity Relative Quantity and other measures You can also report these values by selecting Lane Reports from the Reports menu 4 19 Quantity One User Guide To use known quantities to calculate unknowns you can use the Quantity Standards function see section 6 3 Quantity Standards 4 20 5 Bands After you have defined the lanes on the gel image you can automatically identify and quantitate bands using a set of adjustable parameters Note You can quantitate bands arrays or other objects outside of lanes using volumes See Ch
41. Expose Qro O Acquire Exposure Time sec Can see Exposure Time e automatically changing fo 03 ks O Freeze Fig C 4 Selecting Auto Expose Once an image has reached the specified percentage of saturated pixels it is captured and displayed in the video display window Auto Expose is automatically deactivated and the exposure time appears active in the Exposure Time field C 6 Appendix C ChemiDoc At this point if you are in UV or White image mode Manual Expose will be automatically activated If you are in Chemi mode the Freeze button will be automatically activated Note If you are having difficulty auto exposing your sample you can use Manual Expose to adjust your exposure time directly Most non chemi applications only require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button In UV or White image mode Manual Expose is automatically activated after Auto Expose is complete Step Ill Acquire Image Manual Expose active button appears selected Auto pr ive Exposure Time field active Q mose O ea adjust number of seconds EOS Os zi BEEN Exposure Time sec mx eja O Freeze Fig C 5 Setting a manual exposure With Manual Expose activated you can adjust the exposure time directly by changing the num
42. Hydrate Every 1 0 minutes EX Define hydration area Fig 10 19 Hydrate option 10 22 Excision To determine which area you want to hydrate click Define hydration area then click and drag on the image to create a box To redraw the hydration area click and drag again If you choose not to define a hydration area then the EXQuest Spot Cutter will hydrate the entire cutter stage Wash Enter the wash volume to flush through the cutter tip Specify Options Cut Plate Hydration Wash Wash volume 50 0 ul Fig 10 20 Wash option 10 3 c Specify Cuts Under Specify Cuts determine whether to use Lane Excision or Volume Excision Lane Excision If you select Lane Excision use the Lane tools to define lanes in the image See Section 4 1 Defining Lanes for information using the Lane tools 10 23 Quantity One User Guide Specify Cuts Entire lane Vertical Eg 40 Horizontal 40 Volume overlay ES M 25 I 12 4e Show excisions on image Fig 10 21 Specify Cuts using Lane excision Because of the circular shape of the cutting tip it is necessary to overlap the cuts to some degree to reliably obtain a sample of everything in the lane Use the vertical slider to adjust the percent of overlap Zero will give a single column of cuts and 100 will staggers the spots at approximately 45 degrees Use the horizontal slider to stagger the cuts so that a complete gel piece is o
43. Image mode ehe Rede re E OR ERRORES A 5 Imaging area at A 13 B 12 Invert display tai e EpOS A 9 Liyeamage display rette teet eere pter ee tede te ede eee cles A 4 Manual expose ipee eene eene e A 6 OPUONS EC A 11 B 11 Positioning the sample essent E ene A 4 Saturated pixels highlighting eee A 9 B 9 Saving AM AGES oerte eer ee eet reti ae gebe A 11 B 11 Simulation mode 1 eese eet entente ertet A 2 B 2 UV mode 25 estat ati Me eA ERG SE ee ee E ee A 5 Video C t oce tee pite tee ie Fe edite e ees A 2 Video display window sess A 3 Video printing footer info sessssssseeeeeeneenne A 13 B 13 White light mode eerte Peer cio A 5 Gel Doc XR Acquiring the Imagen neoe ea EE nnne nennen nennen nennen tenens B 5 Annotating Images rinitis ii B 10 Ato expose serae O BEA een B 5 Display Set ngs hinc oia ee ii B 8 Pr dene ee ES B 7 Image moden RR eo gti leet icri ieri B 5 Invert displayecic hehe Aen dd Eee B 9 LAV IMAGE display oen eed sit coves inier ne eere urere eerie B 4 Manu l expose uei erede ei OR B 6 Positioning the sample 5 eec Hr reete reet eee iii B 4 UN Mode a ORL e B 5 White light mode orita e e ecd e Ho etie B 5 Gel Doc XR display window eese ene een enne B 3 Gel Layout form totor J 2 Grab so sos O eat ltd 3 3 GS 800 Imaging Densitometer Index 6 Applic
44. In the event that Quantity One cannot locate the spot cutter a warning dialog box displays Check your connections and click Retry You also have the option of running Manual Excision in simulation mode Note See the spot cutter hardware manual for instructions on installing the platform camera and associated hardware and software 10 6 Excision The Manual Excision tool is designed to guide you through the spot cutting procedure Click on the link for more information about each step Acquire Image In this step you capture an image of your gel or membrane using the spot cutter camera e Specify the Cut Run Options These options can be adjusted for each cut run e Specify Cuts In this step you identify the spots in the image that you want to cut e Performing the Cut Run In this step you perform the cut run When the run is complete you can acquire a confirmation image 10 2 a Acquire the Image Before acquiring an image select the light option appropriate for your gel membrane stain type e White Trans gels and PVDF membranes e UV EPI fluorescent gels Acquire Image C White Trans e e UV EPI fh cquire image Time B secs Fig 10 5 Acquire Image If UV light is selected specify an exposure time in seconds in the field Also make sure that the UV lamp switch on the front of the cutter is on Note For Sypro Ruby stains an exposure time of 20 30 seconds is recommended When you have selected
45. K 4 Appendix K CFR Module K 2 b Secure User Login Quantity One utilizes the Windows security model When you open Quantity One Quantity One determines the user name by the ID of the user currently logged on to that system If you have Require Login on startup checked on the Security tab of the Preferences dialog box then you must enter your Windows password to start Quantity One To view the current user information select Show User Information from the CFR menu The message displays the username full name and group membership for the currently logged in user Note Although Windows XP allows you to switch users without logging off you must first close Quantity One before switching users Windows 2000 does not allow the ability to switch users K 2 c Audit Report The Audit Report included with CFR mode tracks all changes to actual data of images as well as any events associated with analysis K 5 Quantity One User Guide zip xi Time User Description Detail 2003 08 04 09 27 38 Matthew Halsey Open Converted from TIFF Original file name Proteins tif 2003 08 04 09 27 48 Matthew Halsey Save As Old file name Proteins 1sc New file name CFR Proteins 1sc 2003 08 04 09 27 48 Matthew Halsey Save Created backup file Backup of CFR Proteins 1sc 2003 06 0409 27 53 Matthew Halsey New Lane Created Initial Lane Number 2003 06 04 09 27 53 Matthew Halse
46. MAX Fluor 5 MAX2 VersaDac Personal FX ENT Print Export to TIFF Image Export to JPEG Image Exit Ctrl Q Fig 2 7 File menu 2 2 a Opening Images To open a saved image select Open from the File menu or click on Open button on the main toolbar This opens the standard Open dialog box for your operating system 2 7 Quantity One User Guide Macintosh version Select a File Cp Sample Images Colony Counting 1SC MicroSatellite 1SC Proteins 1SC c Disk 1 Eject open O Show all files Windows version Open 21x Look in a Div Database e c E3 Name Sief Type Modified Gi backup File Folder 5 9 2000 3 25 PM Database 1 ddb 109KB TDS DivDB Databas 5 9 2000 3 25 PM Database 2 ddb 26KB TDS DivDB Databas 5 3 2000 2 14 PM Database 3 ddb 33KB TDS DivDB Databas 11 4 1999 10 02 AM E Image 1 1sc 483KB TDS 1 D Scan file 4 12 2000 3 43 PM E Image 2 1sc 589KB TDS 1 D Scan file 11 4 1883 10 02 AM E Image 3 1sc 610KB TDS 1 D Scan file 11 4 1999 10 02 AM E Image 4 1sc 596KB TDS 1 D Scan file 11 4 1833 10 02 AM E Image 5 1sc B22KB TDS 1 D Scan file 5 3 2000 2 15 PM E Image 6 1sc 572KB TDS 1 D Scan file 11 4 1999 10 02 AM E test 1sc 70KB TDS 1 D Scan file 5 3 2000 1 56 PM My Network P Fie name Fl Files of type Loadable Images amp Databases Cancel Fig 2 8 Open dialog box In the dialog box open a file by double clicking on
47. Name field Next click on the buttons next to the Filter Illumination Gain and Binning fields to change these settings Note Note that if you select trans white illumination you will need to place the white light conversion screen on the sample stage A higher Binning setting 2x2 3x3 provides optimal sensitivity for low light applications such as chemiluminescence In this mode the pixels in the camera are binned e g four pixels are combined into one to increase the amount of signal per pixel without increasing noise Note that combining the pixels results in a reduction in the resolution of the image Selecting a higher Gain 4x provides higher sensitivity without reduced resolution however noise will also increase This is useful for faint signals bright spots will saturate Selecting a lower gain 0 5x is useful for brighter images that tend to saturate A gain setting of 1x provides the greatest dynamic range Click OK to implement your changes After you have created an application you can select it from the application tree by selecting Custom and the name you created Appendix I VersaDoc You can edit a custom application by selecting Custom Edit and the name of the application You can also use this feature to create a new custom application from an existing one You can delete a custom application by selecting Custom Delete and the name of the application l 3 Step Il Position Focus Befor
48. Nucleic Acid Gels Protein Gels Blotting Coomassie Blue Densitometry Copper Stain PROS ange SYPRO Red SYPRO Ruby Zinc Stain ac Custom Photographs Clear White EPI 0 5x Gain lxl Bin Fig I 3 The application tree in the VersaDoc acquisition window Note If you select an application that requires trans white illumination you will need to place the white light conversion screen on the sample stage area To exit the tree without selecting press the Esc key or click outside the dialog box Your selection will be displayed below the Select button Custom Applications If your application is not listed if you want to use a user installed filter or if you want to set the gain and bin settings manually you can create and save your own custom applications From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings Quantity One User Guide Nucleic Acid Gels Protein Gels Blotting Densitometry Channel 3 Channel 4 Custom SEEN Edit Delete Colorimetric od Custom Application 1 dF AA Custom Application 2 Create a new VersaDoc Applical x Name Custom Application 3 Filter 530DF70 Illumination Trans UV x 0 5x v Gain Binning m Cancel OK Fig 4 Creating a new custom application Enter a name for your application in the
49. Pages Export Reformat Fig 12 2 Example of a report window The standard report window has buttons for printing the report scrolling through the screen pages of the report and exporting the report to a spreadsheet application Some report windows also have a Reformat button for changing the data display To close a report window click on the Close box in the upper right corner of the window 12 2 Chapter 12 Reports Scrolling If a report has multiple pages the scroll buttons in the report window become active Use them to scroll to the next page previous page first page or last page of the report You can also enter a specific page number in the field to skip to that page Printing Print the report using the Print One Page or Print All Pages commands in the report window Click on either of these buttons to open a smaller version of the standard print dialog described in the next chapter Windows version Detail Report by Lane Pages All First Last Printer mpci HP LaserJet 5 5M Properties Orientation Portrait Landscape Copies DK Cancel Help Macintosh version Quantity One Detail Report by Lane Go Cancel Help Fig 12 3 Print Report dialog Print One Page prints only the current screen page Print All Pages prints all the pages in the report 12 3 Quantity One User Guide Note If you select Print All Pages the number of pages printed may be le
50. Pixel Count ase by 756 Memory Size 264 30 Kb OK Cancel Help Pixel Count and Memory Size reduced Fig 2 11 Reduce File Size dialog box before and after pixel size increase Lower the resolution by entering lower values in the Pixel Count fields or higher values in the Pixel Size fields see the figure for an example Quantity One User Guide Note Since with most 1 D gels you are more concerned with resolving bands in the vertical direction than the horizontal direction you may want to reduce the file size by making rectangular pixels That is keep the pixel size in the y dimension the same while increasing the size in the x dimension When you are finished click on the OK button A pop up box will give you the option of reducing the file size of the displayed image or making a copy of the image and then reducing the copy s size Reducing the file size is an irreversible process For that reason we suggest that you first experiment with a copy of the image Then when you are satisfied with the reduced image delete the original Quantity One x A Confirm reduce resolution Ready to reduce Protein v2 1D Scan to 400 43 169 39 micron resolution The image size will be 268 756 pixels If you choose to reduce in place the descriptions of objects in this scan will be lost Only the scan image itself will be retained Reduce Reduce and Copy Cancel Fig 2 12 Confirm Red
51. Point Molecular Weight and Normalized Rf Note Rf relative front expresses the distance a band has traveled down a lane as a fraction of either the total length of the lane or the vertical distance from the top of the lane to the bottom the calculation method can be specified in the Preferences dialog This provides a generic measure of the positions of bands in lanes Normalized Rf is derived from relative front and includes the results of modeling across the gel that comes when multiple lanes of standards are defined on the image Such modeling is designed to take into account any distortion or smiling across a gel To specify a set of units not on the list click on the New button and specify the unit s parameters in the dialog To edit a set of units select them and click on Edit Chapter 6 Standards and Band Matching Quantity One x Edit Unit Name Short Name Unit Name Scaling Log Linear Order Ascending Descending Delete Done Fig 6 5 Units dialog Note Ascending means that bands of higher molecular weight or isoelectric point are at the top of the gel image and bands of lower molecular weight or isoelectric point are at the bottom of the gel image To select a set of units select them from the list and click on Select then click on OK The Standards dialog will open Here you can enter values for the standards apply them to the bands on the image and save them as a set for future use 6 1
52. Quantity One User Guide Note Freeze is automatically activated if you adjust any of the subsequent controls e g Video Print Image Mode Display controls etc A 5 Step IV Optimize Display The Display controls are useful for quickly adjusting the appearance of your image for output to a video printer Adjusting these controls will automatically freeze the video display and allow you to alter the image within the Gel Doc EQ window Step IV Optimize Display 34 High zess j Low fo IES Gamma 1 00 xj Highlight Saturated Pixels L Invert Display Auto scale Reset Fig A 7 Display controls These controls are similar to those in the Transform dialog box Note The Display controls will only change the appearance of the image They will not change the underlying data High Low Sliders If Auto scale doesn t give you the appearance you want you can use the High and Low sliders to redraw the image yourself In white light mode dragging the High slider handle to the left will make weak signals appear darker In UV mode dragging the High slider handle to the left will make weak signals appear brighter Dragging the Low slider handle to the right will reduce background noise You can also type specific High and Low values in the text boxes next to the sliders Clicking anywhere on the slider bars will move the sliders incrementally A 8 Appendix A Gel Doc Gamma Slider Some
53. Table 1 Recommended Exposure Times and Lenses on page 9 After you have positioned your sample click on the Focus button and look at the image in the acquisition window while adjusting the focus on the camera lens While focusing the camera will limit its focus to a small portion of the sample this will not affect any zoom lens adjustments you may have made See the VersaDoc User Manual for details on focusing When you click on Focus the light inside the camera box automatically switches on To turn the light off while positioning deselect the Light On checkbox When you are finished focusing click on the Stop button 1 4 Step Ill Set Exposure Time The exposure time is the period of time an image is integrated on the CCD The effect is analogous to exposing photographic film to light Setting an Exposure Time Different applications have different optimal exposure times If you are imaging using multiple channels you can select a different exposure time for each channel First select the channel using the tabs then select the appropriate exposure time for that channel See Table 1 Recommended Exposure Times and Lenses on page 9 You can enter an exposure time in seconds directly in the field or use the Arrow buttons to adjust the exposure time in 10 percent increments a Ill Set Exposure Time sec BE eje One Fig I 5 Selecting an exposure time Appendix I VersaDoc The following table
54. Unknown Calibration Not Calibrated OK Help Fig 5 12 Band Information dialog The lane and band number of the band you clicked on are listed at the top of the dialog Enter new numbers in these fields to display information for a different band The band set and band type will be listed if known Other information includes Note Chapter 5 Bands Relative front The distance of the band from the top of its defined lane divided by the total length of the lane The lane length can be determined either by measuring a vertical line from the top of the lane to bottom or if the lane is curved by measuring along the length of the lane Set the preferred measuring method in the Preferences dialog see section 2 5 e Application Note that normalized Rf is derived from relative front however normalized Rf is calculated only for bands that have been modeled using standards or band sets and can change based on the modeling Molecular Weight Isoelectric Point Base Pairs other units This value is determined by the type of standards defined for the gel the band s position in the lane and any modeling performed on the gel using band matching or multiple lanes of standards to compensate for gel distortion Peak density The intensity value of the band s peak Average density The total intensity of the rows of pixels used to generate the profile of the band divided by the number of rows Trace qty The band quantity as m
55. User Guide Volume Regression Curve Dialog Regression Curve Plot Gel name Proteins 1SC Concentration Display Options J Show Name r Regression method Point To Point Linear Cubic Quadratic Logistic Spline OK ok Cancel Pm Print Adjusted Volume r T 4 00 8 00 12 0 16 0 20 0 Regression Equation Conc 64 5 236 1 Vols 12 6 2 33 236 Fig 12 16 Volume Regression Curve window On the curve each standard volume is marked by a red X and each unknown is marked by a blue triangle The X axis is the adjusted volume and the Y axis is the concentration based on the standards you have marked on the image To display the numbers and names of the volumes click on the Show Name checkbox Select the preferred regression method from the list of option buttons The regression equation for the selected method is displayed in the lower left of the window 12 24 Chapter 12 Reports To print the curve click on the Print button To close the window click on the OK button 12 8 VNTR Report The VNTR Report displays the calculated VNTR data see section 11 2 Variable Number Tandem Repeats Select VNTR Report from the Reports menu A pop up box will ask if you want to display the gel image in the report Tandem Repeat Report Proteins rat organs June 18 1998 Mol Wt Raw Rounded Band Type Band KDa Repeat
56. a border is useful for determining the edges of faint images Click the Page Setup button to open the Page Setup dialog box Note that although you can print directly from the Print Settings dialog box by clicking either Print or Print Current Page the print settings affect each image printed using the Print Image command Note The Print Settings dialog box attempts to accurately display the image exactly how it will print However in some cases such as images with large pixel dimensions set to Print Actual Size small differences may be noticed between the preview window and the actual print 13 4 Image Report The Image Report displays the image and basic image information including the image dimensions pixel size date of scan type of imager etc 13 3 Quantity One User Guide To print an image report for a particular gel select Image Report from the File Print submenu This will open a smaller version of the standard printer dialog box Image Report For Proteins Raw 1 D Image Description rat organsO Directory C Program Files Bio Rad New Discovery SeriesiSample Images 1D Filename Proteins 18C Scan Date 29 Feb 1996 00 00 Scanner PDI 4200e Pixel size um X 169 5 Y 169 4 Scan Area mm X 107 3 Y 128 1 Data Range 3 00 OD Scan Pixels X 633 Y 756 Memory Size 469 27 Kb Scan Parameters NO SCAN PARAMETERS AVAILABLE Gel Background aj 3j ESSE je Fig 13 2 Image Report Note TIFF ima
57. a period of ninety 90 days from the date of delivery to you as evidenced by a copy of your receipt This warranty is limited to you and is not transferable During the 90 day warranty period BIO RAD shall a replace any media item not meeting the foregoing warranty and which is returned to BIO RAD or an authorized BIO RAD dealer Authorized Dealer with a copy of your receipt or b if BIO RAD of the Authorized Dealer is unable to deliver a replacement media item which is free of defects in materials or workmanship you may terminate this Agreement by returning the Program and you money shall be refunded The following warranty does not extent to any media item which has been damaged as a result of accident misuse abuse or as a result of service or modification by anyone other than BIO RAD or an Authorized Dealer EXCEPT AS EXPRESSLY SET FORTH ABOVE THE PROGRAM IS PROVIDED AS IS AND NO OTHER WARRANTIES EITHER EXPRESSED OR IMPLIED ARE MADE WITH RESPECT TO THIS PROGRAM INCLUDING WITHOUT LIMITATION THE IMPLIED WARRANTIES OF MECHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE BIO RAD WITHOUT LIMITATIONS EXPRESSLY DISCLAIMS ALL WARRANTIES NOT STATED HEREIN YOU ASSUME THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PROGRAM SHOULD THE PROGRAM PROVE DEFECTIVE YOU AND NOT BIO RAD OR AN AUTHORIZED DEALER ASSUME THE ENTIRE COST OF NECESSARY SERVICING REPAIR OR CORRECTION SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTI
58. an array Resizing an Array To resize an array overlay make sure it is selected then position the cursor over the dot at the center of one of the corner cells Green lines will appear connecting the array frame at the four corners Fig 7 13 Resizing an array Hold down the mouse button and drag the array frame in or out to compress expand the array 7 15 Quantity One User Guide Resizing the Array Cells To resize the individual cells in the array magnify any individual cell and move the cursor over the cell border or corner anchor point in the case of rectangles until it changes to a cursor with an adjustment symbol Hold down the mouse button and drag to move the border in or out All the cells in the array will be resized accordingly Fig 7 14 Resizing an array cell Copying an Array To copy an array within the same image select it then hold down the Ctrl key while dragging it The copy will be created and dragged to the new position To copy an array between images select it then click on the Copy to Clipboard button on the Volume toolbar Open or select the image you want to copy to and click on the Paste from Clipboard button The copied array will be pasted into the new image in the same relative position it was copied from Note If you are pasting into an image with a different pixel size i e resolution you will receive a message that the placement of the copy may not be exact Click on
59. at the top of the dialog 6 25 Quantity One User Guide The calibration curve is generated by plotting the quantities versus the intensities of the known bands The intensities of the bands can be measured in several different ways Click on the Measure button to display a list Quantity One x Measured Attributes Trace Density OD x mm Contour Density OD x mm 2 Peak Density 0D Average Density OD al Fig 6 20 Band attributes to measure for relative quantity The options in the Measure dialog are Trace density The intensity of a band as measured by the area under its intensity profile curve Units are intensity x mm Contour density The intensity of a band that has been defined using the Contour or Draw Band tools see section 5 8 Irregularly Shaped Bands in Lanes It is the sum of the intensities of all the pixels inside the band boundary multiplied by the area of each pixel Units are intensity x mm Peak density The intensity value of a band s peak Average density The total intensity of the rows of pixels used to generate the profile of a band divided by the number of rows Identifying the Known Bands in the Gel The dialog includes three buttons that can be used to select the known bands in the gel To select bands one at a time click on the Band button then click on each band of known quantity Each band will be highlighted If all of the known bands are the same
60. bin setting used for the selected application If you do not have enough computer memory for the specified file size an error message will appear when you attempt to acquire an image Macintosh users can increase the application memory partition See your Macintosh computer documentation for guidance D 15 Quantity One User Guide D 16 Appendix E GS 800 Imaging Densitometer Fig E 1 GS 800 Imaging Densitometer Before you can begin scanning images with the GS 800 Imaging Densitometer your instrument must be properly installed and connected with the host computer See the hardware manual for installation startup and operating instructions PC Only A Note About SCSI Cards The GS 800 is connected to your computer by a Small Computer System Interface SCSI cable To use the GS 800 you must have a SCSI card installed in your PC If E 1 Quantity One User Guide you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or pr
61. can also type specific High and Low values in the text boxes next to the sliders Click anywhere on the slider bars to move the sliders incrementally Log High Low Sliders changes the feedback from the slider handles so that when you drag them the slider markers move a shorter distance in the histogram This allows for finer adjustments when the data is in a narrow range Frequency Distribution Full scale Low High Log In this example the range of data in the image is very limited Low slider remaps background noise to white High slider remaps weaker signals to black Low High magnifies Log scaling enables you to Can better distinguish background area between sliders better distinguish peaks noise from real data y Frequency Distribution Full scale Low High X Log Fig 3 15 Two views of the Frequency Distribution histogram 3 21 Quantity One User Guide Gamma Slider Some images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashion Adjust the Gamma slider handle to expand or compress the contrast range at the dark or light end of the range This is reflected in the Transform Plot and Preview Window 3 8 c Other Features Full Scale and Low High The Full Scale and Low High option buttons adjust how the range of data in the image is displayed in the Frequency Distribution histogram and Transform Plot They do not change how the data is displayed in th
62. eee 1 2 1 3 Gel Quality iii 1 3 1 4 Quantity One Workflow 5 tried ier ttes tete 1 4 1 5 Computer Requirements nette reete rin eere be tpe dieses 1 5 1 6 Installation nee mee et Rees 1 6 1 73 Software Jacense s rhe etn be e donee 1 9 1 8 Downloading from the Internet essere 1 13 1 9 Quantity One Basic 5 onum tdt eee te eire isis 1 14 1 10 Contacting Bio Rad 35 nett tete eee 1 14 General Operation eeeeeeeeeeeeeeeennnnee 2 1 2 1 Menus and Toolbars essent enne 2 1 22 File Commands itte Eee ERU ID eei eerte cias 2 6 2 3 Imaging Device Acquisition Windows essere 2 14 2 4 ERI yaa oie isd Ad 2 15 2 5 Preferences iiss cases eh v ettet o e dien 2 15 2 6 User Settings eg eee omite e Fre OP ER Dp 2 23 Viewing and Editing Images 3 1 3 1 Magnifying and Positioning Tools eee 3 1 3 2 Density LOOMS ct aerei teri hi toe dp etes 3 5 3 3 Showing and Hiding Overlays eee 3 6 3 4 Multi Channel Viewer eseeseeseeeeseeeeen nennen nennen nennen RSE 3 7 3 5 3D VI WE i iet ettet iia 3 10 3 6 Image Stack Tool eB ee 3 13 Quantity One User Guide 3 7 oI oi iita eer 3 14 3 8 Transform x nest REOR 3 17 3 9 Resizing and Reorienting Images seen 3 23 3 10 Whole Image Background Subtraction
63. etc cannot be renamed in the pop up list so be sure to remember which filter you insert into each position in the FX i e 690 nm filter 605 nm BPfilter To use an external laser click on the Laser button and select it from the pop up list i e 488 nm or 635 nm laser Otherwise use the default internal laser 532 1064nm Note The dual laser 532 1064 nm is not available in the FX Pro or the FX Pro Plus systems Click on the PMT Voltage button to select a standard voltage for your custom application or create a custom PMT voltage To select a custom voltage click on the Custom option In the dialog box adjust the slider to select a PMT voltage as a percentage of the maximum High Sample Intensity Quantity One Select a custom PMT voltage Medium Sample Intensity Low Sample Intensity Choose later Custom PMT Voltage ss High Sample Intensity puer pr Low Sample Intensity Low PMT Voltage High PMT Voltage Cancel DK Fig G 6 Selecting a custom PMT voltage Note For voltages above 80 of maximum you will receive a warning message that the high voltage could damage the PMT If you select Choose Later from the list of PMT voltages the choices of sample intensity will be displayed when you select your custom application Quantity One User Guide Finally enter a name for your application in the Name field and click on OK to implement your changes After you have created an application
64. etc as well as all analysis commands will be inactive grayed out To activate Secure mode select Secure mode Once checked you will be prompted for a new Secure mode password which will be required for disabling Secure Mode Note Secure mode only affects images opened on the local machine Images stored on a network can still be opened and modified on other machines where Secure mode is disabled 2 6 User Settings If Quantity One is on a workstation with multiple users each user can have his or her own set of preferences and settings In multiple user situations the preferences and settings are associated with individual user names Under Windows your user name is the name you use to log onto the computer On a Macintosh your user name is the Owner Name on the File Sharing control panel If you do not log onto your Windows PC or do not have a Owner Name on your Macintosh then you do not have a user name and your preferences and settings will be saved in a generic file 2 23 Quantity One User Guide 2 24 3 Viewing and Editing Images This chapter describes the viewing tools for magnifying and optimizing images This chapter also describes the tools for cropping flipping and rotating images reducing background intensity and filtering noise and adding text overlays to images These tools are located on the View Image Window and Edit menus Note The following chapters contain instructions for analyzing X ray
65. films wet and dry gels blots and photographs For the sake of simplicity these are all referred to as gels 3 1 Magnifying and Positioning Tools The magnifying and positioning tools are located on the View menu and Window menu some of these functions are also found on the main toolbar These commands will only change how the image is displayed on the computer screen They will not change the underlying data Quantity One User Guide SPANOS Toolbars View Entire Image Fi zoom Box 2 Grab Ctrl G Zoom In Alt F2 Zoom Out Ctrl F2 Plot Density gt Show Lanes and Bands F5 Hide Overlays F4 Multi channel Viewer Image Stack Tool 3D Viewer Fig 3 1 Viewing functions on View menu and main toolbar Zoom Box Use Zoom Box to select a small area of the image to magnify so that it fills the entire image window Click on the Zoom Box button on the main toolbar or select the command from the View menu Then drag the cursor on the image to enclose the area you want to magnify and release the mouse button The area of the image you selected will be magnified to fill the entire window Chapter 3 Viewing and Editing Images ENE 1 Click on Zoom Box button 2 Drag box on image 3 Boxed region is magnified to fill window Image 4 1D Scan OF x Image 4 1D Scan Oy x Fig 3 2 Zoom Box tool Zoom In Zoom Out These tools work like standard magnifying t
66. function you must normalize the bands in the gel for quantity across all the lanes With the gel open and matched select Normalize from the Match menu and click on a distinct well resolved band that appears in every lane of the gel The quantity of that band in each lane will be set to 100 and the quantities of the other bands in the lane will be normalized to that band If the band is not present in a lane the normalized quantity for that lane is zero 1 Liang P and Pardee A 1992 Science 257 967 970 Quantity One User Guide See section 6 2 d Normalizing for Quantity for more information on normalization 11 1 b Differential Display Searches After you have normalized the gel select Differential Display from the Analysis menu A dialog will open in which you can specify certain parameters 23 Differential Display x Search for Outliers Trends r Differs By 1 Percentage 80 00 el Quantity S CELUM al Normalized Quantity Close Help Fig 11 1 The Differential Display dialog box You can perform two types of searches on bands across a differential display gel an Outliers search or a Trends search Both are determined by the Percentage and Quantity values that you specify in the Differs By field Outliers Select the Outliers button to highlight bands with a normalized quantity that differs from the mean normalized quantity for that band type by the specified percentage and
67. gels are listed in the field to the right of the display window To select an image to display click on a gel name The name will appear highlighted with an arrow and the image will appear in the window Click on another gel name to display that image Buttons for various viewing tools are aligned next to the mage Stack Tool window These commands will change the display of all the images in the stacker at once e g magnifying one image will magnify the same relative area in all the images Using the controls below the list of names you can reorder the images and or scroll through them in the stacker Reordering Images To reorder the images in the stacker first select an image name in the list and then click on the Move displayed image arrow buttons to move it up or down in the list Image Playback Using the controls under Playback you can scroll through the images in the stacker First highlight some or all of the gel names using Shift click or Ctrl click key commands With multiple images selected the Step arrow buttons become active Click on the arrow buttons to scroll through the list of selected gels Alternatively click on the Auto checkbox next to the arrow buttons to begin automatically scrolling through the list You can adjust the auto scroll speed using the Slow Fast slider 3 Colors Select Colors from the Edit menu to open a dialog in which you can adjust the colors of the image as well as windows buttons over
68. ias 6 31 Relative deviation sorene ani eee enne nennen trementem 6 28 Selecting bands of known quantity eese 6 26 Quick Guides sues ida 2 4 ReadyAgarose 96 Plus in inisee pe oeeo re SE E ea ener en enne trennen nens 9 1 Ready Ag rose Wizard esae rei iit ted o e E ect 9 1 Reduce Filesize ok A i aprios 2 12 Registration By fax e mall nap 1 12 By Internet estat 1 11 Registration form io id 1 10 Relative Erotic cari 2 20 5 14 Relative quantity ca 5 15 5 17 Reports ED Analysis ose ee a 12 8 Band Types ME 12 6 Compare Lane Images eese eren rennen 12 11 Idee O 12 4 L ness Ones ee RUE E UG 12 5 Matches DL E 12 5 Phylos netic Tree oe e ee e DU P be ie HERE 12 14 PLUM eti oad ai bed ect ed i e pte tle edet 12 3 Report window features occoooconocccocccoonnconcconononnoconcconnoron conc nronncnnnc enne enn enene ene 12 1 Similarity Matisse 12 19 VNTRA deleite dad de ey 12 25 Index 12 Volume Analysis onere e eet teer 12 20 Revert to Saved x cere eae ne Ie ore IMs 2 10 Rotating Images RO 3 26 Sample Images soe Ob peine nene ten iei 2 9 Saturated pixels highlighting eese enne 3 23 See also individual imaging devices Saving IMAGES sni eed e E e ERES 2 10 Show Hide Volume Label cuna oia 7 7 Similarity of lane based samples comparing see 12 9 Simulation mode see indiv
69. images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated A 1 Gel Doc EQ Acquisition Window To acquire images using the Gel Doc EQ go to the File menu and select Gel Doc EQ The acquisition window for the instrument will open displaying a control panel and a video display window A 2 Appendix A Gel Doc Gel Doc r Step Position Gel Q9 Live Focus IRIS ZOOM FOCUS 4 Open 4 Tele 4 Near Close Y wide Y Fa Show Alignment Grid Step Il Image Mode UV C White Step IIl Acquire Image Auto Manual Qs OE spose t Time sec b ele Q Freeze Step IV Optimize Display High 255 ta fo Gamma 1 00 Xi Highlight Saturated Pixels Step V Analysis Step VI Select Dutput Exposure Status Clnvert Display E Annotate ES Video Print Autoscale Reset Autoscale Reset Analyze E Save Options Help Fig A 2 Gel Doc EQ acquisition window The Gel Doc EQ video display window will open in live mode giving you a live v
70. images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashion Adjusting the Gamma slider handle changes the light and dark contrast nonlinearly Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background and visa versa This will only affect how the image is displayed on the screen not the actual image data Auto scale Clicking on Auto scale will adjust your displayed image automatically The lightest part of the image will be set to the minimum intensity e g white and the darkest will be set to the maximum intensity e g black You can then fine tune the display using the High Low and Gamma sliders described below Reset Reset will return the image to its original unmodified appearance A 6 Step V Analysis The Analysis step of the Gel Doc EQ acquisition window allows you to add annotations and analyze the newly acquired image A 9 Quantity One User Guide Annotate Clicking on Annotate will open a separate image window displaying the captured image The default name for the image will include the date time and user if known The Text Ove
71. in Display option is checked then the all details for each event are displayed in the viewer Otherwise only the first row of each event is displayed Similarly the Expand comments in Display functions the same way The AutoZoom on Detail Image Coordinates option is a convenient way to pinpoint the location an event took place in the image If the box is checked click the coordinates in the report viewer and the image will automatically zoom to the location of the event Note that this tool is only available when a specific location has been recorded in the report K 8 Appendix K CFR Module Printing and Exporting the Audit Report The Audit Report Viewer allows you to print or export the report for use in a spreadsheet application To print the report click either the Print Current Page button or the Print button Print Current Page is only available in print preview mode and allows you to print the currently viewed page The print all pages button opens the Pint dialog box where you can either print all pages or specify a print range Click Page Setup to change the print settings To export the report click the Export button This opens the Export Options dialog box where you can determine the delimiter tab or comma and the export destination save as a file or copy to the clipboard K 2 d Electronic Signature As was previously stated Section 11 3 b 7 of 21 CFR Part 11 defines an electronic signature as a computer dat
72. installation of the application dongle drivers and Roper support Insert the Discovery Series CD ROM into your Macintosh The TDS Mac folder opens on your desktop displaying the installers for The Discovery Series applications Double click on the installer for your application Y w Fig 1 4 Installation program icon Macintosh 1 8 Chapter 1 Introduction Insert the Discovery Series CD ROM The TDS Mac folder opens on your desktop displaying the installers for The Discovery Series applications Double click the installer for your application You must accept the license agreement to proceed with installation The ReadMe contains important information about the HSK Click Continue The installation wizard lists the types of installations in the pull down list If you want to install the application and documentation only select Easy Install from the list If you want to install drivers as well select Custom from the pull down list Remember to also select the application in custom install Note If the computer is going to be used to acquire images from Gel Doc EQ ChemiDoc EQ ChemiDoc XRS or the VersaDoc system then select the driver for your particular imaging device now Install Location identifies where the application will be installed If you want to specify a different location click the pull down button Click Install to proceed Once installation is complete click Quit To return to the installer clic
73. into another program shall continue to be subject to the terms and conditions of this Agreement If you must transfer the Program you must at the same time either transfer all copies whether in printed or computer readable form to the same part or destroy any copies not transferred including without limitations all modifications and portions of the Program contained or merged into other programs You must also reproduce and include the copyright notice on any copy modification or portion merged into another program YOU MAY NOT USE COPY MODIFY OR TRANSFER THE PROGRAM OR ANY COPY MODIFICATION OR MERGED PORTION IN WHOLE OR IN PART EXCEPT AS EXPRESSLY PROVIDED FOR IN THIS AGREEMENT If you transfer possession or any copy modification or merged portion of the Program to another party your license is automatically terminated TERM The license is effective until terminated You may terminate it at any other time by destroying the Program together with all copies modifications and merged portions in any form It shall also terminate upon conditions set forth elsewhere in this Agreement or if you fail to comply with any term or condition of this Agreement Upon such termination you must destroy the Program together with all copies modifications and merged portions in any form LIMITED WARRANTY AND REMEDIES BIO RAD warrants the media on which the Program is furnished to be free from defects in materials and workmanship under normal use for
74. lists the spot number in column one the X coordinates in column two and Y in three then you would enter two for column X and three for column Y Columns must be separated by tabs or by columns Click the browse button to locate the file you want to import Units indicates the measurements of the coordinates and Origin determines starting point for the measurements The image in the Manual Excision Tool displays the placement of the spots as orange circles before you complete the import If the cuts are out of position you can click and drag all cuts at once to better position them If you need to move cuts individually you can do so after import by clicking Add cut then clicking and dragging the cut you want to move When you are satisfied with the placement of the cuts click Done to complete the import Display Options Select the Well check box below the Manual Excision image window to display the well number associated with each cut request Select the Position checkbox to display the position of the cut in millimeters on the gel 10 3 Lane Volume Excision Lane Volume Excision functions much like Manual excision except that you define what to cut based on defined lanes or volumes Before using this tool make sure the spot cutter is connected is switched on and has warmed up then select Lane Volume Excision from the Excision menu The software will connect with the spot cutter platform and camera and the Manual Excis
75. mm mm E m mm Lane Trace Average pixel intensity across sample width gt Average intensity Band Profile Integrate under curve to baseline for band quantitation intensity x mm E Fig 5 2 Illustration of bracket quantitation Chapter 5 Bands When a band is quantitated the average intensity of each horizontal row of pixels within the brackets is calculated Next the number of pixel rows between the top and bottom brackets is determined Taken together these result in an intensity profile for the band Finally the area under the profile curve to the baseline is integrated resulting in units of intensity x millimeters This is the trace quantity of the band Note Because the profile of an ideal band conforms to the shape of a Gaussian curve band profiles can be fitted to a Gaussian model The band quantity can then be quantitated from the area under the Gaussian curve This is the best to resolve overlapping or closely spaced bands in images See section 5 7 Gauss Modeling Bands for details 5 2 Band Detection The Detect Bands command automatically detects the bands in defined lanes based on parameters that you select Note Before detecting bands you should subtract background from the lanes using the Lane Background command see section 4 2 Lane Based Background Subtraction Select Detect Bands from the Band menu or toolbar The Detect Bands dialog will open Quant
76. number and where space permits the band number The Y axis is labeled with quantitative values If the bands in the match group span the entire image window the histogram will not include bars for the bands in the left most lanes because of the space required for labeling the axis To avoid this problem decrease the size of the image using Zoom Out until there is blank space between the left side of the window and the first band of the match group Redisplay the histogram If one or more bars are still not included continue decreasing the magnification until you can see all the data 6 3 Quantity Standards From bands of known quantity you can generate a calibration curve for determining the quantities of all the bands in lanes or cells in lane based arrays To quantitate bands outside of lanes see Chapter 7 Using the Quantity Standards dialog you plot the quantities of the known bands against their intensities to generate a calibration curve You then apply this curve to unknown bands in the current gel as well as other gels To create a calibration curve the quantities of at least two bands must be known The greater the number of known bands and the wider the range of their values the more accurate the calibration curve will be Note The band intensities calculated by Gaussian fitting see section 5 7 Gauss Modeling Bands cannot be used in conjunction with Quantity Standards However you can continue to use the trace inte
77. of the crosshairs in the previous panel Chapter 9 ReadyAgarose 96 PLUS Quantity One E x EA AA NUM ee AA a LER E AA Nn MN NE E a id DA RE RR TNR es de Previous Next mp Size r Postion r Spacing Height Vertical Vertical po Cancel a Help Width Horizontal E Fig 9 2 Resizing and Repositioning the Lanes Use the slider bars at the bottom to change position resize the lanes or modify the vertical spacing of the lanes Note that the sliders move resize or space all the lanes at once When you are satisfied with the location of the lanes click Next Step 3 Arranging the Lane View This panel places the 96 lanes in a new view The default view is 4 x 28 Quantity One User Guide Quantity One E X AAAAMMEB 9 1611122 1 1 3DDDDM 8 101 12 4 E E EEEEEW FFFFFFM 1234567891C11126 5 3458789101126 Arrangement Previous Finish 8x14 Arrangment Previous _ Finish 4 28 Arrangment Cancel E Help 256 Arrangment Create Default Image Fig 9 3 Arranging the Lanes To arrange the lanes in a different view select the appropriate view at the bottom of the panel If you would like Quantity One to create an additional default image with the lanes in their original orientation check the box labeled Create Default Image When you are satisfied with the gel arrangement click Finish to create your new image 10 EXQu
78. on line Help software registration Below the menu bar is the main toolbar containing some of the most commonly used commands Next to the main toolbar are the status boxes which provide information about cursor selection and toolbar buttons 2 1 Quantity One User Guide 2 1 b Main Toolbar The main toolbar appears below the menu It includes buttons for the main file commands Open Save Print and essential viewing tools Zoom Box Grab etc as well as buttons that open the secondary toolbars and the most useful Quick Guides Printing Volumes Molecular Weight and Colony Counting SPS ANORA ED FRSEGRTRET Beso File commands Viewing commands Toolbars Quick Guides Fig 2 1 Main toolbar Tool Help If you hold the cursor over a toolbar icon the name of the command will pop up below the icon This utility is called Tool Help Tool Help appears on a time delay basis that can be specified in the Preferences dialog box see section 2 5 Preferences You can also specify how long the Tool Help will remain displayed 2 1 c Status Boxes There are two status boxes which appear to the right of the main toolbar Draw a box to expand the image inside Fig 2 2 Status boxes The first box displays any function that is assigned to the mouse If you select a command such as Zoom Box the name and icon of that command will appear in this status box and remain there until another mouse function is selected or the mouse i
79. or the entire gel Calibrate Band k Match k Lane k Gel Fig 6 24 Buttons for calibrating bands of unknown quantity If you select Band Match or Lane click on the button and then click on the object in the gel Click on Gel to calculate the entire gel Bands of calculated quantity are highlighted in the image Unapplying the Calibration Curve To undo the quantity calculation for a band match lane or gel use the appropriate button next to Un Calibrate 6 3 d Generating Standard Bands via a Dilution Series One way to generate bands of known quantity is to start with a stock solution and make several dilutions Different dilutions can be loaded into different lanes resulting in a dilution series Quantity One will calculate the values in a dilution series if you enter the known quantity and a dilution factor e g for a solution that has been diluted to 10 times the volume type 1 10 or 0 1 In the Quantity Standards dialog next to the band from the undiluted stock solution enter the known quantity In the Dilution Factor column type stock Next to the remaining bands in the series enter the appropriate dilution factors in the Dilution Factor column 6 30 Chapter 6 Standards and Band Matching Trace Quantity Dilution Rel Band OD x mm Factor Status Dev 6 12 4 042 0 60 stock Known 35 10 13 3 705 oas s mem 12 11 13 2 061 ose s mem 13 8 8 1 561 ozs 4 amp Known 3 12 8 1 059
80. or the specified quantity An outlier is defined as a band with normalized quantity ng that satisfies one of the following criteria 1 Ng gt ratio x mean and ng gt mean quantity 2 Ng lt 1 ratio x mean and Ng lt mean difference Where ratio 1 0 percentage 100 Chapter 11 Differential Displays and VNTRs Trends Select the Trend button to search for increasing or decreasing levels of gene expression across samples represented by trends in normalized quantity across lanes With Trend selected a linear regression of ng versus lane number is computed for each band type The leftmost and rightmost lanes containing that band type are determined The normalized quantities for these lanes are calculated from the regression model If abs leftmost n rightmost_n gt difference and Max leftmost_ng rightmost_ng Min leftmost_ng rightmost nj gt ratio then the band type is flagged as a trend Displaying Results Bands identified as outliers or belonging to a trend are highlighted with a white line in the image If a lane is missing a band assigned to a band type that is flagged as an outlier or a trend the expected location of the band will be highlighted by a white box Use the Normalized Quantity button in the Differential Display dialog to display a histogram of the normalized quantities and the mean quantity for a band type Click on the button then click on the band A graph will display
81. or visit us on line at www bio rad com Help Fig 1 8 Free Trial screen In the Software License screen click on the Free Trial button This will open the Software License Registration Form Enter the required information you will not Quantity One User Guide have a purchase order number or software serial number and can leave these fields blank and click on Submit Via Internet A free trial password will be automatically downloaded to your computer This password will allow you to use the software for 30 days If you decide to purchase the software during that period contact Bio Rad to receive a software package and a Hardware Security Key You can then complete the registration process as described in the previous sections 1 9 Quantity One Basic Quantity One can be run in Basic mode Quantity One Basic does not require a software license The program can be installed and used simultaneously on unlimited numbers of computers Quantity One Basic is a limited version of the flexible and powerful Quantity One The following functionality is active in Basic Mode Image acquisition with Bio Rad imaging devices Transform Crop Flip Rotate Text Tool Volume Rectangle Tool Volume Circle Tool Density Tools ReadyAgarose 96 PLUS Manual Excision Print Export to TIFF and Save 1 10 Contacting Bio Rad Bio Rad technical service hours are from 8 00 a m to 4 00 p m Pacific Standard Time in the U S Phone 800 42
82. percent See section B 9 Options Step Ill Acquire Image Click on Auto Expose gt Aut M button appears depressed Qi O RES Exposure Time sec Can see Exposure Time automatically changing O Freeze Fig B 4 Auto Expose Once an image has reached the specified percent of saturated pixels it is captured and displayed in the display window Auto Expose is automatically deactivated the exposure time appears active in the Exposure Time field and Manual Expose is activated Note If you are having difficulty auto exposing your sample you can use Manual Expose to adjust your exposure time directly Most applications only require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button Manual Expose is automatically activated after Auto Expose has deactivated B 6 Appendix B Gel Doc XR Step Ill Acquire Image Manual Expose active O RU Q ns c button appears selected Expose Acquire Exposure Time field active adjust number of seconds Exposure Time sec ea ela Freeze Fig B 5 Manual Expose With Manual Expose activated use the arrow buttons next to the field to adjust the exposure You can also type in the new exposure time then press ENTER for the change to take effect When the specified exposure time is reached the last captured image will be disp
83. points at either end of the lane line you will delete the entire line Deleting Lanes You can delete both single lane lines and lines from a lane frame Select Remove Lane from the Single Lane submenu or toolbar and click on the lane You will be prompted to confirm the deletion Note If you delete a lane from a group of lanes or a frame select Sort and Recalculate from the Edit menu to renumber the remaining lanes 4 7 Quantity One User Guide 4 1 d Lane Width Lane width is important for band quantition Only the region of the band within the lane sampling width is quantitated so the defined lanes should be slightly wider than the actual lanes in the gel You can adjust the sampling width of all the lanes using the Detect Bands dialog see section 5 2 a Detection Parameters See section 5 1 How Bands Are Identified and Quantified for a full discussion of the effect of sampling width on band quantitation To adjust the width of a single lane select Lane Width from the Lane menu or toolbar and click on the lane line The Sample Width dialog will display the current width of the lane Quantity One x A Enter sample width for lane 6 2 03 mal Cancel Fig 4 8 Sample Width dialog Enter a new width in millimeters and click on the OK button 4 1 e Lane Profile After you have defined a lane you can review the intensity profile of the lane A lane profile provides a quick visualization of the intens
84. progress of each exposure will be displayed in the Exposure Status bar at the bottom of the acquisition window 1 11 Quantity One User Guide Exposure Status i Acquiring Image Fig l 6 Exposure Status bar when acquiring an image Depending on which dark subtraction type you have selected under Options see section I 6 Options a dark image may be acquired immediately following image acquisition If you want to stop an acquisition that is in progress click on the Stop button The current acquisition will be terminated If multiple channels are selected you must click on the Stop button once per channel to stop all acquisitions After an image has been acquired a separate window will pop up containing the new image You can then analyze the image using the analysis functions Optimize Exposure Optimize Exposure allows you to specify an interval over which a series of progressively longer exposures are taken All exposures are then displayed on the screen and you can choose the one that provides the best image Note Multiple exposures will be taken for only the selected multichannel tab Other channels even if enabled will not be used Illumination Flat Fielding will be disabled if you are using Optimize Exposure Click on the Optimize Exposure button A settings dialog box will open in which you can specify the total exposure time starting exposure time and number of exposures The specified numbe
85. provides recommended exposure times for various applications Table 1 Recommended Exposure Times and Lenses Recommended Sample Exposure Lens amp Filter Accessories Used Fluorescent Stain Gel 3 30 sec Zoom IR None Fluorescence End 30 sec 5 min Zoom IR None Label Gel Fluorescent Blot 0 5 5 sec Zoom IR Sample Chemi Tray if sample is small Chemifluorescent 0 5 5 sec Zoom IR None Blot Colorimetric Gel 0 1 1 sec Zoom IR White light con version screen Colorimetric Blot 0 1 1 sec Zoom IR Sample Chemi Tray if sample is small X ray film 0 1 1 sec Zoom IR White light con version screen Weak 5 10 min 50 mm Sample Chemi Chemiluminescence Tray if sample is small Strong 10 sec 2 min 50 mm Sample Chemi Chemiluminescence Tray if sample is small lFor chemi applications the 50mm lens is recommended Always remove the 660 fil ter Quantity One User Guide Note For most applications you can select an exposure time capture an image study it and then adjust the exposure time accordingly Repeat this procedure as many times as necessary to obtain a good image For chemiluminescent samples which degrade over time and emit low levels of light select a high exposure time initially or use the Optimize Exposure command described on page 12 Preview For shorter exposures you can use Preview to test different exposure times Click on the Previ
86. puck beneath the tip Once placed click lower tip This places the puck exactly where it needs to be Once all pucks are placed properly the camera takes an image of the pucks Auto placement If you have previously performed calibration you can have the spot cutter automatically place the pucks for you Simply place the pucks on the cutting stage Check the box labelled Auto place pucks When you click Next the spot cutter takes an image of the pucks on the stage then automatically moves the pucks to their proper positions When the pucks are positioned the spot cutter creates the system position image 10 3 Quantity One User Guide 10 1 6 Calibrating the Plate and Wash Station Positions The second part of the calibration wizard focuses on plate position and wash station position Due to minor imperfections in the construction of the spot cutter it is necessary to fine tune the position of the microtiter plate positions and the wash station Plate Position Calibration To calibrate plate position select a plate type from the list place the plate in the right rear plate position and click Next The spot cutter moves the tip to the location above the Al well hl EXQuest SpotCutter Setup Plate Position Calibration Position the head over well A1 of the right rear container as shown It should be centered over the well and the tip when lowered should be slightly above the well Left R
87. report If you select Diagrams schematic representations of the lanes will be displayed To display the selected data click on the Report button 12 12 Chapter 12 Reports Proteins Compare Images 13 12 100 0 88 3 85 6 57 9 57 2 57 2 57 0 521 35 8 33 7 EET NEST Fig 12 11 Compare Lane Images report The Compare Lane Images report is displayed in a standard report window see section 12 1 Report Window Change the report options by clicking on the Reformat button The Print Report dialog for this report contains special fields for entering a report title 12 13 Quantity One User Guide 12 5 b Phylogenetic Tree Phylogenetic trees are schematic representations of sample similarity To compare the similarity of samples in a phylogenetic tree format select Phylogenetic Tree from the Reports menu You can also select Phylogenetic Tree Quick Guide under the Help menu to guide you through the process of generating a phylogenetic tree In the pop up box select a clustering method for creating the tree See the following pages for information about the different methods Proteins Phylogenetic Tree ES Cluster Method Neighbor Joining cerebellum stem cerebrum liver large Gl stomach small GI spleen lung thymus heart aorta vena cava Fig 12 12 Phylogenetic Tree Note We have found that Ward s method UPGAMA and WPGAMA give the most pla
88. report window see section 12 1 Report Window 12 6 Volume Analysis Report The Volume Analysis Report displays volume data Select Volume Analysis Report from the Reports menu or Volume toolbar The Volume Report Options dialog will open in which you can specify the information that will appear in the report 12 20 Chapter 12 Reports When you click on OK the report will be displayed in a standard report window see section 12 1 Report Window 12 6 a Volume Report Options Volume Report Options Data To Display r Image Display Options Xj Name x Min Value x Display image in report x Type x Max Value x Print image on report x Volume Density res Adi Vol X Mean Backad x Show overlays in image X Volume x Num Pixels Image on 1st page only Concentration X Location rea LY Location p Regression method gt x Mean Valus Width Point To Point Linear X Std Deviation Height Cubic Quadratic Logistic Spline Background Subtraction Method Global Local Show Curve Volume objects to report All objects Selected objects Font Size Medium gt Line Spacing Medium y Modify Report Settings DK Cancel Fig 12 15 Volume Report Options dialog The volume report options are described below Name The name that is automatically assigned to the volume based on its type U Unknown Std Standard B Background and order in
89. that it points in the direction of the new top of the image Note To center the arrow on a particular point on the image e g to align along a particular lane position the cursor on the point and press the F3 key The center of the arrow will shift to the new position To complete the rotation click on the Rotate button in the pop up box Another window will open containing the rotated image and you will be prompted to save this image under a new name or version number If you are not satisfied with the rotated image close the window without saving and start over 3 28 Chapter 3 Viewing and Editing Images Note Because an image is composed of square or rectangular pixels Custom Rotation performs some minor smoothing on the image for rotations other than 90 Also any analysis performed on the image will be lost 3 10 Whole Image Background Subtraction Image background due to gel opacity random signal noise or other factors can interfere with quantitation and data analysis Quantity One has several tools for subtracting background intensity from gel images This section describes whole image background subtraction You can also subtract background from individual lanes see section 4 2 Lane Based Background Subtraction and bands see section 7 6 Volume Background Subtraction Whole image background subtraction is useful for reducing background resulting from the opacity of the carrier medium film gel matrix or bl
90. the Reset button will also erase the count 8 4 Making and Erasing Individual Colonies If automatic colony detection has missed or erroneously detected some colonies you can manually mark or unmark them directly on the image using the buttons under Tools Options rStep 3 Tools options Bl Ignore region el Make Colony Erase Colony Fig 8 4 Colony counting tools To mark a colony click on the Make Colony button then click on the spot on the image that you want to identify as a colony To unmark a colony click on the Erase Colony button then click on the colony on the image that you want to unmark The colony count will change accordingly Quantity One User Guide 8 5 Using the Histogram to Distinguish Colonies The histogram in the Colony Counting dialog is a graphical representation of the signal data in the image You can use the histogram and associated sliders to reduce the number of incorrectly identified colonies and or distinguish between white and blue colonies in the image Colonies Versus Background Noise If there is a clear peak on the left end of the colony counting histogram it is probably due to background intensity or noise in the image For information on subtracting background from entire images see section 3 10 Whole Image Background Subtraction for information on filtering noise from images see section 3 11 Filtering Images If background is being detected as colonies you can use
91. the Transform dialog see Invert Display on page 23 inverts the display of the image However in some cases you may need to invert the actual image data If the image has light bands or spots on a dark background i e the signal intensity of the background is greater than the signal intensity of the sample you need to invert the data before analysis Select Invert Data from the Jmage menu or toolbar This function is reversible You may need to use the Transform controls to adjust the appearance of the inverted image 3 13 Text Overlays To create and display textual notes directly on the image select Text Overlay Tools from the Edit menu or main toolbar This will open the Text Overlay Tools toolbar Text Overlay Tools E Select tool Ty ac N E 2 ud a Text tool Line tool Copy Paste Alignment tools Fig 3 24 Text Overlay Tools toolbar Creating a Text Overlay To create a text overlay click on the Text Tool then click on the image at the spot where you want the text to appear This opens the Text Overlay Properties dialog 3 39 Quantity One User Guide Text Overlay Properties Text color Background color Cancel Fig 3 25 Text Overlay Properties dialog To enter text type in the main field Use the buttons in the dialog to select the properties of the text including format alignment and justification Select the font style font size color of the text and color
92. the band Drag the cursor until the area of the band that you want to define represented by the peak on the intensity trace has been completely enclosed The area of defined band will be highlighted on the trace Chapter 5 Bands Optical Density 0 00 0 50 1 0 Fig 5 7 Creating a band with brackets displayed When you release the mouse button brackets will enclose the band on the image With the bands displayed as lines select Create Band from the menu or toolbar then click on the center of the band of interest A line will appear in the center of the band Fig 5 8 Creating a band with lines displayed Note After you have identified several bands renumber the bands by selecting Sort and Recalculate from the Edit menu 5 11 Quantity One User Guide 5 3 6 Adjusting Bands To reposition a band s boundaries select Adjust Band from the menu or toolbar If the bands are displayed as brackets drag the upper or lower bracket of the band If the bands are displayed as lines drag near the upper or lower boundary of the band that you want to adjust When you drag the band boundary a pop up lane trace will appear and the area of defined band will be highlighted on the trace Drag the band boundary to the correct position 5 3 c Deleting Bands To undetect a band select Remove Band from the menu or toolbar and click on the band If the bands are displayed as brackets a trace of the band will be displayed an
93. the file name To open multiple files first select them using Ctrl click or Shift click key combinations and then click on the Open button Note Due to operating system limitations it is possible to have image files open in two or more locations over a network simultaneously To safeguard your image files keep them in a protected folder or on your local machine You can also open images from other The Discovery Series software PDQuest Diversity Database DNACode Note Chapter 2 General Information Itis possible to move image data between The Discovery Series software applications on different platforms Windows and Macintosh The application comes with a selection of sample images In Windows these are located in The Discovery Series Sample Images 1D directory On the Macintosh they are stored in the Sample Images folder in the Quantity One folder Opening TIFF Images The Open command can also be used to import TIFF images created using other software applications There are many types of TIFF formats that exist on the market Not all are supported by The Discovery Series There are two broad categories of TIFF files that are supported 1 Note 8 bit Grayscale Most scanners have an option between line art full color and grayscale formats Select grayscale for use with The Discovery Series software In a grayscale format each pixel is assigned a value from 0 to 255 with each value corresponding to a particu
94. the left will make weak signals appear darker In Appendix C ChemiDoc UV mode dragging the High slider handle to the left will make weak signals appear brighter Dragging the Low slider handle to the right will reduce background noise You can also type specific High and Low values in the text boxes next to the sliders Clicking anywhere on the slider bars will move the sliders incrementally Gamma Slider Some images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashion Adjusting the Gamma slider handle changes the light and dark contrast nonlinearly Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image Transform command Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background and visa versa This will only affect how the image is displayed on the screen not the actual image data Auto scale Clicking on Auto scale will adjust your displayed image automatically The lightest part of the image will be set to the minimum intensity e g white and the darkest will be set to the maximum intensity e g black You can then fine tune the display using the High Low and Gamma sliders described below Reset Reset will return the image
95. the normalized quantities of that band across the entire gel 11 2 Variable Number Tandem Repeats If your experiments involve the use of microsatellites VNTRs or other repeated elements you can calculate the number of times a repeated element occurs in a band Note You must define the Standards in the gel before you can use this function see section 6 1 Standards To calculate the VNTRs in the bands in a gel select VNTR Calculations from the Analysis menu You can also select the VNTR Quick Guide from the Help menu for guidance on analyzing gels with VNTRs Quantity One User Guide The Tandem Repeat Calculations dialog box will open Quantity One x Tandem Repeat Calculations Band Flanking Region Number of Repeats Repeated Unit Band Size derived from band location Flanking Region Size fio Repeated Unit Size 4 Test for ambiguity ves No Ambiguous if rounded gt o 25 Flag With Leave Blank Done Help Fig 11 2 Tandem Repeat Calculations dialog box At the top of the dialog box is the equation used to calculate the number of times an element is repeated in a band The band size is determined by the position of the band on the gel image In the Flanking Region Size field enter the size in base pairs or other repeated units of the part of the fragment that does not include any repeated elements This would include primer length and the length of any sequences that fall bet
96. the number listed on the form Alternatively you can enter the contents of the form into an e mail and send it to Bio Rad at the address listed in the Registration Form Bio Rad will contact you by fax or e mail in 2 3 days with a full license 1 7 c Entering a Password If you fax or e mail your registration information you will receive a password from Bio Rad You must enter this password manually To enter your password click on Enter Password in the Software License screen If you are not currently in the Software License screen select Register from the Help menu Enter Password 4 To register please contact Bio Rad during Current license US Pacific business hours c ingle system license Phone 1 800 424 6723 2601 in the U S License expires on 24 Aug 200Z 1 510 741 2601 Intl D e r System ID Ask for Software Registration Fax 1 510 741 5885 CMXXXLXNES e mai LSG Software Registration Bio Rad com epoca Please provide the information in the Quantity One OK Not OK completed registration form For network license registrations or software upgrade registrations please submit your request for a new Enter Cancel password by email only Include your system ID purchase order number and the software catalogue number purchased along with the standard registration info in your message text Fig 1 7 Enter Password screen In the Enter Password screen type in your password i
97. the same gel First you select a channel then you select the application under that channel G 2 a Selecting a Channel The four channels are accessed using the tabs under Step 1 Channel 1 is always enabled that is the FX will always scan using the application settings selected under Channel 1 first To enable any of the remaining channels click on a channel tab then select the Enable Channel checkbox and select the application for that channel as described in the following section Appendix G FX Click on the tab to Step Select Application select the channel ov tren Channel 2 Channel 3 Channel 4 Select the checkbox rable Channel to enable the channel rJ Ens Click on the Select _ gt Select button to select the application Densitometry Coomassie Blue Gel Blot Medium 532 1064nm EX Blank 555nm LP 1064nm BL Fig G 3 Enabling Channel 2 Enabled channels have a green check mark on their tabs You do not need to enable Channels 2 4 in sequence For example you can set up your four most common applications using the different channels but only enable Channel 4 for a particular gel Channel 1 would be scanned first Channel 4 second Enabled channels are scanned sequentially The separate scans are displayed and saved as separate images The total scanning time depends on the number and type of enabled applications If you have selected Auto Sav
98. the selected data click on the Report button The report will be displayed in a standard report window see section 12 1 Report Window 12 5 Similarity Comparison Reports Quantity One has three reports for comparing the similarity of lane based samples in a gel Compare Lane Images Phylogenetic Tree and Similarity Matrix Note These reports require band matching see section 6 2 Band Matching Comparison Options Before opening any of these reports select Comparison Options from the Reports menu to specify some similarity settings 12 9 Quantity One User Guide Analysis Options Include All Bands Classified Bands Weighted Yes No ok Fig 12 9 Comparison Options dialog In the Comparison Options dialog select All Bands to include every band in the sample similarity comparison or selected Classified Bands to include only matched bands Next to Weighted select Yes to use both band position and intensity when comparing sample similarity Select No to use only band position when comparing samples The next section describes how weighting is used in the similarity comparion equation Comparison Method The method for computing similarity in Quantity One is the Dice Coefficient The formula for the Dice Coefficient is D S Min sstj sim 200 x y sytt i l dist 100 sim where S and T are vectors representing two lanes in the same band set that are being compared To compute si
99. to its original unmodified appearance C 11 Quantity One User Guide C 6 Step V Analysis The Analysis step of the ChemiDoc EQ acquisition window allows you to add annotations and analyze the newly acquired image Annotate Clicking on Annotate will open a separate image window displaying the captured image The default name for the image will include the date time and user if known The Text Overlay toolbar will also pop up to allow you to annotate your image The image will not be saved until you select Save or Save As from the File menu Analyze Clicking on Analyze will open a separate image window displaying the captured image The default name for the image will include the date time and user if known You can then analyze the image using the other features in the main application The image will not be saved until you select Save or Save As from the File menu C 7 Step VI Select Output The ChemiDoc EQ window has several output options Step VI Select Output Es Video Print Save Fig C 9 Output options C 12 Appendix C ChemiDoc Video Print Clicking on Video Print will automatically send the currently displayed frame either live or integrated to a video printer You can add information about your image to the bottom of the printout by selecting the appropriate checkboxes in the Options dialog box See section C 9 Options Save Clicking on Save will open a separate image
100. to lowest in terms of the dimensions of the resulting pixels in microns Smaller pixels equal higher resolution Each resolution is listed with its typical use In general the size of your pixels should be one tenth the height of your smallest object Specifying Your Own Resolution If you select Oversample in the Options dialog box you can specify your own resolution within the range of 32 169 micrometers With Oversample selected enter values directly in the fields next to X resolution and Y resolution in the main acquisition window E 8 Appendix E GS 800 Step Ill Select Resolution Select x resolution 50 0 Microns Y resolution so o Microns Image file size 0 10 Mb Fig E 8 Entering a custom resolution with Oversample selected Image File Size The size of the scan file for the selected resolution is listed next to Image File Size If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance E 5 Calibration The GS 800 automatically calibrates the densities of scanned images to ensure accuracy and reproducibility of results The GS 800 has built in step tablets for both transmissive and reflective scanni
101. to magnify the image before drawing a boundary If you try to draw a very small boundary the software will think that you are backtracking and erase the boundary When the cursor crosses the line the color of the line will change to indicate that it is a band boundary and a band line will appear on the nearest lane If you keep drawing each time the line crosses itself a new band will be created replacing the old band Note Gaussian modeling and the Plot Band command do not work on drawn bands To list areas and quantities of drawn bands in reports select the Contour Area and Contour Qty report formatting options Display this information on the image using the Band Attributes dialog Editing Band Boundaries To change a drawn band boundary select Edit Band Boundary from the Band gt Draw Band submenu or Contour toolbar and drag the cursor across the previously defined boundary A line will appear When you recross the old boundary the line will change colors and the new boundary will be created 5 25 Quantity One User Guide 5 26 6 Standards and Band Matching After you have defined the lanes and bands in a gel you can identify the standard lanes enter the values of the standards and determine the values of the experimental bands using those standards You can also compare sample similarity by matching bands across lanes Finally you can identify bands of known quantity and use these to generate a calibration c
102. to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button If you are scanning using multiple channels the image created using Channel 1 will be saved using the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you scanned an image using only Channels 1 and 4 the image created using Channel 4 will still be saved as version 2 v 2 Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be prompted to give it a new name This protects the backup file and preserves it from any changes H 15 Quantity One User Guide Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image Transfo
103. trace as defined by the rolling disk As you change the size of the rolling disk the orange line changes following the contours of the profile more or less closely as shown in Fig 4 10 To display the trace with the background removed select Show lane trace with background subtracted The orange line in the trace disappears along with the Quantity One User Guide background and you can visually compare the relative intensities of the bands in the lane 4 2 b All Lanes Off To turn off lane based background subtraction for all the lanes in the image select the All Lanes Off button 4 2 c All Lanes On Same Level Select this option to set the same subtraction level for all lanes The Rolling Disk Size field in the A Lanes section of the dialog displays the rolling disk radius in number of pixels Enter a new value in the field or use the arrows to change the value in 10 percent increments Typical rolling disk sizes range from 50 to 150 As you change the size study the level of background subtraction in the lane trace 4 2 d Subtraction Varies by Lane You can set a different subtraction level for each lane in the gel using the controls in the Selected Lane area of the dialog Any changes you make with these controls will only be applied to the selected lane to select a different lane click on it with the Lane Background command assigned Click on the Lane Off button to turn off subtraction for the selected lane Sel
104. traces for functions such as Plot Lane or Plot Band because the sampling width is only one image pixel Density at Cursor Select Density at Cursor and click on a band or spot to display the intensity of that point on the image It also shows the average intensity for a 3 x 3 pixel box centered on that point Density in Box Select Density in Box and drag a box on the image to display the average and total intensity within the boxed region Plot Density Distribution Select Plot Density Distribution to display a histogram of the signal intensity distribution for the part of the image displayed in the image window The average intensity is marked in yellow on the histogram The histogram will appear along the right side of the image Magnify the image to display the data for a smaller region Quantity One User Guide Plot Cross section Select Plot Cross section and click or drag on the image to display an intensity trace of a cross section of the gel at that point The horizontal trace is displayed along the top of the image and the vertical trace is displayed along the side of the image The intensity at the point you clicked on is displayed as is the maximum intensity along the lines of the cross section Position 33 34 13 37 mm Quantity 0 32 OD Max Quant D 49 OD Click on button then click or drag on the image Fig 3 4 Plot Cross section tool Plot Vertical Trace Select Plot Vertical Trace and clic
105. user defined Normalized qty The trace quantity of a particular band expressed as a percentage of the quantity of a selected band type that is present in the same lane Band type The band type number of a band that has been matched and placed in a band set Band model Displays the modeling lines across the gel that are generated by band matching standards or both These lines are used to compensate for gel distortion or smiling Tandem repeats The number of repeated base pair units in a band that has been analyzed using the VNTR Calculations function see section 11 2 Variable Quantity One User Guide Number Tandem Repeats e Differential Display lIf the band types have been normalized this displays trends in increasing or decreasing expression of a band type across a gel based on its normalized quantity 5 6 Band Information The Band Information dialog displays information about each defined band Select Band Information from the Band menu and click on a band An intensity profile of the band s lane will be displayed with the the selected band highlighted and the dialog will open Band Information for MA00396 1D Scan X Lane 5 Band Set Name 5 Band 17 Band Type Rel Front 0 808 Mol Wt KDa Status Unknown Peak 45 6 CNT Average 31 6 CNT Trace 152 CNT x mm Gauss Model Peak 40 6 CNT Gauss Model Trace 179 CNT x mm Contour N A Relative Oty 3 51 Norm Qty N A Quantity Units Status
106. volume of each well PDQuest uses this setting in conjunction with your cut settings to determine whether you will exceed the well capacity Specify Options Cut Plate Hydration Wash 96 wellMTP 2 00 ml per well 384 well MTP 0 05 ml per well 96 Tubes 1 00 ml per tube Loading order amp 1 2 C amp 1 B1 Fig 10 7 Plate options Load order determines the order in which wells will be filled on the plate Hydrate To keep gels from drying out during long cut runs you can have the EXQuest spot cutter automatically hydrate your gel s during the cut run Check the hydrate box then enter the time in minutes between hydration runs 10 9 Quantity One User Guide Specify Options Cut Plate Hydration Wash x Hydrate Every 1 O minutes El Define hydration area Fig 10 8 Hydrate option To determine the area you want to hydrate click Define hydration area then click and drag on the image to create a box To redraw the hydration area click and drag again Note You must define a hydration area for hydration to function Wash When using the EXQuest with the gel tip EXQuest will flush the tip after each cut is made Enter the wash volume to flush through the cutter tip Specify Options Cut Plate Hydration Wash Wash volume 50 0 ul Fig 10 9 Wash option 10 10 Excision If you are using the membrane tip you cannot adjust the was
107. you only adjust this slider If you want to adjust the display contrast without subtracting background use the Transform command see section 3 8 Transform Before adjustment the image After adjustment background levels are seems clear of background more apparent Background for Protein v2 1D Scan Background los Protein v2 10 Scan Dek Cormon E Dat Centon NE a p pj bestow Soe Bockgourd Bax k Background Stipe KR Fig 3 20 Dark Contrast adjustment reveals true levels of background in the image 3 31 Quantity One User Guide Background Slider To manually adjust the background subtraction levels drag the Background slider to the right You can also move the slider incrementally by clicking on the slider bar or type a specific value into the field next to the bar Objects with signal intensities lower than the subtraction level will be eliminated from the image when you click on OK Background Box Use the Background Box function to define a background area in the gel that is representative of the background in the entire image This method of subtraction is useful for images with uniform backgrounds Click on the Background Box button then drag on a background area of the image The average intensity of the pixels in the box will be used as the background level to be subtracted from the entire image Background Stripe The Background Stripe function is useful for gels in which th
108. 0 e Averaging l 63 C5 C7 JPlaque rStep 2 Adjust Results White Blue Colony count o o Adjusted count o o Count vs Peak density Cutoff White Blue r g Step 3 Tools options 6 Ignore region X Show data area Make Col x Mark white colonies jake Colony el x Mark blue colonies Erase Colony Step 4 Save To Batch File X Batch mode New batch Open batch Load Next cae haa ee ee aa aso aa wl Count Name Colony Counting lsc 1999 05 17 l4hr 5 eese Close Reset Help Fig 8 1 Colony Counting dialog Quantity One User Guide The dialog has been arranged from top to bottom to guide you through the procedure 8 1 Defining the Counting Region First you must define the region you want to count in the Petri dish image Click on the Define Counting Region button in the dialog and position the cursor at the center of dish image Drag the cursor outward As you drag a blue circle will expand on the image this defines the border of the counting region Drag cursor out from 4 center to define region Fig 8 2 Defining a counting region If you make a mistake in defining the counting region click on the Reset button at the bottom of the dialog to start over Position the blue border until it is just inside the interior edge of the Petri dish Chapter 8 Colony Counting Note If the
109. 10 2 EXQuest Spot Cutter wizard 10 1 a System Calibration The wizard guides you step by step through the system calibration System calibration synchronizes the coordinates of the camera and the coordinates of the 10 2 Excision cutter Follow the directions on each panel then click Next to continue If you find you made an error or need to repeat a step for any reason click Back Note Clicking Back requires you to re perform any completed steps Focusing the Camera If this is the first time calibrating the spot cutter or you have replaced the camera you need to focus the camera Check the box labeled Perform Focus Alignment step then click Next If you do not need to focus the camera clear the Perform Focus Alignment step checkbox You can also skip system calibration and go directly to plate alignment provided system calibration was performed earlier and is still valid Flat Field Calibration In the Flat Field Calibration step the spot cutter needs to create an image of the cutting stage Make sure the cutting stage is clean and dry before proceeding Lens Calibration In the Lens Calibration step the spot cutter needs to create an image of the grid placed on the cutting stage Place the calibration grid on the stage before proceeding System Position Calibration This step requires the placement of pucks on the stage in a three by three grid As the gel tip positions above each puck it is necessary to center the
110. 14 to check curve gt 4 5 0 2456 o Ir as 16 m m7 Fig 6 26 Checking an imported calibration curve When you enter the band s value its status will change to Check indicating that it is used to verify the accuracy of the calibration curve and is not used in calculating the curve itself Click on the Show Curve button to display the graph and note that the Check bands are enclosed in diamonds If the Check bands do not fall on or very near the calibration curve we recommend that you do not use the imported standards for this gel 6 32 7 Volume Tools You can use the Volume tools to quantitate bands spots arrays and other image data What is a Volume A volume is the total signal intensity inside a defined boundary drawn on an image To measure the amount of a particular object e g a band or spot you draw a volume rectangle contour free hand or circle around the object and compare the intensity data inside the boundary with the data of other objects or a standard using the Volume Analysis Report and Volume Regression Curve see section 12 6 Volume Analysis Report Volume Sum of the intensities of the pixels within the volume boundary x pixel area Volume units intensity units x mm Volumes are similar to band contours see section 5 8 a Contouring Bands except that they are not dependent on lanes and bands 7 Creating a Volume To create a volume select Volume Tools from the
111. 2 Cancel OK Fig I 8 Options dialog box 6 a Dark Subtraction Type All CCD cameras accumulate electrons that produce a signal that is indistinguishable from light This dark current adds to the noise in your images particularly for long 1 14 Appendix I VersaDoc exposures In most cases you will want to subtract this dark current from your images The settings for subtracting the dark current are in the Options dialog box Click on the Options button in the acquisition window to open this dialog Normal The Normal option button selects the default dark subtraction type In this mode after you acquire an image a dark image of the same exposure length will be taken and this will be subtracted from your image The progress of the dark exposure will be displayed in the Exposure Status bar following the regular image exposure In Normal mode a dark image is only acquired the first time you perform a scan with particular application and exposure settings If you perform subsequent scans with the same settings no dark exposure will be taken Reference If you do not want to perform a dark exposure with each acquisition you can take a reference dark exposure that will be saved and subtracted from all subsequent acquisitions Click on the Referenced button to activate this feature The first time you acquire an image after selecting this option the VersaDoc will take a dark exposure that will b
112. 2 Appendix A Gel Doc as white in the image while the maximum slider defines the pixel value that will appear as black The slider scale is 0 255 with the defaults set to 60 minimum and 130 maximum Imaging Area These fields are used to specify the size of your imaging area in centimeters which in turns determines the size of the pixels in your image i e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less than 1 percent of the pixels in your image saturated Consequently the default value for this field is 0 15 percent Reminder When this checkbox is selected the software will warn you to turn off your transilluminator light when you exit the Gel Doc EQ acquisition window or when your system is idle for more than 5 minutes Video Printing Footer Information The checkboxes in this group allow you to specify the information that will appear at the bottom of your video printer printouts Save Options To automatica
113. 4 will be saved as version 2 v 2 H 2 b Selecting an Application To select an application i e the appropriate filter and other parameters for the type of object you are imaging click on the Select button under a channel tab H 5 Quantity One User Guide Radioisotopes Chemifluorescence Nucleic Acid Stain Protein Stain Fluorophores Microtiter Plate Deep Purple Flamingo Nile Red Sypro Orange Colorimetric Multiplex 8 H vpro Custom i i Sypro ay High Sample Intensity Medium Sample Intensity Radioisotopes Low Sample Intensity Fuii Srreen fTLnouecti Fig H 4 Example of an application tree Ethidium Bromide gel Standard Applications The standard applications and associated settings are listed in a tree that expands from left to right When you select a standard application the software automatically selects the appropriate filter s and laser in the PharosFX for that particular application Standard PharosFX Applications Category Application Fuji Screen Radioisotopes 1 K Screen Kodak Attophos Chemifluorescence ECL Plus Ethidium Bromide Nucleic acid Stains Sybr Gold Sybr Green amp II H 6 Appendix H FX Standard PharosFX Applications Deep Purple Flamingo Nile Red Sypro Orange Sypro Red Sypro Ruby Protein Stain Alexa 488 Alexa 555 Alexa 647 CY2 CY3 CY5 FAM FITC HEX R6G TAMRA Texas Red Fluorophores B Gal FDG DNA Pic
114. 4 6723 510 741 2612 Fax 510 741 5802 E mail LSG TechServ US Bio Rad com For software registration Phone 800 424 6723 in the U S Chapter 1 Introduction 1 510 741 6996 outside the U S Quantity One User Guide 1 16 2 General Operation This chapter describes the graphical interface of Quantity One how to access the various commands how to open and save images how to set preferences and how to perform other basic file commands 2 1 Menus and Toolbars 2 1 a Menu Bar Quantity One has a standard menu bar with pull down menus that contain all the major features and functions available in the software File Opening and saving files imaging device controls printing exporting Edit Preferences other settings View Image magnification and viewing tools tools for viewing image data Image Image transform advanced crop image processing and modification Lane Lane finding tools Band Band finding and band modeling tools Match Tools for calculating molecular weights and other values from standards tools for comparing lanes and bands in lanes Volume Band quantity and array data tools Analysis Colony counting Differential Display VNTR analysis Excision Cut lanes or bands or individual spots using EXQuest Spot Cutter Reports Band and lane analysis reports Phylogenetic Tree Similarity Matrix Window Commands for arranging multiple image windows Help Quick Guides
115. 4 b Optimize Image Once you have acquired an image of your sample you may need to reduce noise or background density in the image Quantity One has a variety of functions to minimize image background while maintaining data integrity 1 4 Chapter 1 Introduction 1 4 c Analyze Image Once a clean image is available you can use Quantity One to gather and analyze your biological data In the case of 1 D gels the software has tools for identifying lanes and defining quantifying and calculating the values of bands Volume tools allow you to easily measure and compare the quantities of bands spots or arrays The colony counting controls allow you to count the number of colonies in a Petri dish as well as perform batch analysis Qualitative and quantitative data can be displayed in tabular and graphical formats 1 4 d Report Results When your analysis is complete you can print your results in the form of simple images images with overlays reports tables and graphs You can export your images and data to other applications for further analysis 1 5 Computer Requirements This software is supported on Windows XP and Windows 2000 or on a Macintosh PowerPC running Mac OS 9 2 2 or Mac OS 10 2 6 The computer memory requirements are mainly determined by the file size of the images you will scan and analyze High resolution image files can be very large For this reason we recommend that you archive images on a network file server
116. 50 dpi Specify perna 300 dpi Transform 1rBit Depth Zoom Linear 8 bit Entire image Current view 16 bit Current view 24 bit RGB rSize Pixels X 155 Y 184 Image 27 85 Kb EJ Expor X Cancel 2 Help Fig 13 3 Export to TIFF Image dialog box There are two basic export options e Export the raw image data for further analysis e Export the displayed view of the image for publishing If you choose to export the displayed view you can export with or without the image overlays 13 6 a Analysis Export Mode To export a TIFF image for further analysis select the Export raw data button The publishing controls in the dialog box will become inactive 13 6 Chapter 13 Printing and Exporting 13 6 b Publishing Export Mode To export a TIFF image that looks like the image as it is currently displayed on the screen select one of the options under Publishing Export View Including Overlays Select this option to include overlays with the exported image The other controls in the dialog box will become inactive Export View Excluding Overlays Select this option to not include overlays with the exported image This is the only mode available if you are exporting images from the Multi channel Viewer see section 3 4 Multi Channel Viewer With this option selected specify a resolution for the TIFF image by selecting 72 dpi typical computer screen resolution 150 dpi 300 dpi standard print
117. A disk radius that is too large will result in poor removal of background A disk radius that is too small may subtract actual data Small Radius Large Radius Fig 4 10 Examples of the background trace for small and large rolling disks The small disk follows the profile trace more closely resulting in more background subtraction Select Lane Background from the Lane menu or toolbar and click on a lane The lane will be highlighted the lane profile will be displayed and the Lane Background Subtraction dialog will open 4 10 Chapter 4 Lanes Lane Background Subtraction Lx riie Proteins lSC r All Lanes All Lanes Off Subtraction Varies By Lane All Lanes On Same Level Rolling Disk Size s 3 r Selected Lane Lane Off Lane On Roling Disk Size so e O Show lane trace with background subtracted vy Done 2 Help Fig 4 11 Lane Background Subtraction dialog In the dialog you can set the same subtraction level for all lanes or specify an individual subtraction level for the selected lane Any changes you make will be automatically applied to the image To close the dialog click on Done 4 2 a Profile Trace When you make changes in the dialog note that the profile trace of the lane also changes In the standard view the original raw trace of the line is shown in black and the orange line represents the background beneath the peaks of the
118. Area dimensions under Options see D 5 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are focusing on you can place a ruler in the ChemiDoc XRS hood so that it is visible in the image D 5 Quantity One User Guide D 4 Step Ill Acquire Image The ChemiDoc XRS control panel has several features for creating image exposures You can take an automatic exposure based on the number of saturated pixels in the image you can enter a specific exposure time or you can take a series of exposures and select the best one Note Exposure refers to the integration of the image on the camera CCD over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Use Auto Expose if you want to take a single exposure but are uncertain of the optimal exposure time Note If you know the approximate exposure time you want 3 seconds you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure During the auto exposure the image is continuously integrated on the camera CCD until it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 75 percent See D 5 Options Step IIl Acquire Image Clic
119. Bands Note These tools are similar to the Volume Contour Tool and the Volume Freehand Tool except that they are lane dependent To quantify objects without defining lanes see Chapter 7 Contoured or hand drawn bands are quantitated based on the signal intensity of all the pixels within the band boundary using the following formula Quantity Sum of the intensity of a pixel x pixel size for all the pixels in the boundary The intensity of a pixel is multiplied by the area of the pixel This is done for all the pixels within the contour or drawn boundary The area of the pixel is determined by the resolution of the image The resulting values have units of intensity x mm PA A Contoured drawn band showing individual pixels with different intensities 0 1 mm One pixel enlarged to show dimensions 0 1 mm Fig 5 15 A contoured band scanned at 100 x 100 micrometers 5 21 Quantity One User Guide The commands for contouring and drawing bands are located on the Contour submenu and Draw Band submenu of the Band menu and on the Contour Tools toolbar Before using these commands magnify the image so that the individual pixels in the band are clearly visible This allows you to position the cursor more accurately 5 8 a Contouring Bands Contour Tools E ONO Cont
120. DNA 100bp Molecular Ruler S Bio Rad DNA 100bp PCR MolecularRuler S Bio Rad DNA 2 5 kb Molecular Ruler S Bio Rad DNA 20bp Molecular Ruler S Bio Rad DNA 500bp Molecular Ruler S Bio Rad DNA AmpliSize Molecular Ruler S Bio Rad DNA Lambda HindIII digest S Bio Rad Protein Broad range S Bio Rad Protein High range S Bio Rad Protein Low range S Bio Rad Protein Polypeptide Cancel Fig 6 3 Selecting a set of standards Sets of Bio Rad molecular weight and base pair standards are installed with the software 6 1 a Selecting Predefined Standards If you are using Bio Rad or other predefined standards select them from the pop up list The Standards dialog will open see section 6 1 c Standards Dialog displaying the values of the standards 6 1 6 Creating New Standards To create a new set of standards select New Standards A dialog will pop up in which you can specify the units 6 3 Quantity One User Guide Quantity One Ea New Standards Units Molecular Weight Ascending Cancel Quantity One x A Select Units Base Pairs Ascending Isoelectric Point Ascending Isoelectric Point Descending Molecular Weight Ascending Normalized Rf Descending Edit New Cancel Fig 6 4 Specifying units Click on the Units button to specify the units This opens a dialog in which you can select from a list that includes Base Pairs Isoelectric
121. ES SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU THIS WARRANTY GIVES YOU SPECIFIC LEGAL RIGHTS AND YOU MAY ALSO HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE BIO RAD DOES NOT WARRANT THAT THE FUNCTIONS CONTAINED IN THE PROGRAM WILL MEET YOUR REQUIREMENTS OR THAT THE OPERATION OF THE PROGRAM WILL BE UNINTERRUPTED OR ERROR FREE YOU ASSUME RESPONSIBILITY FOR THE SELECTION OF THE PROGRAM TO ACHIEVE YOUR INTENDED RESULTS AND FOR THE INSTALLATION USE AND RESULTS OBTAINED FROM THE PROGRAM YOUR SOLE REMEDIES AND BIO RAD S ENTIRE LIABILITY ARE SET FORTH ABOVE IN NO EVENT WILL BIO RAD BE LIABLE TO YOU OR ANY OTHER PERSON FOR ANY DAMAGES INCLUDING WITHOUT LIMITATION ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES EXPENSES LOST PROFITS LOST SAVINGS OR OTHER DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE SUCH PROGRAM EVEN IF BIO RAD OR AN AUTHORIZED DEALER HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES OR FOR ANY CLAIM BY ANY OTHER PARTY SOME STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OF INCIDENTAL OR CONSEQUENTIAL DAMAGES FOR CONSUMER PRODUCTS SO THE ABOVE LIMITATIONS OR EXCLUSIONS MAY NOT APPLY TO YOU GENERAL You may not sublicense assign or transfer the license or the Program except as expressly provided in this Agreement Any attempt to otherwise sublicense assign or transfer any of the rights duties or obligations hereunder is null and void and not merely voidable If you have any questions concerning this Agreement
122. Enter a name for the set in the Parameter Set Name field and click on the Save button Parameter Set Name Gel 12 DF X Load Save Delete Reset Fig 5 5 Parameter set controls in the Detect Bands dialog To load a saved set of parameters click on the Load button and select the set from the list To remove a set of parameters first load it then click on Delete 5 2 c Default Parameters To return all detection parameters to their default values click on the Reset button at the top of the form Quantity One User Guide 5 3 Identifying and Editing Individual Bands To manually identify and edit individual bands in an image use the Create Band Adjust Band and Remove Band commands on the Band menu and toolbar These are also included on the toolbar in the Detect Bands dialog Hal mt ra x es sj Fig 5 6 Create Adjust and Remove Bands buttons Note When editing individual bands it is useful to display the band brackets Select these in the Band Attributes dialog see section 5 5 Band Attributes If your bands are very closely spaced defining them in brackets mode gives you greater control for more precise band definition It also allows you to define overlapping bands 5 3 a Identifying Individual Bands With the bands displayed as brackets select Create Band from the menu or toolbar then click on either the top or bottom boundary of the band in the gel An intensity trace of the lane will pop up next to
123. FX Family PharosFX and PharosFX Plus H 1 Appendix I VOPSAD OG M E E 1 1 Appendix J A ASA J 1 Appendix K AaRed dup K 1 vii Quantity One User Guide viii Preface 1 About This Document This user guide is designed to be used as a reference in your everyday use of Quantity One Software It provides detailed information about the tools and commands of Quantity One for the Windows and Macintosh platforms Any platform differences in procedures and commands are noted in the text This guide assumes that you have a working knowledge of your computer operating system and its conventions including how to use a mouse and standard menus and commands and how to open save and close files For help with any of these techniques see the documentation that came with your computer This guide uses certain text conventions to describe specific commands and functions Example Indicates File Open Choosing the Open command under the File menu Dragging Positioning the cursor on an object and holding down the left mouse button while you move the mouse Ctrl s Holding down the Control key while typing the letter s Right click Clicking the right mouse button Left click Clicking the left mouse button Double click Clicking the left mouse button twice Some of the illustrations of menus and dialog boxes found in this manual are taken from the Windows version of th
124. OFPS sida 12 1 12 1 Report Window eee emet nente eterne ERR der Ead 12 1 12 2 Lane and Match Reports essere 12 5 12 3 AS A noera oost eaen seska enee Eer Pone E ner rO ER Sir SSE 12 6 12 4 1 D Analysis Report een e e e nennen nemen enne 12 8 12 5 Similarity Comparison Reports esee 12 9 12 6 Volume Analysis Report eese ener nre 12 20 12 7 Volume Regression Curve esee nennen 12 23 12 8 MNTR Report jui dees eae nessa tpe de dette ef 12 25 13 Printing and Exporting sitos ra re na lea e cada 13 1 13 Prnt Image o E ESEE 13 1 13 2 Page Setup iege eerte rerit ee Lebe UR e ERR Rig 13 1 13 3 Bunt Settings cts titi 13 1 13 4 Image Report 11 2 e epa Sie Puede e e pps 13 3 13 5 Video Pri ea Rue a TM eines 13 4 13 6 Export to TIFF Image ipee eet ee hee hee 13 5 13 7 Exporting a JPEG Image esee 13 8 Appendix A Gel ppc A 1 Appendix B Gel DOG A n c M B 1 Appendix C GhemiDoc EQ iinianiricia a is C 1 Appendix D ChemiDoc ARS iii D 1 vi Contents Appendix E GS 800 Imaging Densitometer E 1 Appendix F Personal Molecular Imager FX F 1 Appendix G Molecular Imager FX Family FX Pro FX Pro Plus and Molecular pipe E M e P M G 1 Appendix H Pharos
125. OK to complete the paste then position the pasted array manually Ungrouping an Array You can ungroup the individual cells in an array so they behave like normal stand alone volumes 7 16 Chapter 7 Volume Tools With the array selected select the Array Ungroup command from the menu or toolbar This command cannot be undone and you will be prompted to confirm the action The ungrouped array will appear deselected i e displayed in blue You can then move the cells individually and perform all normal volume operations on the individual cells 7 17 Quantity One User Guide 7 18 8 Colony Counting You can use Quantity One to automatically count the number of white blue or plaque colonies in a Petri dish Note For best results when capturing the image of a Petri dish the dish should fill the imaging window Also images with colonies should not have asymmetric pixels Asymmetric pixels can be generated by densitometers and the Reduce File Size command The colony counting function will not work properly on images with asymmetric pixels Select Colony Counting from the Analysis menu to open the Colony Counting dialog Colony Counting Colony Counting 1sc x rStep 1 Define Region and Count J For best results adjust the Gel Doc zoom lens so that the image of the Petri dish fills the image window amp Define Counting Region Drag the cursor from center to edge of dish image Count Sensitivity 10
126. OSE vertiente ie i d eh ee ees C 7 D 7 Options e Reden Hc RORIS C 13 D 11 Positiomng the sample oot emet E C 4 D 5 Saturated pixels highlighting sese C 11 SIM A MR C 13 Simulation mode eee erre re tee eret e EXER US C 2 D 2 UV ANOS TE C 5 Video card it ae AiR eo ie MoE AS Aa C 2 D 2 Video display Window mercado eei e eee tees teles C 3 Video prints i iie eats Ri ne URS C 13 Video printing footer info i need ee deep EE NEU e te eH RH C 15 White light mode dept ei eret merken C 5 Clear Analysis eiii eee ete n e eie e e ide eden 3 42 Closing Images een een iia 2 10 Colony Counting Adj sted Count epa AR e nene mee OPEP 8 9 AXVeraglng ss oce en ido ed eerie ee He etie e eec ted mens 8 4 Background noise a E rennen rere 8 6 Batch TGS e 8 10 Defining counting region oe eee cee cee cneceseeeeceeceeceseeeeeeseesaecaeesaecneeeaees 8 2 Displ ayimg results eee b a rette cede e eee edere 8 4 Histo ra ede eene ap en US 8 6 lgnofimg a regio ie ibid 8 8 Image specifications 0 eee eee e ene EE E EE pe eS Epia ESTS 8 1 Making erasing colonies osere so aee pepesan ees oe iSe eene eeii nEn iH 8 5 Plagues a AS i E 8 3 Saving a count with the image essssseeeeeeeneneen rennen nennen enne 8 9 Saving multiple counts oo lee eee cee eeeeeeeeeeecaeesaecaeceaecaecsseeseseeeeeeeeeeeeesenes 8 10 SAVING tO a Spreadsheet eoo ee eee etidm eins 8 10 Index 3 Quantity One User Guide
127. Quantity One User Guide for Version 4 6 3 Windows and Macintosh P N 10002940 Rev C Quantity One User Guide Bio Rad Technical Service Department Phone 800 424 6723 510 741 2612 Fax 510 741 5802 E mail LSG TechServ US Bio Rad com Notice No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from Bio Rad Quantity One is a registered trademark of Bio Rad Laboratories The Discovery Series is a trademark of Bio Rad Laboratories All other trademarks and registered trademarks are of their respective companies WASTE text engine 1993 1997 Marco Piovanelli Limitations of Liability Bio Rad is not responsible for the misinterpretation of results obtained by following the instructions in this guide Whenever possible you should contact the Technical Services Department at Bio Rad to discuss your results As with all scientific studies we recommend that you repeat your experiment at least once before making any significant conclusions for presentation or publication Copyright 2006 by Bio Rad Laboratories All rights reserved Table of Contents 1 INTOdUCHON ai iia 1 1 1 1 Overview of Quantity One oooooconoconccconconconncononnncnonnnonnnnnnan cnn cnn nro ncnn con ncnn cono 1 1 1 2 Digital Data and Signal Intensity
128. Quest Spot Cutter with a gel head 10 2 d Performing the Cut Run After you have identified all the cuts you want to make and selected the appropriate options you are ready to begin the cut run Click the Begin Resume button Plate Selection The Plate selection dialog box lists all the plates available for the cut run The table shows the plate name barcode ID plate size 96 well 384 well 96 tube wells available on the plate and used wells If this is a new cut run the number of plates listed is determined by the number of wells required for the cut run 10 12 Excision il Plates for Cut Run Wells needed 4 Wells available 96 Use Name Barcode ld Size Order Avail Used Edit Ele zar 001 96 wels 1 A2 96 o a Done EAdd plate X Cancel 2 Help Fig 10 11 Select plates In the Plate selection dialog box you can change the name of a plate and enter a barcode ID directly in the table The barcode helps to properly place the plates in the spot cutter To add a plate click Add plate To remove a plate from the list click Delete If the Use checkbox is cleared the plate will be skipped in the cut run even if it has wells available To skip specific wells in a plate to reserve them for such things as standards click Edit in the table This opens a plate diagram dialog box 10 13 Quantity One User Guide E Plate001 3 Click on wells to skip them during excision
129. Repeat Name 21 289 67 33 67 2 2 278 64 40 64 2 3 226 51 44 5 2 4 206 46 53 qe 2 5 164 35 88 36 2 6 148 3202 32 2 7 128 26 90 27 28 114 23 40 23 29 108 21 99 22 2 10 103 20 65 21 2 11 99 19 63 20 2 12 95 18 65 19 2 13 87 16 82 17 2 14 81 1523 15 245 74 13 53 14 2 16 69 1218 12 247 64 11 00 11 248 60 994 10 2 19 56 8 92 9 2 20 53 833 8 Rounding changed the repeat number more than 0 25 Screen Page 1 of 18 Fig 12 17 Example of a VNTR Report 12 25 Quantity One User Guide The report is displayed in a standard report window see section 12 1 Report Window The report displays the molecular weight the raw repeat number and the rounded repeat number for each band in the gel image The raw repeat number is the number of repeats calculated using the information that you provided in the Tandem Repeat Calculation dialog and is likely to include fractional values The rounded repeat number is the raw repeat number rounded to the nearest whole number An asterisk will appear next to some of the rounded repeat numbers if you selected Test for Ambiguity and Flag with in the Tandem Repeat Calculations dialog The asterisk will appear next to numbers that vary from the raw repeat number by more than the selected ambiguity value 12 26 13 Printing and Exporting The commands for printing and exporting images are located on the File menu Reports are printed from within the individual report windo
130. To display the report click on the Report button This report window includes two additional options Page Setup and Print Preview 12 7 Quantity One User Guide Page Setup 2 x1 Paper Size x Source Automatically Select Orientation Margins inches Portrait Left fi High fi C Landscape Top fi Botto fi Cancel Printer Fig 12 7 Page Setup dialog The Page Setup dialog includes controls for selecting the paper size and orientation paper tray page margins and printer The Print Preview dialog displays the report as it will be printed 12 4 1 D Analysis Report The 1 D Analysis Report displays all the advanced analysis data including band types normalized quantities amount of sample loaded etc for all the lanes in the gel The lanes will also be ranked in similarity to the lane you initially select to generate the report Note This report requires band matching see section 6 2 Band Matching Select 1 D Analysis Report from the Reports menu then click on any experimental lane in the gel A dialog will pop up in which you can select the report data to display 12 8 Chapter 12 Reports Quantity One x Select Report Features Display x Analysis Options Lane X Name x Similarity Category x CPM Loaded Include x Band Data Format Columns Compact x Band Frequencies Cancel Fig 12 8 1 D Analysis Report options To display
131. VersaDoc MP contains blue and white LEDs and optional red and green LEDs l VersaDoc Acquisition Window To acquire images using the VersaDoc go to the File menu and select VersaDoc The acquisition window for the instrument will open The acquisition window opens displaying a control panel and an image display window VersaDoc r Step Select Application of Channel1 Channel2 Channel3 Chamnel4 3 Enable Channel Select SYPRO Orange S20LP Uv TRANS 4x Gain 1x1 Bin r Step Il Position Focus Position Focus 173 Show Alignment Grid zi Lights UN Step Ill Set Exposure Time sec o ela Qe Flat Fielding O Acquire Q Optimize Exposure A Stop r Display Options Highlight Saturated Pixels Image file size 0 50 Mb Options 2 Help p Exposure Status Fig l 2 VersaDoc acquisition window When the VersaDoc window first opens no image will be displayed Appendix I VersaDoc The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to acquiring an image using the VersaDoc 1 Select the application 2 Position and focus the object to be imaged 3 Setthe exposure time 4 Acquire the image I 2 Step Select Application In Step 1 you select the appropriate filters and other imaging parameters for the type of gel blot plate or other obje
132. When you release the mouse button the border changes to a dashed blue line indicating a selected area e To reposition the scanning box you have selected position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move e To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created Quantity One User Guide You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right After you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later G 4 Step Ill Select Resolution The FX acquisition window allows you to scan at 50 100 200 or 800 micrometers These resolutions are listed as option buttons in the control panel Step Ill Select Resolution 50 micrometer 100 micrometer 200 micrometer 800 micrometer Image file size 3 19 Mb Fig G 10 Resolution option buttons The resolution you select should be based on the size of the objects e g bands spots you are interested in For example 50 micrometer resolution should be reserved for images requiring the highest level of detail e g high density in situ samples 1 536
133. XR Print If you are printing to a desktop or digital printer clicking Print sends the currently displayed frame either live or integrated to the printer If you have a video printer connected to your computer Video Print will be displayed in the Select Output window You can add information about your image to the bottom of the printout by selecting the appropriate checkboxes in the Options dialog box See section B 9 Options Save Clicking on Save will open a separate image window displaying the captured image A Save As dialog box will automatically open displaying the default file name for the image which will include the date time and user if known You can then change the file name and storage directory B 8 Exposure Status The Exposure Status bar shows the progress of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical representation of the remaining time before exposure is complete B 9 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your Gel Doc XR system Quantity One User Guide E Gel Doc XR Options Information Reminder Model Gel Doc XR xj Show Light Warning Camera Serial Number BR SIM 1234 Print Footer Info xj Date Time X Exposure Time xj Contrast Settings Imaging Area CM Width 13 60 Height 10 24 Save Options Misc Options Make Backup Copy Y Simple Acqui
134. a compilation of any symbol or series of symbols executed adopted or authorized by an individual to be the legally binding equivalent of the individual s handwritten signature Quantity One allows the user to sign an image thereby locking the file against future changes If a user changes a signed file and attempts to save the file the file automatically receives a new version number and the signed tag is removed from the window title To sign a file in Quantity One with the file open select Sign from the CFR menu A modified version of the Comments dialog box opens Enter any comments you may have and click Sign This opens the Login User dialog box which requires you to enter your user name and password for signature validation K 9 Quantity One User Guide fetosinuser Enter password for electronic signature User Name User Password Domain Domain Hand wv Done X Cancel Fig K 5 Electronic Signature dialog box Once you have signed your file the image window title bar indicates the file has been signed and the event is recorded in the Audit report CFR Proteins Raw 1 D Image Signed Fig K 6 A signed Image If your policies require signature verification by more than one person such as a lead researcher a signed file can be signed again without changing the file version provided the file has not been modified between signatures K 10 Index Numerics
135. a green border Note 7 5 To move the selected volume or volumes position the cursor over the selection and drag To copy within an image hold down the Ctrl key while dragging the selected volume or volumes The copy will be created and dragged to the new position To delete the selected volume or volumes press the Delete key To copy between images click on the Copy to Clipboard button on the Volume toolbar then open or select the image you want to copy to and click on the Paste from Clipboard button The copied volume s will be pasted into the new image in the same relative position it was copied from If you are copying to an image with a different pixel size i e resolution you will receive a message that the placement of the copy may not be exact Click on OK to complete the paste then position the pasted objects manually Volume Standards You can use volumes of known concentration to calculate the concentrations of unknown volumes 7 8 Chapter 7 Volume Tools To classify a particular volume as a standard double click on it This will open the Volume Properties dialog Volume Properties xj U3 r Volume Type Unknown Standard Background r Concentration Edit User Label DK Cancel Fig 7 7 Volume Properties dialog Select the Standard option button then enter the concentration in the Concentration field Do not include units Click on OK to close the
136. a lane frame for the cells in an array You can then specify the cell dimensions and quantitate them using the Quantity Standards function see section 6 3 Quantity Standards 4 16 Chapter 4 Lanes Note You can quantitate arrays outside of lanes using volume arrays see section 7 7 Volume Arrays The first step in defining an array is specifying the number of columns and rows and creating an array frame Got to the Lane menu open the Lane based Array submenu and select Frame Array ane based Array i Frame Array Array Cell Height Array Cell Width Fig 4 15 Lane based Array tools Enter the number of columns in the array and click on OK Quantity One x 9 How many columns are in the array For every column there will be a lane Fig 4 16 Setting number of array columns In the next box enter the number of rows and click on OK Quantity One x How many rows are in the array For every row there will be a cell in each column 10 Cancel Fig 4 17 Setting the number of array rows 4 17 Quantity One User Guide The array matrix will appear on the image Each column will be marked by a red line and each cell will be marked by top and bottom brackets Note If the cells appear marked by lines instead of brackets select Band Attributes from the Band menu and select Brackets in the dialog When you first create the array matrix it will probably not be centered on the c
137. a light option click Acquire Image Note For UV exposures there will be a 15 second delay while the lamps heat up before the exposure begins 10 7 Quantity One User Guide The camera will take a single image of the gel or membrane on the platform then a background image to adjust for image background When the exposure is complete the image will appear in the window u To adjust the brightness and contrast of the image open the Transform window by clicking on the button to the right of the image window 10 2 b Specify Options Under specify Options you can make changes to various settings Cut On the Cut tab you can choose to make multiple cut when possible select the specific cutting tip size and the type of material you are cutting Spebify Options Cut Plate Hydration Wash xj Make multiple cuts when possible Maximum cuts object 3 Cutting tip size 21 0 mm 1 5 mm Material Glass backed gel v Fig 10 6 Cut options If you want to make multiple cuts check the box labelled Make multiple cuts when possible Then enter the maximum number of cuts per object Next select the cutting tip size you are using Quantity One uses this to determine when you have reached the maximum capacity for wells on the plate Finally select the material you are cutting from the drop down list 10 8 Excision Plate On the plate tab select the plate type you are using for this cut run then select the
138. actice scans To enter simulation mode hold down the Ctrl key and select the name of the device from the File menu The title of the GS 800 acquisition window will indicate that it is simulated Note There is no simulated calibration for densitometers E 1 GS 800 Acquisition Window To begin acquiring images go to the File menu and select GS 800 The acquisition window for the densitometer will open displaying a control panel and a scanning window E 2 Densitometer GS 800 Calibrated Transparency Step Select Application Appendix E GS 800 Make Backup Copy Highlight Saturated Pixels Options El Help 4 Select 3 X ray film a 5 T Gray film 7 Filter Red X Green Blue White 9 Light Reflective Transmissive thls l 13 4 Step Il Select Scan Area a T Preview scan a Click and drag in diagram to set scan area dez t Top 0 0 Left 0 0 19 Bottom o o Right o 0 21 23 r Step Ill Select Resolution E 2 ji X resolution 63 5 Microns Select A S OT Y resolution amp 63 5 Microns E 1 29 i T Image file size 0 00 Mb EN 33 B c 4 3 Sto Qacsie i p 35 y Options 37 JAuto Save After Scan Hide Grid 39
139. age Z High Sample Intensity Low Sample Intensity Low PMT Voltage High PMT voltage Cancel OK Fig H 6 Selecting a custom PMT voltage Note For voltages above 80 of maximum you will receive a warning message that the high voltage could damage the PMT If you select Choose Later from the list of PMT voltages the choices of sample intensity will be displayed when you select your custom application Finally enter a name for your application in the Name field and click on OK to implement your changes After you have created an application you can select it from the application tree by selecting Custom and the name you created You can delete the application by selecting Custom Delete and the name of the application H 10 Radioisotopes Chemifluorescence Nucleic Acid Stain Protein Stain Fluorophores Microtiter Plate Colorimetric Multiplex Custom Custom Custom Application 2 532nm EX Blank 530 Fig H 7 Selecting a custom application Channel 3 f Channel 4 Custom Applicafign 1 Custom Application 2 Appendix H FX You can edit a custom application by selecting Custom Edit and the name of the application You can also use this feature to create a new custom application from an existing one Step Select Application uf Channel 1 of Channel 2 w Channel 3 yf Channel 4 X Enable Channel Select Radioisotopes K Scre
140. alue for the first standard band and press the Enter key The cursor will skip to the field below and you can enter a value for the second standard band Repeat this process until all the standard values have been entered Note The values do not need to be entered sequentially They will automatically sort themselves in ascending or descending order depending on how you specified the units You can enter a name for each standard band in the Name column This will appear in subsequent reports and printouts To remove a standard value click on the triangle button at the beginning of the row and select Delete The remaining standards will be renumbered Applying Standard Values to Lanes To apply the values to the standard lanes on the image click on the Apply to Lane button and click on a lane Quantity One User Guide E es E3 5 c E gt ES lt gt E amp m lt gt E Umolassitted Fig 6 8 Click on the Apply to Lane button and click on the lanes containing standards The values will be applied to the bands in the lane you select Click on any remaining standard lanes to apply the same values to them Note In general the more evenly spaced the standard lanes the greater the accuracy of the calculated band values We recommend a minimum of two standard lanes per gel Modeling lines that connect the standard bands in different lanes are used to compensate for any smiling or dist
141. an always select a level capture an image then adjust the level and capture another image For example if you select Low Sample Intensity and the resulting image has too many saturated pixels you will receive a warning message Simply change the setting to Medium Sample Intensity and rescan If you select High Sample Intensity and the resulting image is too faint select Medium or Low and rescan Custom Applications If your application is not listed if you want to use user installed filters or if you want to use an external laser you can create and save your own custom application From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings Radiolsgtopes Chemiluminescence Chemifluorescence Create a new Imager FX Application DNA Stain Gel Name custom Application 1 Protein Stain Gel Fluorophores Filter A Blank Microtiter Plate Filter B Bio Rad Radioisotope Densitometry Custom Filter C 532nm Blocking Laser 532 1064nm PMT Voltage Low Sample Intensity NS Fig G 5 Creating a custom application G 8 Appendix G FX To select a filter including user defined or filter combination click on the buttons for Filters A B and C and make your choice from each pop up list Note Filter wheel C is not available in the FX Pro or the FX Pro Plus systems Note The user defined filters Userl User2
142. and leave them empty e eeooeoeoeoee eeeco eaeno oes 06000000000 06000000000 06000000000 EX Zr rrr rl w Done X Cancel Fig 10 12 Mark wells for skipping Click on a well to mark it for skipping in the cut run Red wells are reserved wells while blue wells are wells with material in them from a previous run Note You cannot change the state of wells that have material in them When you have finished making your plate selections click Done The plate wizard will direct you as to where to place the plate in the spot cutter 10 14 Excision iil Load Plate on Cutter Load Plate001 into the front right position Name Plate001 Plate type 96 wells Front Left Front Right X Cancel Fig 10 13 Plate loading in the EXQuest Spot Cutter As each cut is being made it will be highlighted in yellow on the screen After each cut has been made the cut circle appears in white on the screen Cuts that have not yet been made are circled in green Run Info The Run Info section contains information regarding the current cut run Cuts indicates the total number of cuts in the run including multiple cuts Wells indicates the number of wells to be used Plates indicates the number of plates needed for the run Min well volume indicates the minimum well capacity to avoid overflow based on the number of multiple cuts selected and cutter tip size If the well volume for your 10 15 Quantity One Us
143. ange to retain the same number of pixels in the image 2 2 f Reduce File Size High resolution image files can be very large which can lead to problems with opening and saving To reduce the file size of an image you can reduce the image resolution by reducing the number of pixels in the image You can also trim unneeded parts of an image to reduce its memory size See section 3 9 a Cropping Images 2 12 Chapter 2 General Information This function is comparable to scanning at a lower resolution in that you are increasing the size of the pixels in the image thereby reducing the total number of pixels and thus the file size Note In most cases reducing the resolution of an image will not affect quantitation In general as long as the pixel size remains less than 10 percent of the size of the objects in your image changing the pixel size will not affect quantitation Select Reduce File Size from the File menu to open the Reduce File Size dialog box The dialog box lists the size of the pixels in the image Pixel Size X by Y microns the number of pixels in the image Pixel Count X by Y pixels and the memory size of the image Before liz ma Iz Width X Height Y Pixel Size 169 sa by 169 39 microns Pixel Count 633 by 756 Memory Size 467 33 Kb OK Cancel Help Pixel size in the x dimension increased Reduce File Size After Width X Height Y Pixel Size by 169 39 microns
144. antity One not necessarily the actual gel lane Note that if a lane is straight and vertical both calculation methods will give the same result 2 5 f Devices This tab contains preferences for imaging devices and the EXQuest Spot Cutter Specify the imaging devices that you want to appear on the File menu By default all supported imaging devices are selected clear the checkboxes of the imagers that you do not want to include on the File menu If you have connected the EXQuest Spot Cutter select it under Spot Cutter Click Setup spot cutter to run the spot cutter calibration wizard Note If you are changing the spot cutter settings you need to restart the application before you can perform calibration 2 5 g Security The Security tab of the Preferences dialog box contains the security options for CFR mode and the option for enabling Secure mode CFR Mode Setting the Security preferences for CFR mode requires the user have administrator privileges To locate the Security Preferences select Preferences from the Edit menu and click the Security tab 2 21 Quantity One User Guide Preferences m x Misc Paths Display Toolbars Application Imagers Security Enable 21 CFR Part 11 mode ogram times out if inactive minutes S equire login on startup Automatically create backup for new images Enable Secure Mode to prevent file changes Y OK X Cancel 2 Help Fig 2
145. apter 7 for details The tools for band detection are located on the Band menu and on the Band Tools toolbar Band Match Volume Analysis Report Band Tools Fa S EME HHH y Detect Bands Band Attributes Create Band Remove Band Adjust Band AE Gauss model Bands Remove One Gauss model Remove All Gauss models Band Information Plot Band Bands in Lane Contour gt Draw Band Fig 5 1 Band menu and Band Tools toolbar Note Before detecting bands you should subtract background from the lanes using the Lane Background command see section 4 2 Lane Based Background Subtraction 5 1 Quantity One User Guide 5 1 How Bands Are Identified and Quantified You can automatically identify all the bands in an image using the Detect Bands command see section 5 2 Band Detection or you can mark them individually using the Create Band command see section 5 3 Identifying and Editing Individual Bands Each identified band is defined by brackets above and below the band The width of each set of brackets is determined by the lane sampling width see section 5 2 a Detection Parameters The height of the brackets is determined automatically using a band finding formula together with parameters that you select Lane Sampling Width gt lt epe Bend imm Height me
146. arameter in the dialog you can either type in a new value or use the arrows to increase or decrease the setting by 10 percent Experiment with different settings to find those best suited to your images 5 2 a Detection Parameters Lanes to Detect If the intensities of the bands vary from lane to lane you may need to use different detection parameters on different lanes Specify whether you want to use the detection parameters for all the lanes or a single lane by selecting All or One next to the Lanes prompt If you choose One type the lane number in the field next to the One button When to Detect If you select Auto next to the Detect prompt band detection will occur immediately each time you change a detection parameter You will not need to click on the Detect button located at the bottom of the form To change more than one parameter before detecting choose Manual With the Manual option you can change parameter settings first and then apply them by clicking the Detect button Normalization Normalization is a way to compensate for differences in intensity between lanes It does not normalize for band quantitation The intensity of each lane is determined by the darkest band in that lane For example suppose that in all but one of the lanes the darkest band has an intensity of 50 000 counts In the one light lane the darkest band is only 25 000 counts With normalization band detection will be twice as sensitive when proce
147. ata range in the image This invokes the Auto scale command from the Transform dialog see section 3 8 Transform when an image is first opened in the viewer Note that this setting affects only how the image is displayed in the viewer not the actual data Note If you deselect this checkbox any images currently displayed will remain auto scaled Click on the Transform button in the viewer and click on the Reset button in the Transform dialog to undo auto scaling Buttons for various viewing tools are included in the Multi Channel Viewer Tools such as Zoom Box and Grab will change the display of all the images in the viewer at once Click on the Transform button to open the Transform dialog In the dialog you can adjust the display of each channel independently by selecting the appropriate channel option button Similarly the Plot Cross section command will report the intensity of each channel separately Quantity One User Guide Exporting and Printing Click on the Export button to export a 24 bit TIFF image of the merged view This will open a version of the Export to TIFF dialog see section 13 6 Export to TIFF Image Note that you cannot export data from the Multi Channel Viewer only the current view of the image designated as Publishing Mode in the Export dialog The colors in the viewer will be preserved in the exported TIFF image To print a copy of the merged view to a color or grayscale printer click on the Print button
148. ation Image x File Name Time Printed Fit Image to Page Draw Image Border Acquisition Info Page Number Print Image Actual Size Fig 13 1 Under the Headers and Footers settings select what information you want to include in your prints File name Select File name add the name of the image file to the header of the print 13 2 Chapter 13 Printing and Exporting Acquisition Info If Acquisition Info is selected the date the image was acquired and the data range of the scan is included in the footer information Time printed To add the date and time the image was printed select Time printed Page number Select Page number to add the page number to the footer information The Pagination settings determine allow you to determine whether to print your images actual size or fit the images to the page Fit Image to Page This fits the current view of the image to a single page based on the layout of the image Print Actual Size Select Print actual size to print your images based on the scan size of the gels Note If you are using the Gel Doc EQ ChemiDoc EQ Fluor S or Fluor S MAX you must specify the correct image area size when capturing your images to ensure accurate 1 1 printing You can specify the image area size in the acquisition window for the instrument See the chapter on each imaging device for more information Under Image you can add a border to the image area of the prints Adding
149. ations selecting cocotero oe i E REPRE ERRER E 4 Calibration iiie ette ie et eee E 9 Calibration settings eoe etd eig tee Hte EUER OR Pes E 13 Calibration step tablets sesssesseseeeeeeeeeeeneee eene enne E 10 Filters and light source selecting essere E 6 ODptIODS ein esee ei pe eU e e Monee cte eorr eiue E 14 Oversampling 4 2 cni eot e cep DIO E 14 Preview SCAN eese eO E 7 Resolution Selecting o en eret etie Rien d Mosse teen E 8 Saturated pixels highlighting sese E 15 Scan area selecting oss eo erede e er ee Ph reb iens E 7 Scanning an image sseseseeseeeeeeneenen eene eene en nennen entente terere nee E 13 SCANNING WIDdOW ecce E EUER Ro dore pii ec nennen E 3 SGSLcatrd ner Udemm HERUM E 1 Simulation MOE eite en eee e ee eee n e yes ges E 2 Imagenformation petarda 2 10 Image Report actin nike hts es Raa ee ee 13 3 Image size changlig edee reet der v tees et e hee pee E ede ye 2 11 Im ge Stack Tool mica eer ee sigin asians 3 13 Images Background subtraction eeseseseseeeeeeeeeeeen nennen 3 29 GU M 3 14 Contrasts conocen eene 3 17 enu AM eaves 3 24 Filtering noise from sese nennen ENES 3 33 Inyerting data cie ee eet peintre tp tee dp e oe 3 39 Ma pnit ying ein gene ene Re SS es os E 3 1 Positioning assa bietet tatem Bae dees Oben Done iste d
150. ay overlay will be created and displayed on the image 7 13 Quantity One User Guide 9 e e e e L e O 0000009 0OCOCOO00O eOOOOOO0 Fig 7 11 Array overlay Like regular volumes array volumes are initially displayed without labels To show the labels of the individual wells cells click on the Show Hide Volume Labels button on the toolbar Like regular volumes array volumes are initially labeled U1 U2 U3 etc Note If large volume arrays are slow to display or edit on your computer and the volume labels are showing try hiding the volume labels using the Show Hide Volume Labels command This will increase the processing speed considerably When you create an array overlay it is automatically selected the cells will be displayed with green borders and the Select tool is assigned to the mouse You can then move the array overlay so that it is properly centered on the image resize the cells so they fit the blots wells in the image and resize the overlay so the four corners fit over the four corners of the array on the image To delete the entire array overlay select it and click on the Delete key Moving an Array To reposition an array overlay move the cursor over any individual cell until the cursor changes to a multidirectional arrow and the cell border turns yellow Then hold down the cursor and drag the entire array to a new position 7 14 Chapter 7 Volume Tools Fig 7 12 Moving
151. ay using the High Low and Gamma sliders described below Reset Reset will return the image to its original unmodified appearance B 6 Step V Analysis The Analysis step of the Gel Doc XR acquisition window allows you to add annotations and analyze the newly acquired image B 9 Quantity One User Guide Annotate Clicking on Annotate will open a separate image window displaying the captured image The default name for the image will include the date time and user if known The Text Overlay toolbar will also pop up to allow you to annotate your image The image will not be saved until you select Save or Save As from the File menu Analyze Clicking on Analyze will open a separate image window displaying the captured image The default name for the image will include the date time and user if known You can then analyze the image using the other features in the main application The image will not be saved until you select Save or Save As from the File menu You can export your image as a TIFF image for analysis with other applications You can also export your image as a JPEG image for use in reports and presentations B 7 Step VI Select Output In Select Output you can Print and Save as your output options Step VI Select Output Step VI Select Dutput Pint E Video Print Save Save Digital or Desktop Printer Connected Video Printer Connected Fig B 8 Output options B 10 Appendix B Gel Doc
152. band that you select First select the report from the menu then click on a matched band This report requires band matching see section 6 2 Band Matching AII Matches Report generates a report on all the matched bands in the gel This report requires band matching see section 6 2 Band Matching When you select any of these reports the Report Options dialog will open where you can specify the type and formatting of the data 2 Diversity Database Lane Report x Report Options Locations xj Relative Front x Mol Wt Measurements x Peak Density x Average Density x Trace Oty xj Relative Qty x Gauss Peak Density x Gauss Trace Oty x Contour Area x Contour Oty x Calibrated Qty xj Tandem Repeats x Normalized Oty Database Band Set X Name Band Type x Number x Name Sample x Name Font Size Medium xj Column Spacing Medium xj Line Spacing teaim xj Modify Report Settings Report Cancel Fig 12 5 Report Options dialog 12 5 Quantity One User Guide Select the data to display using the checkboxes Select the Font Size Column Spacing and Line Spacing settings to be used in the report by clicking on the button next to each field and selecting from the list of options To save the report options click on the Modify Report Settings button and enter a name for the report settings in the field To load or delete previously saved settings click on the button next to the Settings to
153. band type click on the Match button then click on one of the bands The matched group of bands will be highlighted 6 26 Chapter 6 Standards and Band Matching fall of the known bands are in one lane click on the Lane button then click on the lane with the known quantities The entire lane will be highlighted Select Band hk Match k Lane k Fig 6 21 Buttons for selecting bands of known quantity In the lower part of the Quantity Standards dialog the lane and band numbers of the selected bands will appear in the Band column The intensity of each band will also be listed Entering the Quantities Enter the quantity of each known band in the Quantity column When you enter a quantity the value of the band in the Status column will change from Unknown to Known Trace Quantity Dilution Rel Band OD x mm Factor Status Dev 6 12 4 042 o6 jme 24 10 13 3 705 o5 jme 2 11 13 2 061 foso jme 13 Ge GAL aa o2 jw 2 12 8 1 059 Pos ee 05 2204 o0 mem 113 EE EE ll Fig 6 22 Entering the known quantities co i J o After you have entered a few quantities the status of the remaining bands in the list may change to O R meaning that the remaining bands are out of the current range of values based on their intensities and what you have already entered 6 27 Quantity One User Guide Alternatively the software may automatically calculate an unknown quantity
154. ber of seconds in the Exposure Time field Type in a number or use the arrow buttons next to the field In UV or White image mode when the specified exposure time is reached the last captured image will be displayed in the ChemiDoc EQ image window The camera will continue to integrate the image on the CCD updating the display whenever the specified number of seconds is reached Quantity One User Guide Once you are satisfied with the quality of the displayed image click on the Freeze button to stop the exposure process The last full exposure will be displayed in the image window r Step IIl Acquire Image Auto Manual Live Expose Expose Acquire Exposure Time sec Click on Freeze to stop 0 03 e s the exposure process COEM 9 Freeze Fig C 6 Freezing the manual exposure In Chemi mode Manual Expose will expose an image over the specified exposure time and then stop automatically Note X Freeze is automatically activated if you adjust any of the subsequent controls e g Video Print Image Mode Display controls etc Live Acquire Live Acquire mode allows you to specify an interval over which a series of progressively longer exposures are taken All exposures are then displayed on the screen and you can choose the one with the best image Click on the Live Acquire button A settings dialog box will open in which you can specify the total exposure time starting exposure time and number of
155. best 6 10 Chapter 6 Standards and Band Matching fits the data points The correlation coefficient provides another measure of curve fit Note Note that point to point semi log is the only method available if you perform band matching on the image because band matching adjusts the positional values of bands in localized areas based on your identification Point to point semi log is appropriate for this kind of localized variation whereas the other methods are not Therefore you should select point to point semi log if you intend to perform band matching required for similarity analysis on the gel Point to point semi log This and the Elder Southern method are especially useful for describing band migration in static field electrophoresis gels This is the only method available if you perform band matching on the image see note above Elder Southern This and the point to point semi log method are especially useful for describing band migration in static field electrophoresis gels At least three standard points are required to use this method Linear This method of least squares polynomial fits is useful for modeling pulsed field electrophoresis gels Quadratic At least three standard points are required to use this method of least squares polynomial fits Cubic At least four standard points are required to use this method of least squares polynomial fits Logistic At least five standard points are required to use th
156. border disappears when you release the mouse button after dragging check to make sure that the Show Data Area checkbox is checked This checkbox is located at the bottom of the dialog If the circle is slightly off center you can reposition it by positioning the cursor on the center target of the circle The cursor will change to a multidirectional arrow and you can drag the entire circle To resize the counting region circle position the cursor on the outer edge of the circle The cursor will change to a bidirectional arrow and you can drag the border in or out 8 2 Counting the Colonies After you have positioned the circle you are ready to detect colonies If you are counting plaques click on the Plaque checkbox Because plaques appear as clear circles on a darker background this checkbox must be selected for proper detection Before counting you may want to adjust the Sensitivity and Averaging parameters described below When you are ready to count click on the Count button Sensitivity The Sensitivity setting determines the minimum signal intensity in the image that will be counted as a colony This is based on the slope of the signal s peak The higher the sensitivity the more colonies will be detected If the sensitivity is set too high background noise will be erroneously detected as colonies If the setting is too low real colonies may be missed The default sensitivity setting is 10 00 If the image ha
157. btained with each cut The lower the number the closer the cuts will be At zero the cut edges overlap Vertical 100 Vertical 20 Horizontal 0 Horizontal 50 Fig 10 22 Use the sliders to adjust cut overlap Note that when using lane excision each individual cut will be placed in a separate well 10 24 Excision Volume Excision If you want to excise individual bands from a gel or blot select Volume excision under Specify cuts Specify Cuts Entire lane Volume overlay E X Show excisions on image Fig 10 23 Specify cuts by volume To create a volume to excise select the Volume rectangle tool then click and drag on the image to make the volume If Make multiple cuts when possible is selected on the Cut options tab the volume will contain up to the maximum number of cuts indicated Note If you select multiple cuts for volumes all cuts for each volume will be placed in the same well Run Info The Run Info section contains information regarding the current cut run Cuts indicates the total number of cuts in the run including multiple cuts Wells indicates the number of wells to be used Plates indicates the number of plates needed for the run Min well volume indicates the minimum well capacity to avoid overflow based on the number of multiple cuts selected and cutter tip size If the well volume for your selected plate is less than the Min well volume then the overflow warning flashes
158. butes User defined category buttons are available in the Standards dialog box Matched Band Set dialog box and Gel Layout dialog box They allow you to categorize the characteristics of your particular gel or any related gel to which you might apply the same set of standards To define a new category click on one of the category buttons A dialog will pop up in which you can select from a list of categories or create a new one Category None I Edit Attribute OK Cancel Fig J 1 Category pop up box To create a new category click on the Edit button This will open another pop up box in which you can enter the name of your new category Quantity One User Guide Edit Category x Category Attribute ef 2 SEIS ELSE bd New Select Delete Done Fig J 2 Edit Category dialog box Type the name of the new category e g Color next to the Category prompt and list attributes of that category in the Attribute fields e g Red Green Blue etc The form will automatically sort your attributes alphabetically within the Attribute fields Categories and attributes can be defined for any characteristics of your gel that would be useful to sort by Typical categories might be Enzyme Primer Probe Type Who etc Once you ve created a category and attributes you can use them in the dialog box Once again click on a category
159. c Standards Dialog The Standards dialog contains the values of the standards and includes tools for applying them to the standard lanes in the image and displaying and adjusting the standards regression curve The dialog opens with a default name for the standards For new standards this is a generic name e g Standards 1 You can enter a new name in the field Quantity One User Guide E Proteins Standard x E Standard Protein Comment Pen mark standards Type S Standard Units Molecular Weight Ascending Enzyme ka Primer ss Category Probe Name KDa 200 000 Z 115 500 Em 000 lee ooo 57 000 54 000 45 000 43 000 30 000 21 000 xl Delete Close Archive Help Fig 6 6 Standards dialog You can enter additional information next to the Comment prompt The category buttons and fields can be used to further define the standards Entering Standard Values If you are creating new standards enter the values of the standard bands in the table in the middle of the dialog Predefined standards already have these values entered Chapter 6 Standards and Band Matching Click here to delete a value and renumber the remaining standards Standard value Fig 6 7 Entering the values of the standards The table has three columns labeled Type Name and the units you previously selected e g KDa pI Rf In the units column type a v
160. ceesecececeeeceseeees 3 7 N Normalized quantity iret vitet eee et 5 15 6 22 Normalized Rf HS Une eene mee eei 6 4 O Opening Imapg s dese eg Dr EP CREER ORE RN UT PIER e treni coded 2 7 Optimizing images See Transform Overlays showing and hiding seen eene 3 6 P A A idea etii i d eei tp e e ee her 13 1 Password entering A enter A EEE eere ten ere ENS eR TR ER ERA 1 12 PCR gel analysis iii 11 1 PRO ee ES Slee deb btes 5 15 Personal Molecular Imager FX Acquiring an image spe e e RE E O cnn nennen nete innen rennen rennen F 6 Control panel et iv ipti reete etos t Ee F 2 Filesize of images ai F 6 lona E HH F 7 Saturated pixels highlighting sese F 7 Saying the 3m g6 siii e eth Iia eI etie EE RUE eS F 6 Scanning window esses rennen eren rennen entren nennen enne F 3 Selecting resol tion sit en e t Re e n e egere F 5 Selecting scan area i e nene edu F 4 Simulation Mode russia E the ee nter tee e aget F 2 PharosEX A te ts ee ee eli ee H 1 Acquirmg tle IMA SE sei esee erre eei eee edet d evt pee eet deny H 14 Index 10 Control panel mitin een pO H 2 Oi vA ABUELA M H 14 Filters 3 eoe est ih Ue Octubre H 16 OPUONS sissies RUE Reo eie anat iE dee H 15 Saturated pixels highlighting sese H 16 Saving the IMAGE eder pe eiecti cet dicet aces H 14 Scanning SN de e pe DP ESO D ROB DR H 3 Selecting an application
161. ch in turn determines the size of the pixels in your image 1 e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images D 5 b Reminder Show Light Warning When this checkbox is selected the software will warn you to turn off your transilluminator light when you exit the ChemiDoc XRS acquisition window or when your system is idle for more than 5 minutes D 12 Appendix D ChemiDoc XRS Note If you are performing experiments that are longer than 5 minutes e g chemiluminescence this should be deselected Show Filter Warning When this checkbox is selected the software will warn you to move the filter slider either to chemi or the UV White position D 5 c Dark Subtraction At the time of installation a reference dark image file was generated and saved to your hard drive The purpose of this file is to reduce dark current noise generated from the CCD Dark current noise is typical of all CCDs and is a result of the accumulation of charge in the absence of light The default time of the reference dark image is ten minutes The default reference file is optimized for ChemiDoc XRS applications If you select to change the reference dark image ensure that th
162. ckbox or a combination of two of the first three Red Green Green Blue Red Blue Filter Color Wavelength Application Examples Red 595 750 nm Coomassie G 250 BCIP NBT Fast Green FCF Methylene Blue Green 520 570 nm Coomassie R 250 Basic Fuchsin Blue 400 530 nm Crocein Scarlet White 400 750 nm Silver Stains Copper Stains Film Fig E 6 Examples of filter colors and applications Next to Light select the appropriate light source e Select Reflective for scanning opaque mediums such as dried gels on filter paper arrays or photographs The scanning dimensions in this mode are 30 cm x 40 cm e Select Transmissive for scanning transparent mediums such as films gels or slides The scanning dimensions in this mode are 29 cm x 40 cm E 6 Appendix E GS 800 E 3 Step Il Select Scan Area Preview Scan Before selecting the particular area to scan you can preview the entire scanning area to determine the exact position of your sample Click on Preview Scan A quick low resolution scan of the entire scanning area will appear in the scanning window Your sample should be visible within a portion of this scan Selecting an Area Using the preview scan as a guide select your scan area by dragging your mouse within the scanning window The border of the scan area you are selecting will be marked by a frame Note The scan area you select must be at least 1 cm wide When you release the m
163. ckground around each volume which is then subtracted from the intensity of each pixel inside the volume One pixel border around the volume Fig 7 8 Local background is calculated from a one pixel border around the volume Any pixels inside the volume that have the same intensity as the background pixels will be reduced to zero thereby eliminating them from the quantitation 7 10 Chapter 7 Volume Tools Global Background Subtraction Note If you select Global Background Subtraction in the Volume Report Options dialog but do not define a background volume as outlined below you will effectively select no background subtraction Global background subtraction calculates a single background intensity for the entire gel This average background intensity is then subtracted from all the volumes in the gel The steps for calculating global background subtraction are 1 Create a volume using one of the volume tools in a representative background region of the image 1 e a region where there is no data and where the average pixel intensity appears to be the same as the background intensity surrounding your data 2 Double click on the volume to open the Volume Properties dialog and select the Background option button E Volume Properties X i 1 B i r Volume Type 4 C Unknown Standard Background gt Concentrat
164. command to identify one or two bands in the lane then click on the Propagate Band Set button and click on the lane to assign the remaining band types to the lane 6 21 Quantity One User Guide Once the reference sample lanes have been modeled identify any unknown bands using the methods outlined in the previous sections 6 2 d Normalizing for Quantity You can normalize the quantities of the bands in a gel to the quantity of a particular identified band that appears in all lanes This is useful if you have loaded different amounts of sample in each lane Note Quantity normalization is required for calculating Differential Display Select Normalize from the Match menu and click on a matched band that appears in every lane If the band is not present in a lane the normalized quantity for that lane will be zero The quantity of that band will be set to 100 in each lane and the quantities of the other bands will normalized to that band You can view the normalized quantities using the Band Attributes dialog or in various reports 6 2 0 Graphs of Match Data You can display graphs of different kinds of data associated with the matched bands The commands for displaying these are located on the Match gt Match Graphs submenu Match Graphs Average o Calibrated Quantity Contour Normalized Quantity Peak Relative Quantity Trace Fig 6 16 Match Graphs submenu From the Match Graphs submenu select a type of graph and cl
165. compensate for gel distortion or smiling Peak density The intensity value of a band peak Average density The total intensity of the rows of pixels used to generate the profile of a band divided by the number of rows Trace qty The quantity of a band as measured by the area under its intensity profile curve Units are intensity x mm Relative qty The quantity of a particular band as measured by its intensity expressed as a percentage of either the total intensity of all the bands in the lane or the total intensity of the lane including the areas between bands The calculation method of Lane or of Bands in Lane is set in the Preferences dialog see section 2 5 e Application Gauss Peak Density The intensity value of a band s Gaussian peak after Gaussian modeling Gaussian Trace Quantity The quantity of a band as measured by the area under its Gaussian fitted profile Contour qty The quantity of a band that has been identified using the Contour or Draw Band tools It is the sum of the intensities of all the pixels within the band boundary multiplied by the area of each pixel Units are intensity x mm Contour area The area in mm inside the boundary of a band that has been identified using the Contour or Draw Band tools Calibrated qty The quantity of a band as calculated from the trace quantity and quantity standards Note that this is different than quantity determined using volumes Units are
166. cquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated G 1 FX Acquisition Window To acquire images using the FX go to the File menu and select FX The acquisition window for the imager will open displaying the control panel for the imager and the scanning area window Appendix G FX M Imager FX xj Step Select Application v Channel1 Channei2 X Enable Channel Select Radioisotopes CS or BI Screen Bio Rad Lowest 532 1064nm EX Blank Bio Rad RI 532mm BL Channel3 Channel 4 r Step Il Select Scan Area Click and drag in diagram to set scan area Toy Left 3 Bottom R Right 14 x Quadrant Mode gt r Step Ill Select Resolution 50 micrometer 100 micrometer 200 micrometer 800 micrometer Image file size 13 73 Mb Acquire 3 Stop Options Auto Save After Scan Highl
167. cribed above they can still be deleted from or modified in the image To make standards read only insert a tilde character in front of the name of the standards then click on the Archive button These standards will always be available under that name in the list of standards All Bio Rad standards are read only Disabling Deleting the Archive To delete the archived standards including Bio Rad standards remove the oneprefs dbs database from the fixed prm folder on your hard drive Under Windows this folder is located in the Bio Rad Program Files The Discovery Series directory On the Macintosh this file is located in The Discovery Series folder in the Preferences folder in the System Folder After you remove this file a new empty oneprefs dbs database will be automatically created the next time you open an image You can use this to begin a new archive 6 2 Band Matching To compare the similarity of samples in a gel using the phylogenetic tree similarity matrix etc you must match bands across lanes using the commands on the Match menu and toolbar Quantity One User Guide Note If you have run standards on the gel you should define them before proceeding as described in the previous section Multiple lanes of standards will facilitate the band matching program however they are not required Match Volume Analysis Reports Match Tools E g en zna Standards E V X i 22 fH SB Standard Curve
168. ct that you are imaging Each set of parameters you select is called an application The VersaDoc supports multichannel sequential imaging This allows you to automatically image the same object using up to four different applications e g to detect different types of stains on the same gel First you select a channel then you select the application under that channel I 2 a Selecting a Channel Note Illumination Flat Fielding will be disabled if you use multiple channels The four channels are accessed using the tabs under Step 1 Channel 1 is always enabled that is the VersaDoc will always capture an image using the application settings selected under Channel 1 first To enable any of the remaining channels click on a channel tab then select the Enable Channel checkbox and select the application for that channel as described below Enabled channels have a green check mark on their tabs Note If you use the Optimize Exposure feature only the application under the selected channel tab will be imaged other channels even if enabled will be ignored Quantity One User Guide You do not need to enable Channels 2 4 in sequence For example you can set up your four most common applications using the different channels but only enable Channel 4 for a particular gel Channel 1 would be scanned first Channel 4 second Enabled channels are imaged sequentially The separate scans are displayed and saved as separate images Th
169. d a pop up box will ask you to confirm the action If the bands are displayed as lines the band line will simply be deleted If the bands are displayed as lines you can delete more than one band at a time Select Remove Band and drag a box around the bands to be removed If you undetect a band it will no longer be counted as a band but its intensity will still contribute to the total lane intensity Note After you undetect bands renumber the bands in the image by selecting Sort and Recalculate from the Edit menu 5 4 Plotting Traces of Bands Fig 5 9 Plot Band and Bands in Lane buttons Plot Band displays an intensity profile of a band Select the command from the menu or toolbar and click on the band of interest 5 12 Chapter 5 Bands Bands in Lane displays an intensity trace of a lane with the defined bands highlighted Select the command from the menu or toolar and click on the lane of interest Fig 5 10 Bands in Lane command 5 5 Band Attributes You can display different types of information about defined bands Select Band Attributes from the Band menu or toolbar to open the dialog Quantity One User Guide Band Attr for Image 1 1D Scan x r Band Labels None Band Number Relative Front Mol wt Peak Density Average Density Trace Qty Relative Oty Gauss Peak Density Gauss Trace Oty Contour Qty Contour Area Calibrated Oty Normalized Oty Ban
170. d Type Band Model Tandem Repeats Diff Display Style Brackets Lines DK Help Fig 5 11 Band Attributes dialog At the bottom of the dialog select how you want to mark defined bands on the image as Brackets or Lines Lines are usually easier to read than brackets in gels with closely packed bands while brackets are better for displaying and editing the boundaries of bands You can also select the band data to display on the image next to each band Note None Band number The sequential number of a band in lane as counted from the top of the lane Relative front The distance of a band from the top of its lane divided by the total length of the lane The lane length can be determined either by measuring a vertical line from the top of the lane to bottom or if the lane is curved by measuring along the length of the lane Set the preferred measuring method in the Preferences dialog see section 2 5 e Application Note that normalized Rf is derived from relative front however normalized Rf is calculated only for bands that have been modeled using standards or band sets and can change based on the modeling Molecular Weight Isoelectric Point Base Pairs other units This value is 5 14 Chapter 5 Bands determined by the type of standards defined for the gel the band s position in the lane and any modeling performed on the gel using band matching or multiple lanes of standards to
171. dialog Standard volumes have the default names S1 S2 S3 etc based on their creation sequence Display or hide volume names using Show Hide Volume Labels command as previously described After you have identified two or more standards you can use the Volume Regression Curve see section 12 7 Volume Regression Curve under the Reports menu to calculate the concentrations of the unknown volumes Note Volumes that fall above the greatest standard value or below the lowest standard value cannot be guaranteed for accuracy To change a standard back to an unknown double click on it then select the Unknown button 7 9 Quantity One User Guide 7 6 Volume Background Subtraction When you draw a volume you will probably include some background pixels inside the volume These background pixels will usually have an intensity value that you do not want to include in the volume quantitation There are two ways of calculating this background intensity local and global The background subtraction method is selected in the Volume Report Options dialog see section 12 6 a Volume Report Options Local Background Subtraction Local background subtraction calculates a separate background intensity for each unknown and standard volume you create For each volume the intensities of the pixels in a 1 pixel border around the volume are added together and divided by the total number of border pixels This gives an average intensity for the ba
172. e The Select template field lists all the templates that are currently open The Automation Manager remembers the files that were open the last time the application was open To create a new template click New A new template appears in the list See Section 3 16 b Step 2 Edit Selected Template for how to modify a new template To open an existing template not currently in the list click Open This opens the standard Open dialog To remove a template from the list click Close 3 16 b Step 2 Edit Selected Template Step 2 Edit Selected Template lists the name of the currently selected template and the list of options available to the template To edit the template check the option s you would like to include and or uncheck the option s you would like to remove from the template 3 43 Quantity One User Guide Use Single Image for Template The Automation Manager allows you to specify a single image as the source for all options you select Checking the Use Single image for template box opens the Select Source Image for this Script item dialog opens x r Select Source Image For This Script Item Proteins test v2 Raw l D KIM n w Done x Cancel 2 Help Fig 3 27 Select an image Make sure the image you select contains the object s you wish to apply to the destination images If you wish to use options from multiple images then uncheck the box labeled Use single
173. e A Rae an tS 3 40 Misure 3 41 TIFF files EXpOfUlfip 25 iecit RU ra iet orit oH E T eiu er 13 5 Iinport ng zu tee eee a UP ODIe ire RENE 2 9 Tile commands tio e eite e t etes 3 4 TOOL Heli 2 2 Toolbars Malti rst bitte eae e ai 2 2 SECON ALY i 5 sciet ii it nisi even hae P nere 2 3 Trace quantity c eed une See a 5 3 5 15 6 26 ON 3 17 AMOS iii 3 20 Controls ip iii dat 3 20 Gamma slider mido ee 3 22 Hich Low Siders itte iran isis 3 20 Highlight Saturated Pixels oooononnnnnnninnnocnnoncononononncnnnonnconncn no nnnnnnnn conc onnnrinnnnos 3 23 PISTOLA a i eret eee ee nen e ni tpe rtg ds 3 20 Invert Display io ess 3 23 U UNIX file names parsing eene enciende 2 17 Index 14 Users multiple meiosis eet ere ratore te tpe pite EEE ep eR 2 23 V VersaDoc ACQUILE e STRE HERRERA E EAE ED EN anna eens I 11 ACGUITING THE mage uen i pr reine ERE Riu e UV YEUSE I 10 BIO ils ARS ea eee en ee a D 4 I 6 Control panel 1 5 tte p rd eet I 3 Custom settings oet ea ede I 5 Dark Subtraction coccooooncnnonononcnonononnnononononnnnnonononnnnnnnononnn narco nono nanaconos D 13 I 14 Expos re tme at uere Aveda eae eee I 8 Exposure times recommended ssssssssseeeeeeneee eene I 9 Galle siete ety ean e en EUR I 6 lll mination Plat Fielding Re Deed eee ie E Hte S I 10 Imaging area size eS io pa ed ipeo rete I 17 Optimize expoSUte a ei eerie ee e eie e ee e
174. e After Scan the image created using Channel 1 will have the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example 1f you scanned an image using only Channels 1 and 4 the image created using Channel 4 will be saved as version 2 v 2 G 2 b Selecting an Application To select an application i e the appropriate filter and other parameters for the type of object you are imaging click on the Select button under a channel tab G 5 Quantity One User Guide Radioisotopes Chemiluminescence Chemifluorescence DNA Stain Gel Protein Stain Gel Sypro Orange Fluorophores Svpro Red Microtiter Plate Nile Red High Sample Intensity Densitometry Medium Sample Intensity Sypro Rub Yp d Low Sample Intensity Fig G 4 Example of an application tree Ethidium Bromide gel Standard Applications The standard applications and associated settings are listed in a tree that expands from left to right When you select a standard application the software automatically selects the appropriate filter s in the FX for that particular application Some applications are inactive in the FX Pro and FX Pro Plus systems Standard FX Applications Category Application CS or BI Screen Bio Rad Radioisotopes K Screen Kodak Fuji Screen Chemiluminesce
175. e RE etie 3 1 Rotating and flipping sseeseeeeeeneeenen nennen nennen enne 3 26 STACKING cuestan iii nrbe fotos 3 13 Textoverlays AO 3 39 Viewing in multiple channels sssini senii 3 7 Imaging devices listing on File menu esee 2 21 Index 7 Quantity One User Guide Imitate ZOOM re nr ER PUR eei Dre EE E A 3 4 Importing TIEF files eet ae Ai a eret dis 2 9 Installation WandOoWS i5 iretur ERREUR EE ERR EHERERE ei 1 7 Invert Image data iniciadas et bie testes Eee 3 39 Image display 5 etse merenti te 3 23 K Keyboard commands e ii 2 6 E Lane frame AUSTIN A aiii 4 4 CLIMA A NA A ee 4 2 Lane Voluime Excision conim cete etre EPT roK tee 10 18 Lane based Arrays AdJUsting inniti aetema eer ee 4 18 Cell height iacet It tete dete os 4 18 Cell wadtb tonnes ime tmeeeepbepeeomeo tee 4 18 Lanes SUIDA EE 4 7 Comparing i see Rp o ads 4 13 DEIS 52e nee quee Ee 4 6 IDA npa EH 4 7 Framing automatically sirosis a E EE R E ennt 4 2 Framing manually n iecit eiecti etr lots ide ce tee ade sence 4 3 Profiling euro id ten e UHR OUO HERREN 4 8 Reports ete to RE UE D e betae ded 12 5 Unadjusting vete teretes 4 7 Width of all lanes iret ee E EIE 5 7 Width of individual lanes eese eene 4 8 Index 8 Eoi v UR C E 1 9 Registration cte Ee pire etae epe 1 10 Tane tool redet ettet DRE EUR Ettore te EDI 3 40 M Macintosh iia tai 1 8 Macintos
176. e a version number v2 v3 v4 etc appended to the base file name The highest version number will be the final exposure If you did not elect to auto save the exposures as they were created then each image will be unsaved Quantity One User Guide To stop the Live Acquire click on the Freeze button or adjust any of the subsequent controls e g Video Print Image Mode Display controls etc Note Exposures captured before freezing will be displayed in image windows Study the different images and select the best exposure s to keep You can then proceed to the next step C 5 Step IV Optimize Display The Display controls are useful for quickly adjusting the appearance of your image for output to a video printer Adjusting these controls will automatically freeze the video display and allow you to alter the image within the ChemiDoc EQ window Step IV Optimize Display High 255 y Low jo pp Gamma 1 00 pem pr xj Highlight Saturated Pixels Invert Display Auto scale Reset Fig C 8 Display controls These controls are similar to those in the Transform dialog box Note The Display controls will only change the appearance of the image They will not change the underlying data High Low Sliders If Auto scale doesn t give you the appearance you want you can use the High and Low sliders to redraw the image yourself In white light mode and chemi mode dragging the High slider handle to
177. e as it is taken Click on OK in the settings dialog to begin taking exposures If you selected Save Images a Save dialog box will open in which you can specify the base file name and location of the exposure files When you click on Save the exposures will be taken The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time When each exposure is complete an image window containing that exposure will open in front of the ChemiDoc XRS window Note that the first exposure will have the base file name the default base file name is the computer user name and a time stamp Each subsequent exposure will have a version number v2 v3 v4 etc appended to the base file name The highest version number will be the final exposure If you did not elect to auto save the exposures as they were created then each image will be unsaved To stop the Live Acquire click on the Freeze button Note Exposures captured before freezing will be displayed in image windows Study the different images and select the best exposure s to keep D 4 a Exposure Status The Exposure Status bar shows the progress of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical representation of the remaining time before exposure is complete Illumination Flat Fielding For UV or white light transilluminatoin applications you should use the app
178. e background changes from top to bottom e g gradient gels Click on the Background Stripe button then drag on a background region to create a rectangular box down the length of the image The average intensity of each horizontal row of pixels in the stripe will be subtracted from each pixel in that row across the entire gel This way if the image has more background at the bottom than at the top more background will be removed from the lower regions of the image Note Make sure that the background stripe runs the entire length of the lanes down the gel The average of the topmost row in the stripe will be subtracted from all rows above the stripe and the average of the bottommost row will be subtracted from all rows below The minimum and maximum intensities in the stripe are displayed next to the Min and Max labels in the box Also the average intensity value for the entire stripe is displayed next to Avg 3 32 Chapter 3 Viewing and Editing Images Completing the Subtraction When you are satisfied with the background subtraction shown in the preview image click on OK Because whole image background subtraction is irreversible you will be prompted to subtract from the original image subtract from a copy or cancel the operation If you choose Copy and Subtract enter the name and or version number for the new copy in the pop up box and click on OK to complete the command 3 11 Filtering Images Filtering is a process tha
179. e band profiles will be stretched and their peaks shifted Printing and Exporting Click on the Print button to print a copy of the Compare Lanes display Click on the Export button to export the data points in the graph to a spreadsheet This will open the Compare Lanes Export dialog 4 15 Quantity One User Guide Compare Lanes Export Number of data points gt gt Points 576 Field Separators Commas 1 2 3 format Tabs Excel format Export Destination File Clipboard Cancel Help Fig 4 14 Compare Lanes Export dialog This Export dialog includes a field for the number of data points to be taken along the length of each lane The default value in this field is the maximum number of data points that are available for the lanes you are comparing Select the export format tab or comma delimited and destination file or clipboard then click on OK Note The exported data will be different depending on whether you have checked Align Band Types Show Gauss Model or neither If the Show Gauss Model checkbox is selected each lane that has been Gaussian fitted will have two columns of data one for the Gaussian fitted profile and one for the regular profile If the Align Band Types checkbox is selected the exported values will reflect the stretched and shifted profiles of those lanes that have been aligned 4 4 Lane based Arrays The lane based array functions allow you to create
180. e effect the next time you open the acquistion window Reset Reference Dark The first time you open the Gel Doc XR acquisition window a reference dark image file is generated and saved to your hard drive The purpose of this file is to reduce dark current noise generated from the CCD Dark current noise is typical of all CCDs and is a result of the accumulation of charge in the absence of light To reset the reference dark image check the Reset Reference Dark checkbox The next time you open the Gel Doc XR acquisition window the software will acquire a new dark reference image Appendix C ChemiDoc EQ Fig C 1 ChemiDoc EQ Before you can begin acquiring images the ChemiDoc EQ system must be properly installed and connected with the host computer See the ChemiDoc EQ hardware manual for installation startup and operating instructions Quantity One User Guide To use the ChemiDoc EQ you will need to have the Bio Rad supplied acquisition board installed in your PC or Macintosh The drivers for this board will be installed when you install the main software application Make sure that your ChemiDoc EQ camera is turned on If the camera is not turned on the ChemiDoc EQ acquisition window will open and an image capture error will be displayed Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active b
181. e image window Select Full Scale to adjust the displays so that they show the full intensity range of the image Select Low High to magnify the range between the Low and High sliders This makes it easier to view the data if it does not occupy the full intensity range of the image Log The Log checkbox changes the way the data is displayed in the histogram so you can better discern subtle changes in signal intensity Image Max Min and Units Image Max and Min display the range of intensity in the gel image The image units are determined by the type of scanner used to create the image For images measured in O D s you can display the maximum and minimum O D values in the image by selecting the Calibrated Quantity checkbox If this box is not selected the maximum and minimum numeric pixel values are displayed 3 22 Chapter 3 Viewing and Editing Images Image Color Click on this button to open a list of color maps which you can define using the Colors command on the Edit menu see section 3 7 Colors Select a color map from the list to change the image in both the Transform window and the image window Invert Display Select the Invert Display checkbox to change light bands on a dark background to dark bands on a light background and visa versa The image data will not change only the display Highlight Saturated Pixels Select the Highlight Saturated Pixels checkbox to highlight areas of saturation in the image in red
182. e listed in the dialog These values are based on any standards you have defined and the band matching If you have not defined standards normalized Rf units are used 6 18 Chapter 6 Standards and Band Matching Name KDa 259 310 277 593 251 421 241 160 225 754 206 128 183 106 163 527 154 097 148 081 x Fig 6 14 Applying and editing band type values Band type value Assign band type to the mouse Delete band type If you change any of the values in the list that will be reflected in the matching on the image Click on the numbered Arrow button next to a band value and click on a band in the gel to identify that band You can enter names for the band types in the Name column These will appear in subsequent reports and print outs To remove a band from the set click on the triangle icon next to the band value Confirm the deletion and the remaining band types will be renumbered Band Set Toolbar The toolbar in the Band Set dialog contains all the commands needed for matching Quantity One User Guide Apply to Lane Create Band Match Propagate Band Set Show band models MENS AA Clear from Lane Remove Band Unmatch Outlier Show band types Standard curve Fig 6 15 Band set toolbar Apply to Lane applies the values in the band set to any lane in the gel Click on the button then click on the lane The bands in that lane that can be matched will change to red Clear from La
183. e positioning and focusing the image select the application that you will be using under Step I Note that the optimal position and focus will be different for a filtered image versus an unfiltered one All standard applications except Chemiluminescence use a filter Position After you have selected your application you are ready to center your gel or other object within the camera frame To do so click on the Position button The VersaDoc will begin capturing a live image and updating it every second With the Position button selected study the image in the acquisition window while you position your object in the center of the sample stage If you have a zoom lens on the camera you can adjust the magnification while you position See the VersaDoc User Manual for details on positioning While you are positioning you can select the Show Alignment Grid checkbox to display a target grid overlay on the image When you click on Position the light inside the camera box automatically switches on To turn the light off while positioning deselect the Light On checkbox When you are finished positioning click on the Stop button Focus Note Before focusing you should adjust the f stop on the camera to the lowest setting i e the maximum aperture This reduces the depth of field allowing you to more accurately focus the camera Then after focusing increase the f stop to the Quantity One User Guide desired setting See
184. e remaining channels click on a channel tab then select the Enable Channel checkbox and select the application for that channel as described in the following section H 4 Appendix H FX Click on the tab to Step Select Application selectthe channel x Channel uf Channel 2 uf Channel3 of Channel 4 Select the checkbox to enable the channel gt xj Enable Channel Click on the Select Select button to select the application Radioisotopes K Screen Kodak Lowest 532nm EX Blank 390nm BP Fig H 3 Enabling Channel 2 Enabled channels have a green check mark on their tabs You do not need to enable Channels 2 4 in sequence For example you can set up your four most common applications using the different channels but only enable Channel 4 for a particular gel Channel 1 would be scanned first Channel 4 second Enabled channels are scanned sequentially The separate scans are displayed and saved as separate images The total scanning time depends on the number and type of enabled applications If you have selected Auto Save After Scan the image created using Channel 1 will have the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you scanned an image using only Channels 1 and 4 the image created using Channel
185. e saved and used to subtract the dark current from all subsequent acquisitions Note The VersaDoc 1000 will take a 60 second reference dark exposure the VersaDoc 3000 and 5000 will take a 180 second reference dark exposure For image exposures that are longer or shorter than the reference dark the reference dark will be scaled accordingly and then subtracted It is recommended that the reference dark exposure time be equal to or greater than the sample exposure time You can change the default reference dark exposure time using the Reset Reference button see below If you deselect the Referenced button and then reselect it the old reference dark exposure will still be available 1 15 Quantity One User Guide Separate reference dark exposures will be taken for images that have different levels of binning or gain Once you have created a reference dark for each level of binning or gain the appropriate reference dark will be used according to the settings of your selected application Reset Reference If you would like a reference dark with an exposure time that more closely matches that of your typical scans click on the Reset Reference button A pop up box will prompt you to enter a new dark exposure time in seconds Quantity One Reset Reference Dark Image Options Dark Exposure Time 30 00 Seconds DK Cancel Fig l 9 Reset Reference Dark pop up box Click on OK to implement your change The new ref
186. e software and some are taken from the Macintosh version Both versions of a menu or dialog box will be shown only when there is a significant difference between the two Quantity One User Guide 2 Bio Rad Listens The staff at Bio Rad are receptive to your suggestions Many of the new features and enhancements in this version of Quantity One are a direct result of conversations with our customers Please let us know what you would like to see in the next version of Quantity One by faxing calling or e mailing our Technical Services staff You can also use Solobug installed with Quantity One to make software feature requests 1 Introduction 1 1 Overview of Quantity One Quantity One is a powerful flexible software package for imaging and analyzing 1 D electrophoresis gels dot blots arrays and colonies The software is supported on Windows and Macintosh operating systems and has a graphical interface with standard pull down menus toolbars and keyboard commands Quantity One can image and analyze a wide variety of biological data including radioactive chemiluminescent fluorescent and color stained samples acquired from densitometers phosphor imagers fluorescent imagers and gel documentation systems An image of a sample is captured using the controls in the imaging device window and displayed on your computer monitor Image processing and analysis operations are performed using commands from the menus and toolbars Ima
187. e specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned H 5 Acquire the Image Once you have selected your application scan area and resolution you are ready to acquire an image Click on the Acquire button There may be a short delay while the image laser warms up then the scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected Note If Warn on Saturated Pixels is checked and the image you are scanning has more than 10 saturated pixels you will receive a warning message If this happens you can go back and select a higher sample intensity in the application tree Saving the Image After the scan is complete a message will appear asking you if you want to keep the scan If you select Yes a separate window will pop up containing the new image Appendix H FX You can then save and analyze the image using the standard menu and toolbar functions H 6 Options Auto Save After Scan To automatically save any scan you create click on the Auto Save After Scan checkbox With this checkbox selected when you click on Acquire a Save As dialog box will open asking you
188. e ted he ier I 12 Optimize uniformity sessi nee cono nn non cra rra crac oracion eene en D 9 nali d RE I 14 Position i eee e pet p Rev I 7 A A eiit net eie trat i ER ER ETTE I 10 Saturated pixels highlighting sse I 18 SAVE ODLIODS iecit Een a I 17 Selecting an application sessi ene I 3 Video PANIN E iet eed eet tee o Ro Re 13 4 View Entire Image eonun 3 4 Viewing images in multiple channels sse 3 7 MNTRAReportz er sppenRepUal e as 12 25 VINTRS calculattng 4 5 ae ert pee e teet eere tae ine oH 11 3 Volumes Ur T 7 12 Background subtraction seesseeeeeeeeeeeeeneee nennen 7 10 Copying between images arc esaeo oposi ouen apenre eapo rennen erre 7 8 Copying within an image e sessesesseeesesseesssserersreeretrtereriserreseeresreresrenterenrereneeet 7 8 Cr notice 7 1 Del ends dee 7 8 Index 15 Quantity One User Guide D SPAY ING ss EN 7 7 Labeling davis epi Sid e aes aces Or be HRS eee 7 5 MOVIDO inier HORE HER HIGHER TRIER ORDRE 7 8 Regresi n CUEVE tete el e bebe ied 12 23 Repott caste nt e eig ened ihe bes sesh Tate ERREUR 12 20 Showing hiding labels esee 7 7 Standards ii RR sit ta n OO RETRO EUR 7 8 IBID Mx PE E 7 5 Windows blur 3 4 Windows file locations ooonccnonnnnoccnocnnoonnnonoconononnnnnncconn non conc non nnne cc terere nne
189. e the area into quadrants There is also an outer box and inner box marked by thicker lines The quadrants are numbered 1 16 left to right and lettered A U top to bottom If you prefer a scanning window measured in centimeters deselect the Quadrant Mode checkbox in the control panel by clicking on it To hide the gridlines click on the Hide Grid checkbox under Options The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to scanning an image using the PharosFX 1 Select the application s 2 Select the scan area Quantity One User Guide 3 Select the resolution 4 Acquire the image H 2 Step Select Application In Step 1 you select the appropriate filters and other scanning parameters for the type of gel blot plate or other object that you are imaging Each set of parameters you select is called an application The PharosFX supports multi channel sequential scanning This allows you to automatically scan the same object using up to four different applications e g to detect different types of stains on the same gel First you select a channel then you select the application under that channel H 2 a Selecting a Channel The four channels are accessed using the tabs under Step 1 Channel 1 is always enabled that is the PharosFX will always scan using the application settings selected under Channel 1 first To enable any of th
190. e the setting to Medium Sample Intensity and rescan If you select High Sample Intensity and the resulting image is too faint select Medium or Low and rescan Custom Applications If your application is not listed or you want to use instrument settings other than the preset options you can create and save your own custom application From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings H 8 Appendix H FX Radioisotopes Chemifluorescence Nucleic Acid Stain Protein Stain Quantity One Create a new Imager FX Application Fluorophores i Name custom Application 1 Microtiter Plate Colorimatr c Fiera Blank Multiplex Fiter B 390nm Bandpass PMT Voltage Low Sample Intensity Fig H 5 Creating a custom application To select a filter including user defined or filter combination click on the buttons for Filters A and B and make your choice from each pop up list Click on the PMT Voltage button to select a standard voltage for your custom application or create a custom PMT voltage To select a custom voltage click on the Custom option In the dialog box adjust the slider to select a PMT voltage as a percentage of the maximum H 9 Quantity One User Guide High Sample Intensity Quantity One Medium Sample Intensity Low Sample Intensity Selecta custom PMT voltage Choose later Custom PMT Volt
191. e time of the reference dark image is of equal or greater duration than your typical sample image When the sample image time is longer than the reference dark image time the ability to detect very faint samples may be compromised To reset the reference dark image time select options in the ChemiDoc XRS acquisition window Select Reset Referenced x Reset Reference Dark Image Options Dark Exposure Time sec 600 o Cancel DK Fig D 12 Reset Reference Dark pop up box Enter the desired time in seconds Select OK You will be prompted to close and re open the acquisition window Place the lens cap on the camera and close the door of the hood See ChemiDoc XRS instruction manual Select OK at the prompt and new reference dark images of the entered time interval will be taken for each of the possible combinations of camera gain and bin settings These will be retained as the reference dark images until the time is reset again D 13 Quantity One User Guide D 5 d Save Options To automatically create a backup copy of any scan you create select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be promted to give it a new name This prot
192. e to generate a flat field corrected exposure For subsequent UV or white light trans exposures you will be prompted to either use the appropriate saved flat field image or acquire a new one D 10 Appendix D ChemiDoc XRS E Quantity One xi AN Illumination Flat Fielding Do you want to use the saved reference image for Image Optimization y Ye X No Fig D 10 Using the saved reference image Note Flat fielding is unavailable for White EPI illumination and chemiluminescence applications D 5 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your ChemiDoc XRS system Quantity One User Guide E ChemiDoc XRS Options 13 xj Information p Dark Subtraction Model ChemiDoc XRS Referenced None Camera Serial Number Reset Referenced BR SIML Save r Imaging Area CM J Auto Save After Scan Width 13 00 Make Backup Copy Height 10 30 r Reminder x amp uto scale Transform After Acquisition x Show Light Warning r Display Show Filter Wami Auto Exposure Threshold iow Filter Warmin x E Saturated Pixels o 00 Cancel OK Fig D 11 Available options in the ChemiDoc XRS acquisition window Click on OK to implement any changes you make in this box D 5 a Imaging Area These fields are used to specify the size of your imaging area in centimeters whi
193. e total imaging time depends on the number and type of enabled applications If you have selected Auto Save After Scan the image created using Channel 1 will have the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you captured an image using only Channels 1 and 4 the image created using Channel 4 will be saved as version 2 v 2 I 2 b Selecting an Application To select an application i e the appropriate filter and illumination source for the type of object you are imaging click on the Select button under a channel tab Standard Applications The standard applications and associated settings are listed in a tree that expands from left to right When you select an application the software automatically sets the appropriate filter in the VersaDoc for that particular application The VersaDoc has four application groups and the VersaDoc MP has six application groups First select your general application then select the particular stain or medium you are using When you select the stain or medium the software automatically sets the appropriate filter S20LP 530DF60 610LP clear or none light type UV white or none and light source Trans Epi or neither in the VersaDoc for that particular application Appendix I VersaDoc
194. eaks at the high end of the range right end of the plot This type of noise is common in electronic cameras with malfunctioning pixels It can also be 3 35 Quantity One User Guide caused by dust or lint in the imaging optics or scratches on photographic film Salt is a type of outlier noise see below Pepper This type of noise appears as specks that are darker than the surrounding background The distribution histogram of this type of noise displays noise peaks at the low end of the range left end of the plot Its causes are similar to those of salt noise Pepper is a type of outlier noise see below Next select one of the following option buttons to describe additional features of the noise Gaussian The distribution histogram of this type of noise has a Gaussian profile usually at the bottom of the data range This type of noise is usually an electronic artifact created by cameras and sensors or by a combination of independent unknown noise sources Uniform noise This type of noise appears in the histogram as a uniform layer of noise across the data range of the image Outlier noise This category of noise includes salt and pepper noise see above The distribution histogram of this type of noise displays noise peaks at the high and low ends of the range After you have identified the type of noise go to the next step Step 2 Select Filter Size Image noise is filtered by means of a filtering window or kernel w
195. easured by the area under its intensity profile curve Units are intensity x mm Gauss Peak Density The intensity value of the band s Gaussian peak after Gaussian modeling Gaussian Trace Quantity The band quantity as measured by the area under its Gaussian fitted profile Contour qty The quantity of a band that has been defined using the Contour or Draw Band tools It is the sum of the intensities of all the pixels inside the band boundary multiplied by the area of each pixel Units are intensity x mm Relative qty The quantity of a band as measured by its intensity expressed as a percentage of either the total intensity of all the bands in the lane or the total intensity of the lane including the areas between bands The calculation method of Lane or of Bands in Lane is set in the Preferences dialog see section 2 5 e Application Normalized qty The trace quantity of the band expressed as a percentage of the quantity of a selected band type that is present in the same lane If the quantity of the band is known you can enter the quantity and units next to the Quantity Units prompt 5 17 Quantity One User Guide To calibrate the band against known quantities click on the Calibration button and select the calibration curve See section 6 3 a Creating and Applying a Set of Quantity Standards for information on calibration curves 5 7 Gauss Modeling Bands If the bands are closely spaced or overlap
196. ect Lane On to turn on subtraction for the lane To adjust the subtraction level for the lane change the value in the Rolling Disk Size field under Selected Lane This field displays the rolling disk radius in number of pixels Enter a new value in the field or use the arrows to change the value in 10 percent increments Typical rolling disk sizes range from 50 to 150 As you change the size study the level of background subtraction in the lane trace When you make changes to the selected lane the Subtraction Varies by Lane button is selected To turn off individual lane subtraction choose either All Lanes Off or All Lanes On 4 12 Chapter 4 Lanes 4 3 Compare Lanes The Compare Lanes graph allows you to superimpose the intensity profiles of any number of lanes from any number of open images Select Compare Lanes from the Lane menu or toolbar then click on the first lane you want to display The Compare Lanes window will open Note The image must have defined lanes for this command to work Compare Lanes n rat organs aE gaa x gt Bd _ Show Gauss Model Done Help amp lign Band Types Fig 4 12 The Compare Lanes dialog The X axis of the graph is the Rf value and the Y axis is the pixel intensity value at each point along the lane Compare Lanes automatically best fits lanes within the display window to maximize the range of intensity values included in the graph Rf values are displayed from 0 0
197. ects the backup file and preserves it from any changes D 5 e Auto scale Transform Auto scale Transform after Acquisition allows the user the option of having the image automatically perform the Auto Scale transform function upon completion of image acquisition This eliminates the need to transform when the acquisition time was too short or the iris not opened enough To enable the auto scale transform function check the box labeled Auto Scale Transform after Acquisition in the Options dialog D 5 f Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less than 1 percent of the pixels in your image saturated Consequently the default value for this field is 0 75 percent Appendix D ChemiDoc XRS D 6 Other Features li Options x Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig D 13 Other ChemiDoc XRS acquisition window features Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image Transform command File Size of Images Image File Size shows the size of the image file you are about to create This size is determined by the
198. ed above C 4 Step Ill Acquire Image For many white light applications you can skip this step and save and print images directly from Live Focus mode For UV light chemiluminescent applications or faint samples the ChemiDoc EQ control panel has several features for creating image exposures You can take an automatic exposure based on the number of saturated pixels in the image you can Quantity One User Guide enter a specific exposure time or you can take a series of exposures and select the best one Note Exposure refers to the integration of the image on the camera CCD over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Use Auto Expose if you want to take a single exposure but are uncertain of the optimal exposure time Note If you know the approximate exposure time you want 3 seconds you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure During the auto exposure the image is continuously integrated on the camera CCD until it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 15 percent See section C 9 Options Step IIl Acquire Image Click on Auto Expose Auto Manual Live button appears selected gt o
199. een Image 5 1D Scan a x Green channel Blue Blue channel w al al la ES EJ w Don amp Pit EsjExport TIFF Help Fig 3 5 Multi Channel Viewer Note The color channel used to display the image in the viewer has no relation to the filter used to capture the image The red green and blue channels are only used to distinguish different images To add another image to the viewer make sure the image is open and click on the pull down button next to the Green or Blue name field Select the image name from the pull down list Add a third image using the same procedure Chapter 3 Viewing and Editing Images 23 Diversity Database x 2 Select image to display 2 Multi Channel Viewer Image 4 1D Scan 1148x486 Channels Image 5 1D Scan 1148x486 Red Image 4 1D Scan v lt clear gt Green Image 5 1D Scan x Cancel l Blue Fig 3 6 Selecting images to display in the viewer To reassign the different images to different channels use the pull down buttons to the right of the name fields Select lt clear gt from the pull down list to remove an image from that channel of the viewer Viewing Options To remove a particular color channel from the display click in the checkbox associated with that channel to deselect it Select the Auto Scale Image When Assigned checkbox to automatically adjust the brightness and contrast of each loaded image based on the d
200. els eem A awl eee 7 5 1 3 Volume Features jicir nce shesceds code iim ee UR RH PREIS 7 7 7 4 Moving Copying and Deleting Volumes eee 7 8 4 5 Volume Standards emi 7 8 7 6 Volume Background Subtraction sese 7 10 DT ON olume ATEAYS os et EAE EE NRBEM SHEER THERE ERE EEUU 7 12 Colony Counting aoc cid ia reca aa 8 1 8 1 Defining the Counting Region essere 8 2 8 2 Counting the Colome s 2 tenere ei d iere 8 3 8 35 Displaying the Results eterne rented 8 4 8 4 Making and Erasing Individual Colonies eee 8 5 8 5 Using the Histogram to Distinguish Colonies sese 8 6 8 6 Ignoring a Region of the Dish eene 8 8 8 7 Saving Resetting the Count seen enne 8 9 8 8 Saving to a Spreadsheet sess 8 10 ReadyAgarose 96 Plus renun 9 1 9 ReadyAgarose Wizatd eec itte et erede etre eee 9 1 EXQuest Spot Cutter 5 oriri ance derraman 10 1 10 1 Calibration and setup emet merenti 10 2 10 2 Manual Excision cece cee ceseesecsseesecesceseeeeeeseseeseseseeeseeeesesesecsaeeneseaeeaees 10 6 10 3 bane Volume EXCISIOD 10 18 Differential Display and VNTRSs 11 1 11 1 Differential Display etti et oe PERDE 11 1 11 2 Variable Number Tandem Repeats sese 11 3 Quantity One User Guide 12 Hep
201. en Kodak Lowest 532nm EX Blank 390mm BP Fig H 8 Application selection and settings Once you select an application the application name and settings appear below the Select button Quantity One User Guide H 3 Step Il Select Scan Area To select a scan area drag your mouse within the scanning window In the scanning window your cursor appearance will change to a cross The border of the scan area you are selecting is marked by a frame Drag cursor ig detine scan area IN Fig H 9 Selecting a scan area If you are in quadrant mode note that the frame locks onto the next quadrant as you drag When you release the mouse button the border changes to a dashed blue line indicating a selected area e To reposition the scanning box you have selected position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move e To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right Appendix H FX Step Il Select Scan Area Click and drag in diagram to set scan area Top EE Left 3 5 Bottom 40 5 Right 31 5 Quadrant Mode Fig 8 10
202. er Guide selected plate is less than the Min well volume then the overflow warning flashes and you will not be able to start a cut run Note Minimum well volume only displays if you are using the EXQuest Spot Cutter with a gel head Ending the Cut Run Click Pause to pause the cut run You will be prompted to confirm the pause Note that the cutter will pause only after the most recent cut has been deposited in a well Any remaining cut requests will remain selected in the image and you can click Begin Resume to complete the remainder of the cuts When all cuts are complete the cutting tip will return to the home position Confirming Cuts When the cut run is completed you have the option of taking a confirmation image If you click Yes PDQuest acquires a temporary image of the gel with the cut spots identified by plate and well number The specification for the EXQuest Spot Cutter spot pick up is greater than 99 effective at picking up a spot that has been cut from a gel However there may be some spots that have not been picked up and these can be re cut The efficiency of pick up on the second cut is again gt 99 so the spot is reliably picked up the second time Use the zoom tools to confirm all spots were cut If there are spots that need to be re cut click Add move cut and then right click the spot in the Manual Excision window When you have identified al the spots that need to be re cut click Begin Resume 10 2 0 Ot
203. eration however not every command is required for every application As with the secondary toolbars you can click on the next to the name of a function to display the Help text 2 1 f Right Click Context Menu With an image open right click anywhere on the image to display a context menu of common commands 2 5 Quantity One User Guide iL view Entire Image Gi Zoom Box Bi Transform s Select Tool Hide Overlays Image Info amp Print Image Ml mage Colors gt Density at Cursor FE Band Attributes Fig 2 6 Selecting Zoom Box from the right click context menu You can select commands from this menu as you would from a standard menu 2 1 g Keyboard Commands Many commands and functions can be performed using keyboard keys e g press the Fl key for View Entire Image press Ctrl S for Save Select Keyboard Layout from the Help menu to display a list of keys and key combinations and their associated commands The pull down menus also list the shortcut keys for the menu commands 2 2 File Commands The basic file commands and functions are located on the File menu 2 6 Chapter 2 General Information Open Ctr o Save Ctri S Save As Save All Ctri Y Close Alt F4 Close All Change Version Revert to Saved Image Info Ctrl I Reduce File Size Gel Doc EQ ChemiDoc EQ ChemiDoc XRS G5 700 GS 710 GS 800 Fluor S Fluor S
204. erence dark will be created when you acquire your next image Because of the high sensitivity of the CCD fluctuations in background radiation and or temperature in the room can affect the level of dark count If you feel that radiation temperature conditions have changed in the room since your last reference dark was created use the Reset Reference button to delete your old reference and create a new one under current conditions None If you do not want to perform dark subtraction select None No dark exposure will be acquired or subtracted 1 16 Appendix I VersaDoc l 6 b Save Auto Save After Scan To automatically save any image you acquire using the Acquire button click on the Auto Save After Scan checkbox in the Options dialog box With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be
205. est Spot Cutter The EXQuest Spot Cutter is a precision instrument that accurately locates and excises protein spots from 1D gels or blots and loads them into microplates for down stream processing and analysis Fig 10 1 EXQuest Spot Cutter The following sections describe how to calibrate the EXQuest Spot Cutter and excise spots using Manual Excision or lane Volume Excision 10 1 Quantity One User Guide 10 1 Calibration and setup Before you can use the spot cutter for the first time you must first calibrate the instrument The calibration wizard helps you to quickly calibrate the camera as well as set the plate and wash station positions The calibration wizard functions the same whether you are using the gel tip or the membrane tip To open the calibration wizard go to the Devices tab of the Preferences dialog box make sure you have selected the EXQuest Spot Cutter and click Setup Spot Cutter hl EXQuest SpotCutter Setup EXQuest Calibration Wizard Detected EXQuest SpotCutter with Gel head Last calibrated on 22 Nov 2004 12 53 You will need the calibration grid template and 9 calibration pucks If you have not aligned and focused the camera place the focusing template on the gel tray insert the tray into the EXQuest SpotCutter and check the box below Y Perform focus alignment step C Skip to plate position step Click Next when ready yl X O c a 67 i t iz Cancel Fig
206. ew button to create a preview exposure and display it in the acquisition window Note The camera on the VersaDoc must be at the correct operating temperature before capturing images The temperature adjustment can take several minutes after the camera is turned on depending on your VersaDoc model and the ambient room temperature See your hardware manual for details A preview scan takes only half as long to create as a real scan because the preview scan does not capture a dark image The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the dialog box You cannot save preview scans If you want to stop a preview scan that is in progress click on the Stop button I 5 Acquire the Image You can acquire a single exposure for each channel based on the time selected in Step III Set Exposure Time or take a series of exposures for a particular channel over a specified interval Optimize Exposure Illumination Flat Fielding For applications using the UV or white light transillumination you should use the appropriate reference plate to ensure a uniform intensity in the image This will compensate for normal variations in image pixel intensity that occur with a transilluminating light source 1 10 Appendix Il VersaDoc To enable this feature select the Flat Fielding checkbox Note Flat fielding is only available in single channel single exposure mode If you are using multiple channels o
207. exposures Appendix C ChemiDoc Chemi Doc Live Acquire Live Acquire Settings Total Exposure Time sec 10 00 Starting Exposure Time sec 5 o0 Number of Exposures 5 l Save Images Cancel DK Fig C 7 Live Acquire settings Note You should specify no more than 10 exposures in the Live Acquire Settings dialog to avoid excessive build up of image background in later exposures The fewer the exposures the less background will be added to the image See the Release Notes for additional instructions on reducing background in images captured using Live Acquire Select the Save Images checkbox if you want to automatically save each exposure as it is taken Click on OK in the settings dialog to begin taking exposures If you selected Save Images a Save dialog box will open in which you can specify the base file name and location of the exposure files When you click on Save the exposures will be taken The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time When each exposure is complete an image window containing that exposure will open behind the ChemiDoc EQ window When the full exposure time has lapsed all the image windows will move in front of the ChemiDoc EQ window Note that the first exposure will have the base file name the default base file name is the computer user name and a time stamp Each subsequent exposure will hav
208. ey The image will shift so that point is at the center of the image window Imitate Zoom To magnify the same area on multiple images at the same time use the Imitate Zoom command on the Window menu First adjust the magnification in one of the images Then with that image window still selected select Imitate Zoom The zoom factor and region of the selected image will be applied to all the images Note Imitate Zoom only works on images with similar dimensions Tiling Windows If you have more than one image open the Tile commands on the Window menu allow you to arrange the images neatly on the screen Select Tile to resize all the windows and arrange them on the screen left to right and top to bottom Select Tile Vertical to resize all windows and arrange them side by side on the screen Select Tile Horizontal to resize all windows and stack them top to bottom on the screen Chapter 3 Viewing and Editing Images 3 2 Density Tools The density tools on the View gt Plot Density submenu and the Density Tools toolbar are designed to provide a quick measure of the data in a gel image Density Tools E3 pm n El T y e rE sg Plot Density Density at Cursor Density in Box Plot Cross section Ee Plot Density Distribution Plot Vertical Trace Show Lanes and Bands Hide Overlays F4 Fig 3 3 Density tools on the menu and toolbar Note The density traces will be slightly different than the
209. f any of the selected lanes includes Gaussian modeling see section 5 7 Gauss Modeling Bands If you select this checkbox the Gaussian fitted profiles will be superimposed on the regular lane profiles The Gaussian profiles are displayed in white Align Band Types The Align Band Types checkbox is active if any of the selected lanes includes defined band types see section 6 2 Band Matching Chapter 4 Lanes If this checkbox is selected the profiles of all bands that have been identified as the same band type will be stretched and superimposed on one another so their peaks align This is useful if the same band appears as peaks in slightly different positions in different lanes and you want to align the peaks to confirm that they are all the same band type Note This command only changes the lane profiles as they are displayed in the Compare Lanes dialog and will not affect image data in any way aeu ase lt q an asa Bere Heb Mon Bond Types Done Hep Aim Band Types Unaligned Aligned Fig 4 13 Two band types as they appear in two lanes before alignment and after Note that this function will not align band types from different band sets e g Band Type 1 in Band Set A and Band Type 1 in Band Set B will not be aligned However the same band types from different images will be aligned Note The Rf values in the X axis will no longer be accurate if Align Band Types is selected since som
210. ference Temporary File Location Temporary image files are normally stored in the TMP directory of your The Discovery Series folder The full path is listed in the field To change the location of your temporary files type in a new path or click Browse and select a new directory To return to the default TMP directory click Default 2 5 c Display Click on the Display tab to access the following preferences Zoom Zoom determines the percentage by which an image zooms in or out when you use the Zoom In and Zoom Out functions This percentage is based on the size of the image 2 17 Quantity One User Guide Pan 96 Pan determines the percentage by which the image moves side to side or up and down when you use the arrow keys This percentage is based on the size of the image Band Style Bands in your gel image can be marked with brackets that define the top and bottom boundaries of the band or they can be marked with a dash at the center of the band Indicate your preference by clicking on the Brackets or Lines button This setting can be temporarily changed in the Band Attributes dialog box However all newly opened images will use the preferences setting Auto Imitate Zoom When this checkbox is selected the magnifying and image positioning commands used in one window will be applied to all open windows This is useful for example if you want to compare the same band of group of bands in different gels magnify
211. ges can be magnified annotated rotated and resized They can be printed using standard and video printers All data in the image can be quickly and accurately quantitated using the Volume tools The lane based functions can be used to determine molecular weights isoelectric points VNTRs presence absence and up down regulation of bands and other values The software can measure total and average quantities determine relative and actual amounts of protein and count colonies in a Petri dish The software can cope with distortions in the shape of lanes and bands Lanes can be adjusted along their lengths to compensate for any curvature or smiling of gels Image files can be shared among all The Discovery Series software Images can also be easily converted into TIFF format for compatibility with other Macintosh and Windows applications 1 1 Quantity One User Guide 1 2 Digital Data and Signal Intensity The Bio Rad imaging devices supported by Quantity One are light and or radiation detectors that convert signals from biological samples into digital data Quantity One then displays the digital data on your computer screen in the form of gray scale or color images A data object as displayed on the computer is composed of tiny individual screen pixels Each pixel has an X and Y coordinate and a value Z The X and Y coordinates are the pixel s horizontal and vertical positions on the image and the Z value is the signal intensi
212. ges do not contain all the tagged information that would normally be included in an image file for example imaging device scan date image color etc For this reason the Image Report may list this information as Unknown for imported TIFF files 13 5 Video Print Select Video Print from the File gt Print submenu to print images and reports to a video printer 13 4 Chapter 13 Printing and Exporting Note X Video printing requires installation of the video board and cable that come with the Gel Doc EQ and ChemiDoc EQ systems The video board and cable can also be ordered separately Settings for the Mitsubishi P9QOW P91W Video Printer There are three settings for the Mitsubishi P9OW P91W video printer Set Contrast to zero Brightness to zero and Gamma to 5 The dip switches should stay in the orientation in which they are shipped Pin 1 is up on and Pins 2 10 are down off 13 6 Export to TIFF Image You can export images in TIFF format for analysis and publishing using other applications Select Export to TIFF Image from the File menu to open a dialog box in which you can specify the export parameters 13 5 Quantity One User Guide E Export FX Etbr Raw 1 D Image As TIFF x r Export Mode Analysis export raw data Publishing export view excluding overlays export view including overlays Resolution dpi 72 dpi Same as scan izs3 4 gt 1
213. h memory assigned to Quantity One essere 1 6 Magnifying Images 3 nere ege iine He eee Fre feeds te eere tte nen 3 1 Manual EXcISIOD oS Rame eS 10 6 Match graphs RE trc UR e re Pte dida 6 22 Matching Displaying band ty pes cases eerte entity ed 6 16 Displaying modeling lines essere 6 16 Manually matching bands eene 6 16 Match commands ue ee bert eerte ate rte eere Peine ent 6 16 Match STPS iia 6 22 Matched band sets susoni eee ete ERR 6 17 Rep e ici 12 5 Maximizing the application window essere 2 17 Memory alloWwahCe sitos eee pe RE eiue 2 16 Menus 3s UERBO REO RR HP RUE 2 1 Modeling lines Displaying sese ERE EROR EDIT ere EAS t 5 15 Molecular Imager FX Acquiring the mage eee eter ERA seinen re tees G 13 Control Panel cc tet eei daga G 2 Filesize of images ia G 13 ODttOnBS erret tate p Ee ien er lesione te reor eA RUN een G 14 Saturated pixels highlighting sess G 14 Saving themate ioo RR epe tesi te nies e ERN G 13 Scanning WIndows r a a E nennen nn nro nc treten enne G 3 Selecting an appli Caton secs eroe etie eere He e Pe Een G 4 Selecting resolution siie e ee eE E E E enne ethe nete tenens G 12 Selecting Scan areire i E RE E eedem R G 11 Simulation mode sss esee ener ene nr enr oaro G 2 Index 9 Quantity One User Guide Multi channel viewer cccccccseessssesececececcececceccecececesevecscsescscsnsescscs
214. h volume as the tip is washed in the three membrane wash stations 10 2 c Specify Cuts In Specify Cuts click Add move cut then click on spots in the image to identify which spots to cut To make multiple cuts of large spots if selected on the Cut tab of the Specify Options section click and drag over the spot in the image Specify Cuts F Add move cut YJ Remove cut xj Snap to peak Fig 10 10 Specify cuts Use the Zoom Box tool on the right of the Basic Excision Tool to magnify each spot When clicking on spots in the image select the Snap to Peak checkbox to automatically center the cut circle on the spot peak calculated from the spot intensity in the image To remove a cut click Remove cut and click on a cut selection in the image Run Info The Run Info section contains information regarding the current cut run Cuts indicates the total number of cuts in the run including multiple cuts Wells indicates the number of wells to be used Plates indicates the number of plates needed for the run Min well volume indicates the minimum well capacity to avoid overflow based on the number of multiple cuts selected and cutter tip size If the well volume for your 10 11 Quantity One User Guide selected plate is less than the Min well volume then the overflow warning flashes and you will not be able to start a cut run Note Minimum well volume and the overflow warning only display if you are using the EX
215. he 1 For a complete explanation of the calculations and assumptions used to generate these dendrograms please refer to Sneath and Sokal Numerical Taxonomy San Francisco W H Freeman amp Company 1973 2 Vogt and Nagel Clinical Chemistry 38 2 182 198 1992 12 15 Quantity One User Guide method of computing the new distance matrix in step 5 In the discussion that follows let p q be indices indicating two clusters that are to be joined into a single cluster k be the index of the cluster formed by joining clusters p and q 1 be the index of any remaining clusters other than cluster p q or k n the number of samples in the p th cluster ng the number of clusters in the q th cluster n the number of clusters in the k th cluster formed by joining the p th and q th cluster n n n p q doq the distance between cluster p and cluster q Single Linkage This is also called Nearest Neighbor or Minimum Method d i min d d Complete Linkage This is also called the Furthest Neighbor or Maximum Method d max d d Single and Complete linkage are good algorithms for indicating outlier clusters UPGAMA Unweighted pair group method using arithmetic averages This is also called Weighted Average Linkage 12 16 Chapter 12 Reports WPGAMA Weighted pair group method using arithmetic averages This is also called Average Linkage d 05 dj 05 dy WPGAMA is a special case of UPGAMA tha
216. he camera CCD over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Auto Expose will take an exposure whose time length is determined by the number of saturated pixels in the image This is useful if you are uncertain of the optimal exposure length A 5 Quantity One User Guide Note If you know the approximate exposure time you want 3 seconds you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure During the auto exposure the image is continuously integrated on the camera CCD until it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 15 percent See section A 9 Options Step IIl Acquire Image Click on Auto Expose _ M button appears depressed o See Qro Exposure Time sec Can see Exposure Time automatically changing o o 3 l O Freeze Fig A 4 Auto Expose Once an image has reached the specified percent of saturated pixels it is captured and displayed in the video display window Auto Expose is automatically deactivated the exposure time appears active in the Exposure Time field and Manual Expose is activated Note If you are having difficulty auto exposing your sample you can use Ma
217. her Options Prime Pump Gel head only The spot cutter should be primed when it is first installed anytime the water bottle is filled when the cutting head is changed and for the first cut run of the day Click 10 16 Excision Prime pump until no large air bubbles are visible in the tubing For an empty system this usually takes 3 prime cycles Save To save the image taken by the spot cutter click the Save button in the Manual Excision Tool window You will be prompted to enter a name and specify a location for the image before saving Import If you have a cut list saved to a text file you can import it into the Manual Excision Tool This is useful if you have a large number of cuts for similar gels Note You can only import a cut list to an image that does not have any cuts added However after you import cuts you can add additional cuts to the image Click Import to open the Import Excision Coordinate List dialog box E Import Excision Coordinate List C Skip first line of file Column 3X 2 File test spots txt m Unis mm C cm inches Origin Upper left Lower left 3 All cuts may be dragged at once with the mouse Y Done X Cancel 2 Help Fig 10 14 Importing a cut list If the first line of your file is header information select Skip first line of file Next enter which column the coordinates are located in the file For example if your file 10 17 Quantity One User Guide
218. hich is measured in pixels This filtering window slides across the image processing the pixels within It The available filter dimensions range from 3 x 3 to 9 x 9 pixels To select an appropriate size magnify a background region of the image so that you can see the individual pixels The filter size you select should be larger than the average noise feature but smaller than the data features Note A smaller filter will alter the image less than a larger filter Large filters can result in better suppression of noise but can also blur desirable features in the image 3 36 Chapter 3 Viewing and Editing Images Step 3 Begin Filtering After you have completed the selections the filter name and size will be displayed at the bottom of the Filter Wizard dialog To being filtering click on the OK button Because filtering is an irreversible process you will be prompted to filter the original image filter a copy of the image or cancel the operation If you choose Copy and Filter enter a name and or version number for the new copy in the pop up box and click on OK 3 11 b Selecting a Filter Directly If you know the type and size of filter you want you can select it directly from the Image Filter List submenu The submenu includes all the available filters The types of filters are Weighted Mean This filter is useful for reducing Gaussian noise It calculates the weighted mean of the pixels within the filtering wind
219. ick on a matched band The bands in the matched group will be displayed along with the selected graph 6 22 Chapter 6 Standards and Band Matching Band Type 3 Average 0300 OD 42 5 2 6 1 7 1 9 1 10 1 12 2 13 3 142 Fig 6 17 Example of a match graph Average displays a histogram of the average densities of the bands in a band type Calibrated Quantity displays a histogram of the quantities of the bands in a band type as calculated from a calibration curve see section 6 3 Quantity Standards Contour displays a histogram of the intensity of each contoured band in a band type This function only works with contoured bands Normalized Quantity displays a histogram of the normalized quantities of the bands in a band type See section 6 2 d Normalizing for Quantity for instructions on how to normalize for quantity Peak displays a histogram of the peak intensities of the bands in a band type Relative Quantity displays a histogram in which each bar represents the quantity of the band in a lane as a percentage of either 1 the total intensity data in the band s lane or 2 the total intensity of all the bands in the band s lane The calculation method of Lane or of Bands in Lane is set in the Preferences dialog Trace displays a histogram of the trace quantities of the bands in a band type group 6 23 Quantity One User Guide Each bar of the histogram is labeled on the X axis with the lane
220. ideo display of your sample If no image is visible make sure the camera is on check the cable connections make sure the iris on the camera is not closed and make sure that the protective cap is off the camera lens Also check to see that the transilluminator is on and working The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are six basic steps to acquiring an image using the Gel Doc EQ 1 Position and focus the gel or other object to be imaged 2 Select Mode 3 Acquire the image A 3 Quantity One User Guide 4 Optimize the display 5 Analysis 6 Select the output A 2 Step Position Gel The Gel Doc EQ window will open in live mode giving you a live video display of your sample In this mode the Live Focus button will appear selected and frames will be captured and displayed at about 10 frames per second depending on the speed of your computer You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note Newer versions of the Gel Doc EQ feature a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens These buttons will not be visible if you are connected to older versions of the Gel Doc EQ without the motorized zoom lens
221. idual imaging devices Size of images changing sess eneen en nooo enne emeenneenenenen s 2 11 Software Cn see etes HERR Be recie eate ten E Diete 1 9 Sort and Recalculate sess rene e nenne 3 42 Standards Applymg s nen eben 6 7 Xr E eve 6 12 Archive disabling deleting sese 6 13 lop en 6 1 Calculated values viewing oonoonocnnccnonnconcnnccnonnnonocnnonnnnnnonn nono conc rnnc nn cra eene 6 12 CIO iia aiii ida 6 3 Del a a de ts e e a ed i 6 12 Dialog ti ida 6 5 Entering values rom 6 6 Modeling s oi rere te Ee tiene 6 9 Opening existing use E Ae nre 6 12 Prede ed uii ee epp etd eerte rete o t te 6 1 Read only uus oo ER 6 13 Regression CUEVE eiii 6 9 Regression curve display options ooooconccconnoncconconoconcnnnnanonn conc cnn cranc nn con nora conos 6 11 Regression Models eoe eee ide 6 10 Removing standards from lanes sse 6 9 NIgnupD RE 6 12 Status DOXES desea eG IUD EA Rupee 2 2 Index 13 Quantity One User Guide Ni MH D 4 Subtract Background See Background subtraction T Tandem repeats c o eR AUR III S 5 15 Tandem repeats calculating eres tette deti nie eres 11 3 Technical Service contacting nennen ener 1 14 Text Overlays Creating ne UU NEUES 3 39 Ed A E 3 41 Linetool sicher eae ASG Rae S
222. if it is between two known quantities and enter a value for it in this case the Status column will indicate that the quantity has been calculated Calc In either case you can type a quantity directly into the Quantity column and the status will change to Known Relative Deviation of Known Quantities After three values have been entered the relative deviations of those bands are automatically calculated and displayed in the Rel Dev column The relative deviation is calculated from the known value that you entered and the back calculated value from the calibration curve If the deviation value is too high you can exclude a band from the calibration curve Click on the arrow button next to the problem band In the pop up box select Remove to remove the band from the Quantity Standards dialog All the information about that band will be deleted Alternatively select Outlier from the pop up list to retain the information about the band in the calibration file but exclude it from the calibration curve 6 3 b Calibration Curve Interpolation and Extrapolation There are two methods for calculating the calibration curve Point to Point generates a curve in which each data point is connected directly to the next regardless of the shape of the resulting curve e Linear Regression using the method of Least Squares generates a smooth curve that is the best fit of the values you provided Select the preferred option next to Inte
223. ifferent lanes These lines are based on the positions of the known green bands and any standards you may have defined To display the band type modeling lines select Show Band Models from the Match menu or toolbar 6 2 a Editing the Results of Band Matching After you have matched bands automatically you can manually indentify the remaining unknown bands as new or existing band types Use the tools on the Match menu and toolbar to manually identify new bands unmatch bands or identify bands as outliers from the band set Note The modeling lines are designed to give you guidance on identifying new bands e To identify an unknown yellow band as a new band type select Match and click on the unknown band e To match a band to a particular band type select Match and first click on the identified red or green band Then click on the red or yellow band you want to match The band will appear green known and the modeling line will change to reflect the match e Ifa matched red band in a lane is in fact a new band select Match hold down the Shift key and click on the red band e To change a matched band to unknown yellow select Unmatch and click on that band e To remove a green band from the modeling select Outlier and click on that band An X will appear through the band indicating that the software is ignoring it when auto matching bands across the gel image Chapter 6 Standards and Band Matching Manually match ba
224. ight Front Back Raise Lower Lower head to adjust eject height m x O amp o 7 67 o t ho Cancel Fig 10 3 Plate position for a 384 well plate 10 4 Excision The sliders allow you to adjust the tip by moving it along X and Y coordinates Click Lower to lower the tip into the well This gives you a clear idea of where the tip is in relation to the center of the well The tip height slider adjusts the height of the tip above the well when ejecting material into the well This should be slightly above the lip After calibrating one type you have the option to return to the plate selection panel to calibrate another type If you anticipate using only one type of plate you can skip calibrating the other types If at a later time you find you need to use another plate type you can return to this step without performing system calibration Wash Station Calibration In the Wash Station Position step the tip moves to the location above the Wash station If you are doing Membrane tip calibration the tip moves to just above Wash Station 1 The sliders allow you to adjust the tip by moving it along X and Y coordinates Click Lower to lower the tip This gives you a clear idea of where the tip is in relation to the center of the wash station The tip height slider adjusts the height of the tip in relation to the wash station The tip should be low enough to effectively wash the tip Calibration Summary
225. ight Saturated Pixels Make Backup Copy Hide Grid E Warm On Saturated Pixels Info Help nx eor E RE Er EFOTT OR T ee es OS eat ai T2 SE ED ee Sg Fig G 2 FX acquisition window The default scanning window is marked by grid lines that divide the area into quadrants There is also an outer box and inner box marked by thicker lines The quadrants are numbered 1 16 left to right and lettered A U top to bottom If you prefer a scanning window measured in centimeters deselect the Quadrant Mode checkbox in the control panel by clicking on it To hide the gridlines click on the Hide Grid checkbox under Options The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to scanning an image using the FX 1 Select the 2 Select the application s Scan area Quantity One User Guide 3 Select the resolution 4 Acquire the image G 2 Step Select Application In Step 1 you select the appropriate filters and other scanning parameters for the type of gel blot plate or other object that you are imaging Each set of parameters you select is called an application The FX now supports multi channel sequential scanning This allows you to automatically scan the same object using up to four different applications e g to detect different types of stains on
226. ill not be saved until you perform a Save or Save As operation E 13 Quantity One User Guide E 7 Other Options Oversample This feature allows you to scan at the maximum resolution of the scanner and then use spatial averaging to create an image with lower resolution This can result in better images at lower resolution however it takes longer to scan To turn on oversampling click on the Options button in the acquisition window and select the Oversample checkbox in the options dialog With oversampling on you can specify your own resolution within the range of the densitometer by entering values directly in the fields next to X resolution and Y resolution in the main acquisition window Auto Save After Scan To automatically save any scan you create select the Auto Save After Scan checkbox With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy If you have selected Auto Save After Scan you can also automatically create a backup copy of any scan you create Click on the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read o
227. image for template As you check each option in step two you will be prompted to select a source image for that option Note The source image must be open when the option check box is selected Once you have finished checking options from a source the source image can be closed When you are satisfied with your changes click Save or Save As to save it as a new template If this is a new template you must enter a new name for the template when you click Save To change the name of a template click Save As and enter a new name 3 44 Chapter 3 Viewing and Editing Images Default Automation The default automation template is applied to the currently active image when you select Analysis Apply Default Automation To set a new default template highlight the desired template in the Automation Manager and click Set as Default To run the default automation template on the currently active image select Apply Default Automation from the Analysis menu Once a default template is chosen The Automation Manager does not need to be open to be applied nor do you need to select a default each time Quantity One opens as it remembers the default from the last open session of the application 3 16 c Step 3 Select Images This portion of the Automation Manager lists the currently open images Select the images to which you wish to apply the selected template Use ctrl gt click and shft gt click to select more than one image or click Select A
228. in methods of optimizing the image Auto scale High and Low sliders and a Gamma slider You can use these controls to adjust the way the software transforms raw image data into the visual display Chapter 3 Viewing and Editing Images Transform Plot Preview Window Transform Proteins3 Raw 1 D Image Modified Note that transform opprations change only the view of the data noft the data itself Transform Plot Frequency Distribution Full scale Low High Log T 3 p 200 Image Display ins E S 23 Max 253 Invert display Log High Lbw Sliders JCalibrated Quantiy raw Min 21 Cabras Always auto scale Gamma lm y OK X Cancel Help Auto scale Reset Blige color Frequency Distribution histogram Auto scale Fig 3 14 Transform dialog 3 8 a Transform Subwindows Preview Window The Preview Window shows a smaller view of the same image that is displayed in the main image window Changes in the controls are automatically reflected in the Preview Window They are only applied to the main image when you click on OK 3 19 Quantity One User Guide You can use viewing tools such as Zoom Box Grab and View Entire Image in the Preview Window just as you can in the main image window to focus on particular regions of interest Frequency Distribution Histogram The Frequency Distribution histogram shows the total data range in the image and the amount of data at each po
229. in the text box on the image Also on the image you should see the marked white colonies gold triangles change to blue colonies blue squares Note If the blue colonies are not marked on the image check to make sure that the Mark Blue Colonies checkbox at the bottom of the dialog is checked Quantity One User Guide 8 6 Ignoring a Region of the Dish If a particular region of your Petri dish is damaged and you do not want to consider the colonies if any that appear there in the final count you can exclude that region of the dish from the calculations Click on the Ignore Region button then position the cursor on one edge of the region you want to ignore Drag the cursor on the image defining the full region you want to ignore Totals White adj 1072 Blue adj 0 i Excluded 30 5 Fig 8 7 Marking a region to ignore As you drag you will create a pie slice marked with red cross hatching Any colonies in this region will not be considered in the final count When you have defined the region release the mouse button If you want to change the size of the ignored region position the cursor on the edge of the pie slice near the rim of the blue circle The cursor will change to a bidirectional arrow and you can drag the edge of the pie slice Chapter 8 Colony Counting Colony Count and Adjusted Count After you have defined a region to ignore two different counts will appear in the dialog the colony count a
230. including warranty service you should contact BIO RAD LABORATORIES INC 1000 Alfred Nobel Drive Hercules CA 94547 This Agreement shall be governed by the laws of the state of California as applicable to contracts between residents of California which are entered into and performed entirely within California YOU HAVE ACKNOWLEDGED THAT YOU HAVE READ THIS AGREEMENT UNDERSTAND IT AND AGREE TO BE BOUND BY ITS TERMS AND CONDITIONS YOU FURTHER AGREE THAT IT IS THE COMPLETE AND EXCLUSIVE STATEMENT OF THE AGREEMENT BETWEEN US WHICH SUPERCEDES ANY PROPOSAL OR PRIOR AGREEMENT ORAL OR WRITTEN AND ANY OTHER COMMUNICATIONS BETWEEN US RELATING TO THE SUBJECT MATTER OF THIS AGREEMENT
231. indicating that the volume is deselected To reselect the volume click on it again If you move the cursor over the volume selected or not the border changes to gold After you create a volume you can view the volume data area density etc by selecting the Volume Analysis Report from the Reports menu Tips The volume you draw should completely surround the data you want to quantitate You should also adjust for background intensity You may want to experiment with several different volumes drawn around the same object before selecting the one that gives you the best quantitation data Displaying Volumes To display previously created volumes after opening an image select Volume Tools from the Edit menu or main toolbar 7 7 Quantity One User Guide If you have concealed all overlays using Hide Overlays click on any button in the Volume toolbar to display the hidden volumes 7 4 Moving Copying and Deleting Volumes You can move copy or delete a single volume or group of volumes within an image You can also copy and paste volumes between images First select the volume s Click on the Select Tool button on the Volume toolbar To select a single volume click on it To select multiple volumes either drag a box around them or hold down the Shift key while you click on them one at a time When you drag to select a group of volumes make sure that you completely surround all the volumes Each selected volume will have
232. ing resolution Same As Scan or any resolution that you enter in the Specify field up to the resolution of the scan Under Transform you can specify a Linear transform or preserve the Current View which is automatically log transformed Images with a higher Bit Depth e g 16 bit or 24 bit RGB can be compressed to a lower bit depth e g 8 bit during export Note TIFF images are automatically exported from the Multi channel Viewer in 24 bit RGB mode to preserve the colors displayed in the viewer Finally if you are only displaying part of the image due to magnification or repositioning you can preserve the Current View or export the Entire Image 13 6 c Exporting the Image The size of the pixels in the image and the file size of the image are listed at the bottom of the dialog When you are ready to export click on the Export button In the Save As dialog box the default file name will have a tif extension and the file type will indicate that this is a TIFF image 13 7 Quantity One User Guide 13 7 Exporting a JPEG Image The Export to JPEG Image command allows you to export an image as a JPEG image The Export to JPEG Image is primarily for exporting your images for use in reports presentations etc rather than a TIFF image because a JPEG image is considerably smaller in size than a TIFF image Unlike TIFF images JPEG images cannot be opened by TDS applications To export an image as a JPEG open the File men
233. int in the range In a typical scan there is a signal spike at the left gray end of the histogram due to background noise Transform Plot The Transform Plot is a logarithmic representation of how the raw pixel data are mapped to the pixels of the computer screen 3 8 6 Transform Controls Auto scale Click on the Auto scale button to optimize the image automatically The lightest part of the image will be set to the minimum intensity e g white and the darkest will be set to the maximum intensity e g black This enhances minor variations in the image making fine details easier to see You can then fine tune the display using the High Low and Gamma sliders described below High Low Sliders If Auto scale doesn t give you the appearance you want use the High and Low sliders to redraw the image manually Drag the High slider handle to the left to make weak signals appear darker Drag the Low slider handle to the right to reduce background noise As you drag the sliders the slider markers on the Frequency Distribution histogram will move Everything to the left of the Low marker will be remapped to minimum intensity while everything to the right of the High marker will be remapped to maximum intensity Using the histogram you can position the markers at either end of the data range in the image and use the low slider to cut off the spike of background noise 3 20 Chapter 3 Viewing and Editing Images You
234. ion dialog box will open 10 18 Excision Acquire Image C White Trans e UV EPI racauire image Time secs Specify options Cut Plate Hydration Wash n mi LU UM BEL Cutting tip size e 1 0mm C 15mm Material PVDF membrane 1 Specify Cuts Entire lane Vetical 30 Horizontal r 10 Volume overlay aj 1 de x Show excisions on image Run Info Cuts Wells 0 Plates Show x Well Position mm Al cy e amp al il Zoom on cut EM Close CQ Hep Fig 10 15 Lan Volume Excision In the event that Quantity One cannot locate the spot cutter a warning dialog box displays Check your connections and click Retry You also have the option of running Lane Volume Excision in simulation mode Note See the spot cutter hardware manual for instructions on installing the platform camera and associated hardware and software The Manual Excision tool is designed to guide you through the spot cutting procedure Click on the link for more information about each step Acquire Image In this step you capture an image of your gel or membrane using the spot cutter camera e Specify the Cut Run Options These options can be adjusted for each cut run e Specify Cuts Select whether to cut lanes or volumes e Performing the Cut Run In this step you perform the cut run When the run is co
235. ion 1 e Edit User Label DK Cancel Fig 7 9 Defining a background volume object The average intensity of the pixels in the background volume will be calculated and subtracted from each pixel in all standard and unknown volumes Any pixels inside the volumes that have the same intensity as the average background will be reduced to Zero thereby eliminating them from the quantitation 7 11 Quantity One User Guide If you create more than one background volume all the pixels in those background volumes will be used to calculate the average background Background volume s will have default names B1 B2 etc based on their creation sequence You can display hide volume names using the Show Hide Volume Labels command Note If the region you identified as background has a higher average intensity value than a data object that object will have a negative adjusted volume in the Volume Analysis Report If this happens select a new background region that has less intensity than the data object Displaying the Results of Background Subtraction The Volume Analysis Report see section 12 6 Volume Analysis Report will display both the unadjusted volume and the volume with background subtracted adjusted volume of standards and unknowns so you can see exactly how much intensity was subtracted 7 Molume Arrays The Volume Array Tool on the Volume menu and toolbar can be used to quantitate dot blots slot blots and othe
236. is method of nonlinear least squares curve fitting Cubic Spline At least five standard points are required to use this beta cubic spline method Display Options The following options will change how the curve graph is displayed Show Standard Points displays the standard data points on the graph The standard points in that lane will be marked on the graph as triangles Note that known band types will appear marked as standards 6 11 Quantity One User Guide Show Calculated Points displays the calculated points on the graph The calculated points in the lane will be marked on the graph as circles Perform Log Transform changes the shape of the curve from linear to log This will not change the calculated values Show Numerical Data of Points displays the value of each band on the graph next to its corresponding point Display Correlation Coefficient displays the correlation coefficient for the linear quadratic and cubic regression models Note The correlation coefficient is a measure of how well the regression model fits the data It is the square root of the proportion of total variation that can be explained by the regression model A correlation coefficient of 1 000 would indicate 100 percent certainty of fit 6 1 e Displaying Calculated Values To view the calculated values of all the bands in the gel image select Band Attributes from the Band menu then select the value to be displayed molecular weights base pairs e
237. ith this mode selected the data will be measured in linear intensity units White Light Select this mode for reflective and transmissive samples With this mode selected the data will be inverted so that darker areas will be of higher intensity levels B 4 Step Ill Acquire Image For many white light applications you can skip this step and save and print images directly from Live Focus mode For UV light or faint samples you can take an automatic exposure based on the number of saturated pixels in the image or you can enter a specific exposure time Note Exposure refers to the integration of the image on the camera CCD over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Auto Expose will take an exposure whose time length is determined by the number of saturated pixels in the image This is useful if you are uncertain of the optimal exposure length Note If you know the approximate exposure time you want you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure B 5 Quantity One User Guide During the auto exposure the image is continuously integrated on the camera CCD until it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 15
238. ity One User Guide Detect Bands MicroSatellite 1D Scan Ea Parameter Set Name Load Save Delete Reset Lanes All C One Detect Auto Manual Normalize Yes C No Shadows Accept Reject Bands All C Limit 2 Band tools butt gt included in the dialog Fo d az E 3 Sensitivity 10 000 t Detect Bands MicroSatellite 1D E e ME 1709mm al 4 x E xj El zl Min Density 5 00 el Sens 10 000 Noise Filter 4 0 Width 1 70922 t Shoulder Sens 1 00 el Dens 5 00 t Size Scale 5 ely Detect Close Help Detect Close Help El Click here to toggle format Fig 5 3 Detect Bands dialog short and expanded formats with default values When you first open the Detect Bands dialog all bands will be automatically detected based on the default parameters in the dialog and the bands will appear marked on the image The bands can be marked by either lines or brackets depending on the settings in the Band Attributes dialog see section 5 5 Band Attributes Note If you have already manually identified bands using Create Band Adjust Band etc the Detect Bands function will overwrite them You should use Detect Bands first and then manually add adjust or remove bands as needed Chapter 5 Bands The Detect Bands dialog has a short format and an expanded format toggle between them by clicking the toggle box in the lower right corner To change a p
239. ity One is very tolerant of an assortment of electrophoretic artifacts Lanes do not have to be perfectly straight or parallel Bands do not have to be perfectly resolved However for accurate lane based quantitation bands should be reasonably flat and horizontal Lane based quantitation involves calculating the average intensity of pixels across the band width and integrating over the band height For the automatic band finder to function optimally bands should be well resolved Dots that appear as halos rings or craters or that are of unequal diameter may be incorrectly quantified using the automatic functions 1 3 Quantity One User Guide 1 4 Quantity One Workflow The following steps are involved in using Quantity One Acquire Image v Optimize Image v Lane and Band Analysis Volume Analysis Colony Counting v Report Results Fig 1 2 Quantity One workflow 1 4 a Acquire Image Before you can use Quantity One to analyze a biological image you need to capture the image and save it as an image file This may be done with one of the several Bio Rad imaging instruments supported by this software the Molecular Imager FX and Personal Molecular Imager systems the GS 800 Imaging Densitometer the Gel Doc EQ ChemiDoc EQ and ChemiDoc XRS Gel documentation systems and the VersaDoc The resulting images can be stored in files on a computer hard disk network file server or removable disks 1
240. ity of your sample data and is also useful for determining the level of background in the gel Select Plot Lane from the Lane menu or toolbar then click on a lane A lane profile graph will be displayed 4 8 Chapter 4 Lanes Optical Density 0 00 10 Background Intensity peaks Fig 4 9 Profile of a defined lane In the lane profile bands are represented by peaks and background intensity is represented by the baseline region below the peaks The profile is generated by calculating the average intensity of each horizontal row of pixels across the specified width of the lane To close the lane profile click on Hide Overlays on the main toolbar 4 2 Lane Based Background Subtraction After defining lanes we strongly recommend that you perform lane based background subtraction This is the best method for removing background intensity from lanes and is required for Gaussian modeling of bands Lane based background subtraction uses a rolling disk method of subtraction named for a hypothetical disk that rolls along underneath the lane profile removing different intensity levels along the length of the lane The size of the disk determines how much background will be subtracted A large disk will follow the profile trace less closely touching fewer points along the trace 4 9 Quantity One User Guide and removing less background A smaller disk will more closely follow the profile trace removing more background
241. k Continue Uninstalling The Discovery Series from a Macintosh Insert the Discovery Series CD ROM The TDS Mac folder opens on your desktop displaying the installers for The Discovery Series applications Double click the installer for your application In the installer screen select Uninstall from the pull down list 1 7 Software License When the software opens for the first time you will see a Software License screen that shows the current status of your software license With a new HSK or network license you receive a 30 day temporary license Your license will expire on The temporary license is designed to give you time to purchase the software if you have not already done so 1 9 Quantity One User Guide Software License Welcome to Quantity One Current License Check License Single system license License expires on 24 Aug 2002 Check License On line Enter Password If your registration has been updated with Bio Rad this will update your password and service will continue Registration Form without interruption Exit Quantity One Run Run Run the program as usual Help Fig 1 5 Temporary license screen During the 30 day period the Software License screen will appear every time you open the software To use the software during this period click on the Run button Network license holders can click on the Check License button at any time during the 30 day period to activate thei
242. k on Auto Expose Auto Manual Live button appears selected gt D Expose O Expose O Acquire r Exposure Time sec Can see Exposure Time ANTT automatically changing gt gt 03 o Freeze Fig D 5 Selecting Auto Expose D 6 Appendix D ChemiDoc XRS Once an image has reached the specified percentage of saturated pixels it is captured and displayed in the video display window Auto Expose is automatically deactivated and the exposure time appears active in the Exposure Time field Note If you are having difficulty auto exposing your sample you can use Manual Expose to adjust your exposure time directly Most non chemiluminescent applications only require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button Manual Expose is automatically activated after Auto Expose is complete Step Ill Acquire Image Manual Expose active button appears selected Auto pr ive Exposure Time field active Q mose O ea adjust number of seconds EOS Os zi BEEN Exposure Time sec mx eja 9 Freeze Fig D 6 Setting a manual exposure With Manual Expose activated you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field Type in a number or use the arrow buttons
243. k or drag on the image to plot an intensity trace of a vertical cross section of the image centered on that point 3 3 Showing and Hiding Overlays To conceal all plots traces info boxes and overlays on an image select Hide Overlays from the main toolbar or View menu Chapter 3 Viewing and Editing Images Note Click once on Hide Overlays to conceal the overlays Click twice to deassign any function that has been assigned to the mouse To redisplay the lane and band overlays select Show Lanes and Bands from the View menu or main toolbar 3 4 Multi Channel Viewer The Multi Channel Viewer can display different types and levels of fluorescence in a gel that has been imaged at different wavelengths You can merge the data from up to three different images of the same gel Note The gel images being compared must be exactly the same size When changing image filters be careful not to move the gel If the images are not exactly the same size you can use the Crop tool see Section 3 9 a Cropping Images to resize them With at least one image open select Multi Channel Viewer from the View menu The first open image will be displayed in the viewer window using the Red channel and the image name will be displayed in the field at the top of the viewer Quantity One User Guide 2 Multi Channel Viewer Jof x r Channels Display r Transform Red Image 4 1D Scan xj Red channel x Auto scale image when assigned Br
244. kbox is selected the software will warn you to turn off your transilluminator light when you exit the ChemiDoc EQ acquisition window or when your system is idle for more than 5 minutes Note If you are performing experiments that are longer than 5 minutes e g chemiluminescence this should be deselected Video Printing Footer Information The checkboxes in this group allow you to specify the information that will appear at the bottom of your video printer printouts Quantity One User Guide Save Options To automatically create a backup copy of any scan you create select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be promted to give it a new name This protects the backup file and preserves it from any changes Simple Acquisition Mode The Simple Acquisition mode option allows you to reconfigure the ChemiDoc EQ acquisition window to simple mode In simple mode the acquisition window contains the same steps as the advanced acquisition window with the exception of Step IV Optimize display and Step V Analysis Manual exposure is also disabled in simple mode Appendix C ChemiDoc E Chemi Doc x r Step Position Gel
245. lar shade of gray 16 bit Grayscale Bio Rad s Molecular Imager FX and Personal Molecular Imager and Fluor S and VersaDoc imaging systems use 16 bit pixel values to describe intensity of scale Molecular Dynamics and Fuji imagers also use 16 bit pixel values The Discovery Series understands these formats and can interpret images from both Bio Rad and Molecular Dynamics storage phosphor systems The program can import 8 and 16 bit TIFF images from both Macintosh and PC platforms TIFF files that are not supported include 1 1 bit Line Art This format is generally used for scanning text for optical character recognition or line drawings Each pixel in an image is read as either black or white Because the software needs to read continuous gradations to perform gel analysis this on off pixel format is not used 24 bit Full Color or 256 Indexed Color These formats are frequently used for retouching photographs and are currently unsupported in The Discovery Series 2 9 Quantity One User Guide although most scanners that are capable of producing 24 bit and indexed color images will be able to produce grayscale scans as well 3 Compressed Files The software does not read compressed TIFF images Since most programs offer compression as a selectable option files intended for compatibility with The Discovery Series should be formatted with the compression option turned off 2 2 0 Saving Images To save a new image or an o
246. lation 1 7 Quantity One User Guide The installer program guides you through the installation The installer creates a default directory under Program Files on your computer called Bio Rad The Discovery Series to select a different directory click Browse The application program will be placed in the Bin folder inside The Discovery Series folder An additional folder for storing sample images is also located in The Discovery Series folder User profiles will be created and stored in the Documents and Settings folder for each user The installer places a shortcut to the application and user guide on your desktop and creates a The Discovery Series folder in Programs on your Windows Start menu After installation you must reboot your computer before using an imaging device Note If you are installing in a Windows 2000 environment you must start the application before allowing any other user access to the application Uninstalling The Discovery Series from Windows If you need to uninstall The Discovery Series for any reason go to Add Remove Programs in the Control Panel Highlight the application you want to remove and click Remove Note Uninstalling The Discovery Series software does not remove any system files in the Documents and Settings folder located in Application Data If you want to remove these files as well you must do so manually 1 6 c Installing the Discovery Series on a Macintosh The Mac install process involves
247. layed in the Gel Doc XR image window The camera continues to integrate the image on the CCD updating the display whenever the specified number of seconds is reached Once you are satisfied with the quality of the displayed image click on the Freeze button to stop the exposure process The last full exposure will be displayed in the image window Step Ill Acquire Image Q Auto Expose Click on Freeze to stop poss me Hast the exposure process y Q Freeze Fig B 6 Freezing the exposure Q Manual Acquire Note Freeze is automatically activated if you adjust any of the subsequent controls e g Print Image Mode Display controls etc B 7 Quantity One User Guide B 5 Step IV Optimize Display The Display controls are useful for quickly adjusting the appearance of your image for output to a printer Adjusting these controls will automatically freeze the display and allow you to alter the image within the Gel Doc XR window Step IV Optimize Display High 4095 i Low 0 SSS Gamma 1 0 r uf xj Highlight Saturated Pixels x Invert Display Auto Scale Reset Fig B 7 Display controls These controls are similar to those in the Transform dialog box Note The Display controls will only change the appearance of the image They will not change the underlying data High Low Sliders If Auto scale doesn t give you the appearance you want you can use the High and Low sliders to
248. lays and other features of the display 3 14 Chapter 3 Viewing and Editing Images Colormap Name st andard Load Delete Color Group Light Overlay Colors White Overlay Light Yellow cum EXE e Reset Cancel Help Fig 3 9 Colors dialog Selecting a Color Group In the Colors dialog click on the Color Group button to select the colors of a particular group of objects e g pop up boxes image colors etc A Select Color Group Light Overlay Colors Dark Overlay Colors Matte Colors Popup Colors Fixed Colors Image Colors Cancel Fig 3 10 List of Color Groups Click on a color group in the list to select it Quantity One User Guide Changing a Color After you have selected the color group to change click on the specific color button In the Color Edit dialog adjust the RGB values of the color you selected Color Edit cia 11 Gea 255 7 1 E 1 Cancel Fig 3 11 Color Edit dialog Saving Selecting a Defined Set of Colors After you have changed the colors within color groups you can save these settings for future use on other images The Colormap Name field displays the name of a defined set of colors and color groups There are several predefined colormaps or you can create your own To select a predefined colormap click on the Load button Chapter 3 Viewing and Editing Images
249. ld image with changes select Save from the File menu In Windows new images will be given a 1sc extension when they are first saved Save As can be used to save a new image rename an old image or save a copy of an image to a different directory The standard Save As dialog box for your operating system will open To save all open images select Save All from the File menu or click on the button on the main toolbar 2 2 C Closing Images To close an image select Close from the File menu To close all open images select Close All You will be prompted to save any changes before closing 2 2 d Revert to Saved To reload the last saved version of an image select Revert to Saved from the File menu Because any changes you made since last saving the file will be lost you will be prompted to confirm the command 2 2 0 Image Info Image Info on the File menu opens a dialog box containing general information about the selected image including scan date scan area number of pixels in the image data range and the size of the file Type any description or comments about the image in the Description field 2 10 Chapter 2 General Information 23 Image 1 1D Scan Ea C Program Files File Eri i The Discovery Series Info Sample Images Div Database Image l lsc Description Scan date September 1 1993 Scanner Desktop Scan area mm X 112 8 Y 35 9 Scan pixels X 1130 Y 361 Pixel size um X 99 8 Y
250. le lane If you select One type the number of the lane in the field Gauss model ma00196 1 EQ Lanes All One OK Help Fig 5 14 Gauss model Bands dialog Click on OK A status box will display the progress of the modeling Reviewing the Results Bands that have been Gauss modeled will appear as normal bands in the image To view the results of Gaussian fitting magnify a few bands in a modeled lane using the Zoom Box tool then select Bands in Lane or Plot Band from the Band menu and click on the lane Plot Band displays the Gaussian curve superimposed on the profile of the selected band Bands in Lane displays the Gaussian profiles of all the bands in the lane as shown in Fig 5 13 Quantity One User Guide The Band Information dialog displays information about the Gaussian peak and trace quantity for modeled bands that you click on In the dialog you can compare the Gaussian values to the regular band detection values These quantities can also be displayed in the Lane Report and All Lanes Report Note The quantities determined by Gaussian fitting cannot be used to in conjunction with Quantity Standards see section 6 3 Quantity Standards However you can use the original trace quantities in calculating Quantity Standards after you have Gauss modeled the bands Adjusting Bands in a Gauss modeled Lane If you use any of the individual band commands Create Band Delete Band Adjust Band in a lane that ha
251. listed under the Help menu four of these are also available on the main toolbar Help Quantity One Basic Quick Guide 7 7 Tzs E Printing Quick Guide Eb mm E Volumes Quick Guide Band Analysis Quick Guide Colony Counting Quick Guide Differential Display Quick Guide Phylogenetic Tree Quick Guide VNTR Quick Guide Fig 2 4 Quick Guides listed on Help menu and main toolbar The Quick Guides are similar in design to the secondary toolbars but are application specific Each Quick Guide contains all of the functions for a particular application from opening the image to outputting data 2 4 Chapter 2 General Information EA g Volumes Quick Guide Select Volumes 1 Select Scanner Quick Guide from zs main toolbar Open i 2 Zoom Box Question marks 13 Transf open on line Transform Commands are numbered iB Help to indicate sequence for el 4 Volume Rect Tool preparing the image creating volumes and ef 5 Volume Free hand Tool outputting data 2 6 Volume Contour Tool Select Tool Print Image E 2 2 2 3 2 3 2 2 2 2 Bi Volume Analysis Report Key commands Keyboard commands Ctrl click copy a volume box Shift click rotate a volume box Fig 2 5 Example of a Quick Guide Volumes In their expanded format the Quick Guide commands are numbered as well as named The numbers provide a suggested order of op
252. ll appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox F 7 Quantity One User Guide Warn on Saturated Pixels The Warn on Saturated Pixels option is checked by default Uncheck this box to disable this warning F 8 Appendix G Molecular Imager FX Family FX Pro FX Pro Plus and Molecular FX Fig G 1 Molecular Imager FX Before you can begin acquiring images using any of the Molecular Imager FX family of products the particular instrument must be properly installed and connected with the host computer See the corresponding FX hardware manual for installation startup and operating instructions Quantity One User Guide Note The FX should be turned on and the initialization sequence completed before the host computer is turned on except in the case of certain Power Macintosh configurations See the hardware manual for more details PC Only A Note About SCSI Cards The FX is connected to your computer by a Small Computer System Interface SCSI cable To use the FX you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device a
253. ll prevent the possibility of two users accessing the same file Also a regular backup procedure should be in place Electronic Signature Section 11 3 b 7 of 21 CFR Part 11 defines an electronic signature as a computer data compilation of any symbol or series of symbols executed adopted or authorized by an individual to be the legally binding equivalent of the individual s handwritten signature Quantity One allows the user to sign images thereby locking the files against future changes If a user changes a signed file and attempts to save the file the file automatically receives a new version number and the signed tag is removed from the window title Secure User Login Section 11 3 b 4 defines a closed system as an environment in which system access is controlled by the persons who are responsible for the content of electronic records that are on the system Quantity One utilizes the Microsoft Windows 2000 and XP Professional security model as the secure user login K 2 Using The Discovery Series in CFR Mode Access Levels Before CFR mode can be utilized the system must be correctly configured by the network administrator There are four levels of access to Quantity One in CFR Mode K 2 Appendix K CFR Module Guest A user with guest access has the ability to open and view files but a guest cannot change data nor can a guest save a file Tech The tech level user has the ability to acquire images tra
254. ll receive a warning message If this happens you can go back and select a higher sample intensity in the application tree Saving the Image After the scan is complete a message will appear asking you if you want to keep the scan If you select Yes a separate window will pop up containing the new image You can then save and analyze the image using the standard menu and toolbar functions Quantity One User Guide G 6 Options Auto Save After Scan To automatically save any scan you create click on the Auto Save After Scan checkbox With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button If you are scanning using multiple channels the image created using Channel 1 will be saved using the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you scanned an image using only Channels 1 and 4 the image created using Channel 4 will still be saved as version 2 v 2 Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox With this checkbox selected whe
255. ll to select all the images in the list 3 16 d Step 4 Apply Selected Template Click Apply to apply the selected template in step 1 to the selected images in step 3 3 45 Quantity One User Guide 3 46 4 Lanes Before you can use many of the analysis functions you must first define lanes and bands on the gel image This chapter describes the tools for defining lanes Note If you want to compare bands across lanes using standards or band matching the lane lines should be approximately the same length with their starting points aligned across the top of the image This is important for calculating the relative mobility of the bands If gel wells are visible in the image you should center the start points of the lane lines on the wells and position the ends of the lanes slightly below the last band for best results 4 1 Defining Lanes You can define lanes individually or as part of a frame The functions for doing this are on the Lane menu and toolbar I E Band Match Vo Frame Lanes Auto Frame Lanes Edit Frame b p gms utri oR iE Single Lane b Lane Width Lane Background Plot Lane Compare Lanes Lane basedAray Fig 4 1 Lane menu and toolbar In Quantity One lanes are defined by red lane lines overlaid on a gel The lane lines can be created individually see section 4 1 c Single Lanes or they can be created as part of a lane frame 4 1
256. lly create a backup copy of any scan you create select the Make Backup Copy checkbox A 13 Quantity One User Guide With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be promted to give it a new name This protects the backup file and preserves it from any changes Simple Acquisition Mode The Simple Acquisition mode option allows you to reconfigure the Gel Doc EQ acquisition window to simple mode In simple mode the acquisition window contains the same steps as the advanced acquisition window with the exception of Step IV Optimize display and Step V Analysis Manual exposure is also disabled in simple mode Gel Doc x r Step I Position Gel Q Live Focus IRIS Z00M FOCUS 4 Open T Near Close Fa Step Il Image Mode UV C White e JE rStep Ill Acquire Image c Q fee Step IV Select Output E Video Print Save Options 2 Help Fig A 10 Simple Acquisition Window Appendix A Gel Doc To change the acquisition window to Simple Acquisition Mode check the box marked Simple Acquisition Mode The change will take effect the next time you open the acquistion window A 15 Quantity One User Guide
257. log box displaying the plate il Plate001 3 Click on wells to skip them duting excision and leave them empty 9 10 11 12 w Done X Cancel Fig 10 25 Mark wells for skipping Click on a well to mark it for skipping in the cut run If you are continuing an unfinished cut run some of the wells may already be marked as reserved 10 27 Quantity One User Guide Note Reserved wells from unfinished cut runs may have material in them Be sure there is no material in the well before you mark it as unreserved When you have finished making your plate selections click Done The plate wizard will direct you as to where to place the plate in the spot cutter iil Load Plate on Cutter Load Plate001 into the front right position Name Plate001 Back Right Plate type 96 wells Front Left Front Right X Cancel Fig 10 26 Plate loading in the EXQuest Spot Cutter As each cut is being made it will be highlighted in yellow on the screen After each cut has been made the cut circle appears in white on the screen Cuts that have not yet been made are circled in green 10 3 e Other Options Prime Pump The spot cutter should be primed when it is first installed anytime the water bottle is filled when the cutting head is changed and for the first cut run of the day Click 10 28 Excision Prime pump until no large air bubbles are visible in the tubing For an empty system this usually takes 3 pri
258. main toolbar or the Edit menu These commands are also located on the Volume menu 7 1 Quantity One User Guide ANTES Analysis Reports Wi MIU LIAEESUS Lx a Select Too b Ref me EV S Volume Contour Tool Volume Free hand Tool Volume Rect Tool Volume Circle Tool Volume Array Tool Array Ungroup Tool Show Hide Volume Labels Copy To Clipboard Paste From Clipboard Volume Analysis Report Fig 7 1 Volume tools Note When using any of the following tools be careful to completely surround the data you want to quantitate You should also adjust for background intensity see section 7 6 Volume Background Subtraction You may want to experiment with several different volumes drawn around the same object before selecting the one that gives you the best quantitation data Volume Contour Tool Use the Volume Contour tool to quickly create a volume boundary that follows the outer edge of the object you want to quantify To use this tool first magnify the object then click on the Volume Contour button Using the tool e Click on a pixel at the edge of a band or other object to create a contour that encloses pixels of equal or greater intensity e Drag to create a contour that changes as you move over pixels of different intensity Drag from inside the object outward until the contour follows the outer edge of the object When you release the mouse button the v
259. me cycles Save To save the image taken by the spot cutter click the Save button in the Manual Excision Tool window You will be prompted to enter a name and specify a location for the image before saving Display Options Select the Well Number check box below the excision image window to display the well number associated with each cut request Select the Cut Position checkbox to display the position of the cut in millimeters on the gel 10 29 Quantity One User Guide 10 30 11 Differential Display and VNTRs Quantity One has tools for studying gene expression using differential display analysis and counting VNTRs or other repeated elements in gels These functions are located on the Analysis menu 11 1 Differential Display Differential display is a popular technique using mRNA and PCR amplification to identify genes that are differentially expressed between and among cell types The mRNA in a cell is amplified using RT PCR and resolved on a DNA sequencing gel where it can be compared with mRNA from related cell types to determine differential gene expression The Differential Display tool in Quantity One facilitates the side by side comparison of the bands in a sequencing gel by highlighting the up or down regulation of bands across lanes Note You must match the bands in the gel image before using this function See Chapter 6 for more information 11 1 a Normalization Before using the Differential Display
260. milarity a vector is constructed that represents the bands identified in the lane The vector depends on the comparison options see above selected If the 12 10 Chapter 12 Reports search was done on classified matched bands only then the vector S contains B elements S s1 s2 s3 sB where B is the number of band types in the lane s band set The values for s1 s2 s3 sB have the following values Weighting Off Search Sj 1 if the i th band type is found in the lane sj O if it is not found Weighting On Search If the band set has a normalizing band type then Sj The normalized density of the band assigned to the i th band type sj 0 if the lane does not have a band assigned to the i th type Otherwise si The quantity of the band assigned to the i th band type sj O if the lane does not have a band assigned to the i th type 12 5 a Compare Lane Images The Compare Lane Images report displays the lanes in decreasing order of similarity to a lane that you select Select Compare Lane Images from the Reports menu then click on a reference lane Select the report features in the pop up box 12 11 Quantity One User Guide Select Report Features Display Images Diagrams Band Type Band Weighting xj Band Type Ruler x Ref Sample on each page Cancel Fig 12 10 Compare Lane Images options If you select the Images option actual images of the lanes will be displayed in the
261. mplete you can acquire a confirmation image 10 19 Quantity One User Guide 10 3 a Acquire the Image Before acquiring an image select the light option appropriate for your gel membrane stain type e White Trans gels and PVDF membranes e UV EPI fluorescent gels Acquire Image C White Trans DRE e UV EPI la Acquire image Time secs Fig 10 16 Acquire Image If UV light is selected specify an exposure time in seconds in the field Also make sure that the UV lamp switch on the front of the cutter is on Note For Sypro Ruby stains an exposure time of 8 10 seconds is recommended When you have selected a light option click Acquire Image Note For UV exposures there will be a short delay while the lamps heat up before the exposure begins The camera will take a single image of the gel or membrane on the platform then a background image to adjust for image background When the exposure is complete the image will appear in the window u To adjust the brightness and contrast of the image open the Transform window by clicking on the button to the right of the image window 10 3 b Specify Options Under specify Options you can make changes to various settings 10 20 Excision Cut On the Cut tab you can choose to make multiple cut when possible select the specific cutting tip size and the type of material you are cutting Sp tity Options Cut Plate Hydration Wash x Make multiple cuts
262. n the field 1 12 Chapter 1 Introduction Once you have typed in the correct password the OK light next to the password field will change to green and the Enter button will activate Click on Enter to run the program 1 8 Downloading from the Internet You can download a trial version of the software from Bio Rad s Web site Go to The Discovery Series download page at www bio rad com softwaredownloads and select from the list of applications Follow the instructions to download the installer onto your computer then run the installer After installation double click on the application icon to run the program The software will open and the Software License screen will be displayed Note If you attempt to start the downloaded program and receive an Unable to obtain authorization message you will need a Hardware Security Key to run the program Contact Bio Rad to obtain a key Software License 2 Welcome to Quantity One 30 day Free Trial Free Trial Allows you to fully use the package to create and analyze the images of your choice You will need DASS to fill out the Registration Form and submit it to Bio Rad _Enter Password Basic mode Allows you to acquire new images open existing images Registration Form perform basic editing functions print export to TIFF and Save Click Help for further information Contact y your local Bio Rad office for information on purchasing Ext Quantity One a full user license
263. n them individually When dragging to select a group of objects make sure that you completely surround all the objects to be selected Each selected overlay line will have a green border e To move the selected object s position the cursor over the selection and drag e To copy within an image hold down the Ctrl key while dragging the selected object s The copy will be created and dragged to the new position e To delete the selected object s press the Delete key e To copy between images click on the Copy to Clipboard button then open or select the image you want to copy to and click on the Paste from Clipboard button The copied object s will be pasted into the new image in the same relative position they were copied from Note If you are pasting into an image with a different pixel size 1 e resolution you will receive a message that the placement of the copy may not be exact Click on OK to complete the paste then position the pasted objects manually Viewing Previously Created Text Overlays Lines Previously created text overlays and lines will appear on the image when you open the Text Overlay Tools toolbar If you have concealed all overlays using the Hide Overlays command section 3 3 Showing and Hiding Overlays click on any of the buttons on the Text Overlay Tools toolbar to redisplay the text 3 41 Quantity One User Guide 3 14 Erasing All Analysis from an Image To delete all analysis and overlays f
264. n you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be promted to give it a new name This protects the backup file and preserves it from any changes Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image Transform command G 14 Appendix G FX Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox Warn on Saturated Pixels The Warn on Saturated Pixels option is checked by default Uncheck this box to disable this warning Quantity One User Guide G 16 Appendix H PharosFX Family PharosFX and PharosFX Plus Fig H 1 PharosFX Before you can begin acquiring images using any of the PharosFX family of products the particular instrument must be properly installed and connected with the host computer See the corresponding PharosFX hardware manual for installation startup and operating instructions Quantity One User Guide Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the cont
265. nalyze the image 6 Select the output B 2 Step Position Gel The Gel Doc XR window will open in live mode giving you a live display of your sample In this mode the Live Focus button will appear selected and frames will be captured and displayed You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note The Gel Doc XR features a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens Step Position Gel Q Live Focus IRIS ZOOM FOCUS 4 Open 4 Tele 4 Near Y Close Y wide Y Far J Show Alignment Grid Fig B 3 Camera control buttons in the acquisition window You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging Area dimensions under Options see section B 9 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are focusing on you can place a ruler in the Gel Doc XR cabinet so that it is visible in the image B 4 Appendix B Gel Doc XR B 3 Step Il Select Image Mode The Image Mode option buttons allow you to set the type and scale of your data UV Select this mode for fluorescent samples W
266. nce Chemi Screen Bio Rad ECL Plus Chemifluorescence Attophos Ethidium Bromide DNA Stain Gel Sybr Green amp II Sybr Gold G 6 Appendix G FX Standard FX Applications Sypro Orange Sypro Red Protein Stain Gel Nile Red Sypro Ruby Alexa 488 Alexa 532 Alexa 546 FITC Fluorophores FAM CY3 HEX R6G Texas Red DNA Sybr Green I Protein Nano Orange TENE ssDNA Oligreen Microtiter Plate DNA Picogreen B Gal FDG GUS FDG Coomassie Blue Gel Blot Copper Stain Gel Blot Densitometry Silver Stain Gel Blot X Ray Film Grey Type INot supported in the FX Pro and FX Pro Plus systems 2Not supported in the FX Pro systems First select your general application next select the particular stain or medium you are using and finally 1f appropriate select the intensity of your samples Note Some applications require an external laser If you choose one of these without having an external laser attached you will receive a warning To exit the tree without selecting press the ESC key Your selection and settings will be displayed below the Select button G 7 Quantity One User Guide Sample Intensity Many FX applications require that you select a sample intensity High Medium or Low from the application tree This is simply a rough estimate of how much sample is visible in your gel or other object If you are unsure of the level of intensity of your sample you c
267. nd drag To rotate the box click on it to select it then hold down the Shift key while dragging an anchor point The volume will pivot around its center This is useful if the object is lying at an angle for example if the gel is smiling Volume Circle Tool Use the Volume Circle Tool to create a circular boundary around an object such as a spot To use this tool click on the Volume Circle button then position the cursor at the center of the object to be quantified and drag outward As you drag a circle will appear When you release the mouse button the volume is created Fig 7 5 Volume circle 7 4 Chapter 7 Volume Tools The volume circle should completely surround the data you want to quantify To resize the circle click on it to select it then position the cursor on the circle border and drag 7 2 Volume Labels Volume labels help to identify the type of the volumes you create As you create each volume it is given a default Auto label The default labels are U1 U2 U3 etc The U stands for unknown as distinguished from standard Std and background B volumes The number indicates the sequence in which the volume was created To change the volume type double click the volume to open the Volume Properties dialog box volume Properties x u4 r Volume Type Unknown Standard Background r Concentration r Label Edit User Label Use Auto Rename Label
268. nd the adjusted count The Colony Count is the number of colonies that appear in the defined circle minus those in the ignored region The Adjusted Count is an estimate of the total colony count in the Petri dish it uses the known colonies to extrapolate the number of colonies that might have appeared in the ignored region if it had not been damaged The adjusted count is calculated based on the area of the ignored region and the density distribution of colonies in the rest of the circle 8 7 Saving Resetting the Count A colony count can be saved to the image and or a separate spreadsheet file Saving to the Image Any count you perform is automatically stored with the image To save the count with the image exit the Colony Counting dialog by clicking on the Close button and use the Save commands under the File menu to save the image To view the count data again simply open the image and open the Colony Counting dialog To save a count or multiple counts to a spreadsheet file see the following section Resetting the Count Click on the Reset button to clear the Colony Counting dialog and any changes you have made to the image This command cannot be undone Quantity One User Guide 8 8 Saving to a Spreadsheet The Batch File controls allow you to export colony data from an image or multiple images to a Microsoft Excel spreadsheet for review and comparison To activate these controls click on the Batch Mode checkbox x
269. ndix K CFR Module Audit Trail Comment Editor x Enter your comments here w Done X Cancel Fig K 3 Comment Editor Audit Report Options The Audit Report Options dialog box allows you to customize the Audit Report view Click the Options button to open the Audit Report Options Dialog box K 7 Quantity One User Guide Audit Report Options xj r Category Filters p Entry Field Choices xj Save x Time X Signature xj User X Acquire Image x Description X Image Operations x Detail x Overlay x Comment x Lanes x File x Bands x Application Version x Match x Analysis x Expand Details In Display xj Expand Comments In Display X amp utaZoom On Detail Image Coordinates w Done X Cancel 4j Help Fig K 4 Audit Report Options The left hand column of the Audit Report dialog box lists the category filters If a box is checked all events of that type display in the Audit Report To hide a category in the report viewer clear the check box next to the corresponding category The right hand column lists the columns the report viewer displays To hide a column clear the check box next to the corresponding column name Note These options only affect what is displayed in the report viewer not what is recorded As previously stated all changes to image data and events associated with analysis are recorded If the Expand Details
270. nds until all the bands in the image are identified The gel should have no yellow unknown bands and the modeling lines should intersect at or near the middle of the bands across the gel If the modeling lines are not parallel the lanes may be warped or distorted and thus difficult to compare using the automatic analysis features 6 2 6 Band Set Dialog The Band Set dialog contains the values of all the experimental bands in a gel a tool bar for band matching and other information about the bands To open the dialog select Matched Band Set from the Match menu or toolbar and click anywhere on the image The dialog will open with a default name for the band set e g Band Set 1 Enter a new name at the top of the dialog and add any comments or category attribute information you want to associate with the band set 6 17 Quantity One User Guide Proteins Band Set Oy x E EE Band Set Bana get 3 Comment Type E Experimental Units Molecular Weight Ascending Tolerance 3 008 Normalization Band Type 23 Enzyme category mcd category l a i category mem a ss ss s el sil is 21 Type Name KDa 1k 289 310 2 3 277 593 3k 251 421 4h 241 160 5h 225 754 BR 206 128 7k 183 106 8k 163 527 3k 154 097 10k 148 081 First Lane Delete Close Help Fig 6 13 Band Set dialog List of Band Types The values of the individual bands ar
271. ne removes the identified values from any lane in the gel Click on the button then click on the lane to be cleared The bands in the lane will change to yellow Create Band and Remove Band are standard band commands that have been included in the toolbar for convenience see section 5 3 a Identifying Individual Bands Match is used to identify a new band Click on the button then click on the unknown yellow band to identify it and add it to the set Unmatch removes the identification of a band Click on the button then click on the matched band to change it to unknown Propagate Band Set applies all the values in the set to the bands in a lane based on a few identified bands in the lane Click on this button then click on the lane The bands in that lane that can be matched will change to green to indicate their known status Outlier excludes a known green band from the band set model However the band will still be marked as known Show Band Types displays all the red green and yellow bands on the image with the band type numbers next to the matched bands Show Band Models displays the modeling lines across the gel image 6 20 Chapter 6 Standards and Band Matching Standard Curve displays the Standards Regression Curve see section 6 1 d Standards Regression Curve Click on the button then click on any lane in the image Other Band Set Functions The units of the bands are displayed in the top half of the dial
272. next to the field When the specified exposure time is reached the last captured image will be displayed in the ChemiDoc XRS image window D 7 Quantity One User Guide Step Ill Acquire Image Auto Manual Live Expose Expose Acquire Exposure Time sec Click on Freeze to stop o 03 elo the exposure process o Freeze Fig D 7 Freezing the manual exposure Live Acquire Live Acquire mode allows you to specify an interval over which a series of progressively longer exposures are taken All exposures are then displayed on the screen and you can choose the one with the best image Click on the Live Acquire button A settings dialog box will open in which you can specify the total exposure time starting exposure time and number of exposures Optimize Exposure x r Settings Total Exposure Time sec 10 0 Starting Exposure Time sec 1 0 Number of Exposures 5 O Save Images Cancel OK Fig D 8 Live Acquire settings Note You should specify no more than 10 exposures in the Live Acquire Settings dialog to avoid excessive build up of image background in later exposures The D 8 Appendix D ChemiDoc XRS fewer the exposures the less background will be added to the image See the Release Notes for additional instructions on reducing background in images captured using Live Acquire Select the Save Images checkbox if you want to automatically save each exposur
273. ng e The transmissive step tablet calibrates transmissive scans from 0 to 3 0 O D e The reflective step tablet targets reflective scans from 0 to 2 0 O D The first time you use the GS 800 you must select some settings to ensure accurate calibration Click on the Options button in the main acquisition window This will open the Densitometer Options dialog box E 9 Quantity One User Guide Densitometer Options r Calibration On the GS 700 this requires that the calibration overlay x Calibration On E xi Calibration On be installed O Calibrate Before Every Scan Recalibration E The Densitometer will be re calibrated Interval minutes after this time has expired hrak calibration report will be printed each time the ES Densitometer is calibrated Calibrate Now Edit Step Tablet Data Handling The Densitometer will be told to scan at its maximum resolution and spatial averaging will be used to produce the desired resolution This gives better spatial fidelity and lower noise although it takes longer to scan Oversample A modified logarithmic scaling system will be used to reduce Save Scaled Data the size of the data by 50 It introduces a constant relative error of less than 3 Cancel Help Fig E 9 Densitometer Options dialog box Note that calibration is always on for the GS 800 E 5 a Step Tablet Values The built in transmissive and reflective step tablets on
274. ng Anchors To remove an anchor point select Unadjust Anchors from the Edit Frame submenu or toolbar and click on the anchor The anchor will disappear and the adjusted lanes will straighten out 4 1 c Single Lanes You can define individual lanes using the single lane tools These are located on the Lane gt Single Lane submenu or on the Lane toolbar 4 6 Chapter 4 Lanes HOPE ete kl oR EB Single Lane Create Lane Adjust Lane Unadjust Lane Remove Lane Lane Width Lane Background Fig 4 7 Single Lane tools Note You can use the single lane commands on any lane within a frame however the lane will be detached from the frame To mark an individual lane select Create Lane and drag a line from the top to the bottom of the gel lane The lane line will be marked in red Repeat this procedure to manually mark all the lanes you want to analyze in the gel Note Ifthe lane numbering gets out of sequence select Sort and Recalculate from the Edit menu to renumber the lanes Adjusting Single Lanes You can adjust the position of any lane line Select Adjust Lane from the Lane Single Lane submenu or the Lane toolbar and either drag one of the existing anchor points or click anywhere on the lane to create a new anchor point and drag it into position To undo any lane adjustments select Unadjust Lane from the submenu or toolbar and click on an anchor point to remove it If you remove the anchor
275. ng and review To view or edit the default reflective values make sure that the Reflective checkbox is selected in the main acquisition window then click on the Options button In the Densitometer Options dialog click on the Edit Step Tablet button The Step Tablet Values for GS 800 Reflective dialog box will open When you are finished viewing editing the reflective step tablet values click on OK Diffuse Versus Specular O D In the step tablet form you enter O D as diffuse density and then the software automatically calculates the specular density Specular density is a measure of the light that passes directly through a medium Diffuse density includes light that is scattered as it passes through the medium Step tablet values are given in diffuse density but are measured by the scanner in specular density and therefore must be converted according to the specular diffuse O D ratio This conversion does not affect quantitation q Specular Light passes directly through medium lt q Medium gel film etc rd N lt q Diffuse Light is scattered by medium Fig E 11 Specular and diffuse density Diffuse density values are converted to specular optical density units according to the following formula Specular OD 1 4 Diffuse OD Appendix E GS 800 E 5 b Other Calibration Settings After you have entered the step tablet values you can immediately calibrate by clicking on the Calibrate Now b
276. nly files If you attempt to save a backup file you will be promted to give it a new name This protects the backup file and preserves it from any changes Appendix E GS 800 Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Transform command on the main toolbar or Image menu Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox E 15 Quantity One User Guide E 16 Appendix F Personal Molecular Imager FX Fig F 1 Personal Molecular Imager FX Before you can begin acquiring images using the Personal Molecular Imager FX the instrument must be properly installed and connected with the host computer See the Personal FX hardware manual for installation startup and operating instructions Note The Personal FX should be turned on and the initialization sequence completed before the host computer is turned on except in the case of certain Power Macintosh configurations See the hardware manual for more details F 1 Quantity One User Guide PC Only A Note About SCSI Cards The Personal FX is connected to your computer by a Small Computer System Interface SCSI cable To use the Personal FX you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME
277. nsform images crop flip rotate use the text and line tools perform basic analysis print and export The tech level user also has the ability to save and sign files User At this level all functions are available to the user However the user cannot change the CFR settings Administrator The administrator level has all the capabilities of the full user as well as the ability to make changes to the CFR settings CFR Image Files For files to be opened in CFR mode the files must have been created on a CFR system either through acquisition or importing a TIFF file Any files created on a non CFR system cannot be opened To open a file created on a non CFR system open the file on a non CFR system and export it to TIFF You can then import it in CFR mode To ensure accuracy remember to select the analysis option as your export mode when you export your non CFR images Note Due to operating system limitations it is possible to have image files open in two or more locations over a network simultaneously To safeguard your image files keep them in a protected folder or on your local machine K 2 a Setting Security Preferences Setting the Security preferences for CFR mode requires the user have administrator privileges To locate the Security Preferences select Preferences from the Edit menu and click the Security tab K 3 Quantity One User Guide x Misc Paths Display Toolbars Application Imagers Security
278. nsity i e the area under a band s intensity profile to calculate Quantity Standards after you have Gauss modeled your bands 6 3 a Creating and Applying a Set of Quantity Standards Select Quantity Standards from the Analysis menu A pop up box will prompt you to create a new curve or load a saved calibration curve 6 24 Chapter 6 Standards and Band Matching Quantity One Ea A Calibration Curves Create New Cancel Fig 6 18 Loading a quantity calibration curve Select Create New to open a blank Quantity Standards dialog Quantity Standards for Proteins 1D ScaQty Stds x Name Cal 1 Units Origir Description Measure Trace Units OD x mm Select Band k Match k Lane he Calibrate Band M Match k Lane k Gel Un Calibrate Band he Match k Lane he Gel Interpolation Point to Point Linear Regression Extrapolation Yes C No Trace Quantity Dilution Rel Band OD x mm Factor Status Dev 6 12 4 042 0 60 b Knowm 43 2 10 13 3 705 0 53 p Known 4 11 13 2 061 0 30 p Known 3 8 8 1 561 0 22 p Known 2 12 8 1 059 0 15 p Knowm 13 8 7 0 2204 0 03 p Enowm 10 El Bal Show Curve Import Curve DK J Delete J Help Fig 6 19 Quantity Standards dialog The dialog will open with a default name for the quantity standards e g Cal 1 Enter a new name specify the quantity value units e g ug and enter any descriptive information in the appropriate fields
279. nsity changes from an absolute value to a percentage This percentage is the fraction of the signal intensity of the darkest band in the lane that will be detected as a band For example if the darkest band in a lane is 50 000 counts and the Min Density is set to 25 000 counts when you turn on Normalize the Min Density will switch to 5096 i e a band must be at least half as dark as the darkest band in the lane Noise Filter The Noise Filter is used to minimize the number of small fluctuations in the image i e noise that are called bands while still recognizing larger features 1 e real bands This filter becomes especially important at higher Sensitivity levels The Noise Filter value refers to the size of the filter in pixels e g a value of 2 50 equals a filter size of 2 50 x 2 50 pixels Features smaller than the filter size will not be recognized as bands Entering a noise filter size of zero turns it off completely The default value is 4 00 If band detection detects doublets as single bands decrease the Noise Filter setting and or increase the Sensitivity You can also try decreasing the Size Scale parameter instead of the Noise Filter to improve the detection of closely spaced bands However if you decrease both the Noise Filter and the Size Scale the fuzziness around bands may be mistakenly detected as separate bands Shoulder Sensitivity Normally band detection tries to distinguish shoulders as separate bands
280. nt any changes you make in this box Clicking on Defaults restores the settings to the factory defaults DAC Settings Note The default DAC settings are highly recommended and should be changed with caution C 14 Appendix C ChemiDoc These sliders may be used to adjust the minimum and maximum voltage settings of your video capture board The minimum slider defines the pixel value that will appear as white in the image while the maximum slider defines the pixel value that will appear as black The slider scale is 0 255 with the defaults set to 60 minimum and 130 maximum Imaging Area These fields are used to specify the size of your imaging area in centimeters which in turns determines the size of the pixels in your image i e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less than 1 percent of the pixels in your image saturated Consequently the default value for this field is 0 15 percent Reminder When this chec
281. nual Expose to adjust your exposure time directly Most applications only require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button Manual Expose is automatically activated after Auto Expose has deactivated A 6 Exposure Time field active adjust number of seconds r Step Ill Acquire Image Exposure Time sec Appendix A Gel Doc Auto Manual Expose Expose Manual Expose active button appears selected Fo ala 9 Freeze Fig A 5 Manual Expose With Manual Expose activated you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field Type in a number or use the arrow buttons next to the field When the specified exposure time is reached the last captured image will be displayed in the Gel Doc EQ image window The camera continues to integrate the image on the CCD updating the display whenever the specified number of seconds is reached Once you are satisfied with the quality of the displayed image click on the Freeze button to stop the exposure process The last full exposure will be displayed in the image window Click on Freeze to stop the exposure process o Freeze r Step Ill Acquire Image Manual Expose Auto Expose a Time sec Fig A 6 Freezing the exposure A 7
282. numbers have been concealed e g by the Hide Overlays command you can redisplay them by selecting VNTR Display Quantity One User Guide 11 6 12 Reports Quantity One can display and print a variety of analysis reports You can format the reports to include different kinds of data The available reports are listed under the Reports menu Reports Window Help Lane Report All Lanes Report Band Types Report Match Report All Matches Report 1 D Analysis Report Compare Lane Images Phylogenetic Tree Similarity Matrix Comparison Options Volume Analysis Report Volume Regression Gurve VNTR Report Fig 12 1 Reports Menu 12 1 Report Window Many of the reports share the same basic report window 12 1 Quantity One User Guide Click here to close the report window x Proteins Report of x Detail Report by Lane Proteins November 28 2001 Name cerebellum Band Set Band Set 3 Band Relative Mol Wt Peak Average Trace Relative Type Front KDa OD OD OD x mm Gty 0 005 291 70 x 0 47 0 395 1 0 0 014 279 88 0 26 0 476 1 2 0 058 227 52 0 42 1 060 2 7 0 079 205 92 0 33 0 832 2 1 0 103 183 11 0 11 0 146 0 4 0 126 163 79 0 20 0 412 1 1 Molecular Weight Calculation Method Point to Point Known x Extrapolated Screen Page 17 of 135 ela da re Be eje Controls for scrolling through screen pages Print One Page Print All
283. o a dashed blue line indicating a selected area e To reposition the scanning box you have selected position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move e To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created F 4 Appendix F Personal FX You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right After you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later F 3 Step Il Select Resolution The Personal FX acquisition window allows you to scan at 50 100 200 or 800 micrometers These resolutions are listed as option buttons in the control panel r Step Il Select Resolution 50 micrometer 100 micrometer 200 micrometer 800 micrometer LII za Image file size 6 09 Mb Fig F 4 Resolution option buttons The resolution you select should be based on the size of the objects e g bands spots you are interested in For example e 50 micrometer resolution should be reserved for images requiring the highest level of detail e g high density in situ samples 1 536 well microplates high densi
284. oas s mem 13 2204 o os fa fino 9 Fig 6 25 Entering a dilution series co I J o The quantity of each band will be automatically calculated 6 3 0 Importing a Calibration Curve A calibration curve created for one gel can be applied to other gels Make sure that the new gel you want to quantitate and the gel with the existing calibration curve are both open Click on the new gel to quantitate Select Quantity Standards from the Analysis menu and click on Create New as previously described In the new Quantity Standards dialog click on the Import Curve button and select the existing calibration curve from the list When you make the selection the values for the curve will be displayed in the new Quantity Standards dialog Each standard value will be labeled Import in the Band column Checking the Imported Curve If the quantity of one or more bands in the new image is known you can verify the accuracy of the imported calibration curve Go to Select in the Quantity Standards dialog click on Band then click on a known band Its lane number band number and intensity will be displayed in one of the standard values fields 6 31 Quantity One User Guide Trace Quantity Dilution Rel Band OD x mm Factor Status Dev Import 4 042 4 04 gt known 13 Import abrir 1 82 b known 4 Import 1 610 1 68 known 2 Import 0 4163 0 39 D known 17 Band used Import 0 2879 o p Joutzier
285. od Global or Local Note If you select Global and have not defined a background volume no background subtraction will be performed on the image The Image Display Options affect how the image is displayed and or printed on the report You can choose whether to report on all volume objects All objects or only those objects you have selected Selected objects Select the regression method for calculating the Volume Regression Curve To display the curve click on the Show Curve button 12 22 Chapter 12 Reports Select the Font Size and Line Spacing settings to be used in the report by clicking on the button next to each field and selecting from the list of options Saving the Report Options To save the report options click on the Modify Report Settings button at the bottom of the dialog In the pop up dialog enter a name for the report settings in the field To load or delete previously saved settings click on the button next to the Settings to Load or Delete field and select from the list of saved settings Then click on Load or Delete 12 7 Volume Regression Curve If you have defined at least two standard volumes on the image you can display a regression curve for calculating the quantities of the unknowns Note Volumes that fall above the greatest standard value or below the lowest standard value cannot be guaranteed for accuracy Select Volume Regression Curve from the Reports menu 12 23 Quantity One
286. of the background in the text box using the pull down lists After you have typed the text click on OK The text will appear on the image at the spot where you originally clicked Editing a Text Overlay To edit a text overlay make sure the Text Tool or Select Tool is selected and then double click on the overlay to open the Text Overlay Properties dialog The existing text will be displayed and can be edited Line Tool You can use the Line Tool to draw a line between text and an image feature or between any two points of interest on the image Click on the Line Tool button then drag on the image to create the line To resize or adjust a line make sure the Line Tool or Select Tool is selected and then position the cursor on one end of the line marked by a circle and drag 3 40 Chapter 3 Viewing and Editing Images To add arrowheads to a line make sure the Line Tool or Select Tool is selected and then double click on the middle of the line A dialog will pop up with options to add arrowheads to one or both ends of the line Moving and Copying Text and Lines You can move copy or delete a single text overlay or line or a group of overlays and lines within an image You can also copy and paste between images First you must select the object s Click on the Select Tool button To select a single overlay or line click on it To select multiple objects either drag a box around them or hold down the Shift key and click o
287. og as is the matching tolerance used Tolerance is the minimum spacing between unique bands that you specified when you created the band set The Normalization button allows you to pick a specific identified band that appears in all the lanes to normalize the relative quantities of the other bands against see section 11 1 Differential Display for more information Click on the resize button in the lower right corner to reconfigure the dialog to its smaller palette version which displays only the tool buttons and band type buttons To delete the band set click on the Delete button To close the band set click on OK Note that the band set is saved when you save the image 6 2 c Tips for Gels Without Standards If you are not using standards we recommend that you load at least two lanes per gel with a reference sample containing many if not most of your experimental bands of interest Select the Match command from the menu or toolbar and click on this reference sample lane to create a new band set Then select Matched Band Set from the Match menu to open the Band Set dialog and apply that band set to other reference sample lanes using the Propagate Band Set command Propagate Band Set is a feature that not only simplifies identifying bands it allows the software to do some optimizations that will significantly speed up modeling Choose another reference sample lane that you want to apply the band set to Start by using the Match
288. ogreen DNA Sybr Green I GUS FDG Protein Nano Orange ssDNA Oligreen Microtiter Plate Coomassie Blue Gel Blot Copper Stain Gel Blot Silver Stain Gel Blot X Ray Film Grey Type Colorimetric Diamond DIGE CY2 DIGE CY3 DIGE CY5 Emerald Sypro Ruby Multiplex 1 Supported in the PharosFX Plus systems only First select your general application and then select the particular stain or medium you are using Finally select the intensity of your samples H 7 Quantity One User Guide Note Some applications require an external laser If you choose one of these without having an external laser attached you will receive a warning To exit the tree without selecting press the ESC key Your selection and settings will be displayed below the Select button Sample Intensity All PharosFX applications require that you select a sample intensity High Medium or Low from the application tree This is simply a rough estimate of how much sample is visible in your gel or other object Note If you are using the PharosFX Plus and select Radioisotopes you do not need to select a sample intensity If you are unsure of the level of intensity of your sample you can always select a level capture an image then adjust the level and capture another image For example if you select Low Sample Intensity and the resulting image has too many saturated pixels you will receive a warning message Simply chang
289. ol panel and a video display window B 2 Appendix B Gel Doc XR Gel Doc XR Step Position Gel Live Focus IRIS ZOOM FOCUS POpen P Tele 4 Near Close A wide Y Far 3Show Alignment Grid Step Il Image Mode CUY White r Step Ill Acquire Image Auto Manual o Expose Q Acquire Exposure Time sec EE Q Freeze Step IV Optimize Display High lt Low o Gamma 1 0 xj Highlight Saturated Pixels Step Y Analyze Step VI Select Output Exposure Status xj Invert Display r Fee Annotate 2 Pit L AutoScale R lo Scale eset Options 2 Help Analyze Save Fig B 2 Gel Doc XR acquisition window The Gel Doc XR display window will open in live mode giving you a live display of your sample If no image is visible make sure the camera is on check the cable connections make sure the iris on the camera is not closed and make sure that the protective cap is off the camera lens Also check to see that the transilluminator is on and working The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are six basic steps to acquiring an image using the Gel Doc XR 1 Position and focus the gel or other object to be imaged 2 Select an illumination mode 3 Acquire the image B 3 Quantity One User Guide 4 Optimize the display 5 A
290. older versions of the ChemiDoc EQ without the motorized zoom lens r Step Position Gel IRIS ZOOM FOCUS t Open T Tele Near Close 4 Wide rs O Show Alignment Grid Fig C 3 Live Focus button and camera control buttons You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging Area dimensions under Options see section C 9 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are Appendix C ChemiDoc focusing on you can place a ruler in the ChemiDoc EQ cabinet so that it is visible in the image C 3 Step Il Image Mode The Image Mode option buttons change the type and scale of the data as well as the behavior of the ChemiDoc EQ when acquiring an image UV Select this mode for fluorescent samples With this mode selected the data will be measured in linear intensity units White Light Select this mode for reflective and transmissive samples With this mode selected the data will be measured in uncalibrated optical density uOD units Chemi This mode is designed for chemiluminescent samples With this mode selected the data is measured in linear intensity units however the data is inverted so that samples will appear dark on a light background Also Chemi mode changes the behavior of the Auto and Manual Expose functions as describ
291. olume is created 7 2 Chapter 7 Volume Tools Fig 7 2 Volume contour The contour should completely surround the data you want to quantify To edit the contour position the cursor on the border The cursor will change to a pencil tool Drag across the line a new white line will appear When you recross the old line a new contour will be created Volume Free Hand Tool Use the Volume Free hand Tool to manually draw a volume boundary First magnify the band or other object you must be able to see the individual pixels Then click on the Volume Free hand button and use the cursor to draw a line around the object When the line crosses itself a free hand volume is created Fig 7 3 Volume free hand If you make a mistake while drawing backtrack with the mouse The line you draw should completely surround the data you want to quantify To edit the volume position the cursor on the border and drag across the line a new white line will appear When you recross the old line a new free hand volume will be created 7 3 Quantity One User Guide Volume Rect Tool Use the Volume Rectangle Tool to create a volume box around an object Click on the Volume Rect button then drag a box around the object to be quantified When you release the mouse button the volume is created Rp Fig 7 4 Volume rectangle To resize the box click on it to select it then position the cursor on one of the corner anchor points a
292. olumns and cells in the actual image In the next step you will adjust the position of the matrix Adjusting the Array Matrix The Add Adjust Anchors tool will be automatically assigned to the mouse after you create the frame otherwise select it from the Lane Edit Frame submenu Position the cursor on the corner points of the frame and drag them into position so that the red lines run down the middle of the array columns and the top and bottom brackets are centered on the array cells see section 4 1 b Editing the Frame for guidance on adjusting frames If necessary the Adjust Lane command see Adjusting Single Lanes on page 7 and Adjust Band command see section 5 3 b Adjusting Bands can be used to adjust the placement of columns and cells within the frame Reducing Background in the Array After you have positioned the array you should reduce lane background using the Lane Background command see page 4 2 Lane Based Background Subtraction Lane background will affect quantitation of the cells Setting Array Cell Height and Width Now you should adjust the cell brackets so that they completely enclose the cells in the array Select Array Cell Height from the Lane Lane based Arrays submenu and enter the height in millimeters of the array cells 4 18 Chapter 4 Lanes E Quantity One x Enter the height in millimeters to use for A all cells Every cell will be changed to reflect the new size 40 00
293. om the submenu and choose the settings to be deleted from the list 3 9 b Flipping and Rotating Images Use the image flipping and rotating commands to reorient lanes and bands for proper analysis Note These actions will erase any analysis you have performed on the image You will be prompted to confirm the changes 3 26 Chapter 3 Viewing and Editing Images Flipping To flip the image right to left select Horizontal Flip from the mage menu or toolbar To flip the image top to bottom select Vertical Flip 90 Rotations Select Rotate 90 Left Rotate 90 Right or Rotate 180 from the Image gt Rotate menu or mage toolbar to perform the specified rotation Custom Rotation Use the Custom Rotation command to rotate the image in increments other than 90 Select Custom Rotation from the Image gt Rotate submenu or Image toolbar A green plus sign will appear next to the cursor Click on the image and a circular overlay with an orange arrow will appear A pop up box will indicate the angle of rotation in degrees and radians 3 27 Quantity One User Guide Rotate Image 1 v3 E3 Angle 11 6 degrees radians 0 202 Rotate Cancel Fig 3 18 Custom rotation the arrow points in the direction of the new top of the image To perform the rotation position the cursor on the arrowhead and drag As you drag the arrow will rotate and the angle in the box will change Position the arrow so
294. om the drop down list A higher Binning setting 2x2 3x3 provides optimal sensitivity for low light applications such as chemiluminescence In this mode the pixels in the camera are binned e g four pixels are combined into one to increase the amount of signal per pixel without increasing noise Note that combining the pixels results in a reduction in the resolution of the image D 4 Appendix D ChemiDoc XRS Click OK to save and add the new custom application to the custom list The new application is automatically selected as your application D 3 Step Il Position Gel Click the Live Focus button and frames will be captured and displayed at about 10 frames per second depending on the speed of your computer You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note ChemiDoc XRS features a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens Step Position Gel Q Live Focus IRIS Z00M FOCUS Open 4 Tele 4 Near Close wide Y Fa J Show Alignment Grid Fig D 4 Live Focus button and camera control buttons You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging
295. on on the image using the Band Attributes dialog 5 8 b Drawing Tools Contour Tools ele of ete g Contour b Draw Band Density in Region Draw Band Boundary Edit Band Boundary Fig 5 18 Drawing tools Density in Region Density in Region displays intensity information for any area on an image 5 23 Quantity One User Guide Select Density in Region from the Band Draw Band submenu or Contour Tools toolbar and use the cursor to draw a line around a region of interest a Area 47027 090 mm 2 Volume 1585500015 503 CNT x mm 2 Average 33714 610 CNT Fig 5 19 Density in Region tool When you close the border a pop up box will display information about the enclosed area For very small regions magnify the region before using this command Drawing Band Boundaries Note To use the drawing tools at least one lane must be defined on the image Also magnify the region you want to draw in using Zoom Box Use Draw Band Boundary to draw the boundary of a band manually Select the command from the Band Draw Band submenu or Contour toolbar and drag the cursor around the region that you want to define as a band A boundary line will appear 4 Fig 5 20 Draw Band Boundary tool If you make a mistake and need to retrace part of the band boundary backtrack with the cursor the path will be erased and you can redraw it 5 24 Chapter 5 Bands Note Be sure
296. on procedure There are three basic steps to acquiring an image using the ChemiDoc XRS 1 Select the application 2 Position and focus the gel or other object to be imaged 3 Acquire the image D 3 Quantity One User Guide D 2 Step Select Application To set the appropriate parameters for the type of object you are imaging click on the Select button under Select Application You have the option of selecting a UV White or Chemiluminescence application You can also select a custom setting Once you select an application the name of the application and its settings appear in the Select application step D 2 a Custom Applications The Bio Rad installed applications have pre set gain and bin settings If the settings for the available applications do not meet your needs you have the option of creating a custom application which allows you to set your own binning settings To use a custom application click Select and choose custom Next select a custom application from the list or select Create to create a new application This opens the custom dialog Create a new A Name Custom Application 1 Illumination None gt Gain amp Binning 2x 3x3 gt Cancel OK Fig 4 3 Creating a custom ChemiDoc XRS application Enter a name for the application From the Illumination drop down list select an illumination mode You can select UV white or no illumination Next select Gain and Binning settings fr
297. on the microtiter plate format When acquiring the gel image zoom in such that there is minimum blank space around the gel This is extremely important with low resolution imagers Once the gel image is acquired the ReadyAgarose 96 plus feature uses a step by step approach to analyze the image The First step is to align the gel image Quantity One then places lane overlays over each lane You can then make slight adjustments to the size and position of the lane overlays The final step allows you to determine the lane format There are three lane formats from which to choose 8 x 14 4 x 28 default or 2 x 56 format includes standard lanes 9 1 ReadyAgarose Wizard Once you have acquired your image select Ready Agarose 96 plus from the Analysis menu This opens the Ready Agarose 96 plus Wizard Step 1 Positioning the Image The first panel of the wizard displays your image with four red crosshairs Quantity One User Guide Quantity One d x Please move the cross hair overlays over the gel corner triangles tevious Next mp Cancel 7 Help Fig 9 1 Positioning the Image Click and drag each crosshair to place them over the four triangles visible in the gel holder The ideal position is at the tip of the triangle When you have correctly positioned the crosshairs click the Next button Step 2 Resizing Repositioning and Spacing the lanes In the next panel the image displays 96 lanes based on the position
298. ont of the VersaDoc window Subsequent exposures are tiled in front of the VersaDoc window Note that the first exposure will have the base file name the default base file name is the computer user name and a time stamp Each subsequent exposure will have a version number v2 v3 v4 etc appended to the base file name If you are using the 1 13 Quantity One User Guide default base file name the time stamp may change in the course of the series of exposures in this case the base file name will change and the version numbering will reset for subsequent exposures The highest version number will be the final exposure If you did not elect to auto save the exposures as they were created then each image will be unsaved To stop the image acquisitions click on the Stop button Note Exposures captured before stopping will be displayed in image windows Study the different images and select the best exposure s to keep 1 6 Options Click on the Options button to open the Options dialog box VersaDoc Options gt x 7 Information gt r Dark Subtraction Type Model VersaDoc 5000 Normal Referenced None Firmware Version 1 0 Reset Referenced Hardware Version 1 0 rSave Auto Save After Scan White EPI Light Available UV EPI Light Available UY Trans Light Available is r Displ r Imaging Area CM Pa WANT UU Width iz Auto scale Transform After Acquisition Height 5 1
299. ools in other applications Click on the Zoom In or Zoom Out button on the main toolbar or select from the View menu The cursor will change to a magnifying glass Click on an area of the image to zoom in or out a defined amount determined by the setting in the Preferences dialog see section 2 5 Preferences Grab This tool allows you to change the position of the image in the image window Select Grab from the main toolbar or View menu The cursor will change to a hand symbol Drag the cursor on the image to move the image in any direction Arrow Keys You can also move the image inside the image window by using the Arrow keys on the keyboard Click on an arrow button to shift the image incrementally within the window The amount the image shifts is determined by the Pan setting in the Preferences dialog see section 2 5 Preferences Quantity One User Guide View Entire Image If you have magnified part of an image or moved part of an image out of view select View Entire Image from the main toolbar or View menu to return to the original full view of the image Centering an Image You can center the image window on any point in an image quickly and easily using the F3 key command This is useful if you are comparing the same region on two gel images and want to center both image windows on the same point Position the cursor on the point on the image that you want at the center of the image window then press the F3 k
300. operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated F 1 Personal FX Acquisition Window To acquire images using the Personal FX go to the File menu and select Personal FX The acquisition window for the imager will open displaying a control panel and the scanning area window F 2 Appendix F Personal FX x Step Select Scan rea aS Click and drag in diagram to set scan area Top Left s E Bottom R Right 14 e xj Quadrant Mode D E rStep Il Select Resolution F 50 micrometer G 100 micrometer H 200 micrometer P 800 micrometer Image file size 13 73 Mb K Orie A Acquire 5 Stop oj Ni ptions N Auto Save After Scan Highlight Saturated Pixels o
301. or above the Max values will be collapsed to fit in the window For non Neighbor Joining trees use the Clusters option to define the number of clusters 0 to 18 in the tree The separate clusters will be identified in the tree by letters 12 5 c Similarity Matrix The Similarity Matrix report compares the similarity of all the lanes to one another If there are N lanes in the gel then the similarity matrix is an N by N matrix that is computed using the Dice Coefficient as described in section 12 4 1 D Analysis Report The matrix has the following properties e The diagonal elements always have values of 100 This is because a lane is always 100 percent similar to itself e The matrix is symmetrical Mij Mii Select Similarity Matrix from the Reports menu to open this report 12 19 Quantity One User Guide Population 1 Band Frequencies Population 1 of Database 1 October 7 1998 Bandset expt Units Molecular Weight Normalization Band Type 13 Samples 24 Band Frequency KDa Unweighted 96 Weighted 96 5945 99 16 7 5 3 5687 71 50 0 22 7 5477 47 79 2 38 6 5243 58 58 3 29 7 4838 55 70 8 16 6 4640 99 8 3 ta 4520 08 0 0 0 0 4352 12 58 3 22 6 4179 56 83 3 37 6 3943 85 54 2 27 1 3628 10 100 0 66 7 2950 30 45 8 12 7 2655 65 100 0 100 0 1 2 3 4 5 6 7 8 Screen Page 1 of 1 ald ee Page E oa Fig 12 14 Similarity Matrix report The Similarity Matrix report is displayed in a standard
302. or 3D viewer This allows Quantity One to emulate a graphics driver to render a 3D image See Section 2 5 c Display for further information 3D Viewer n Options Display Mode Lighting v Scale Factor t Close 4 Reset View Help Fig 3 7 3D Viewer 3 5 a Positioning the Image Windows For Windows operating systems use your mouse to reposition and rotate the image 3 11 Quantity One User Guide Rotate the image Left click and drag to rotate the image Reposition the image Right click and drag to reposition the image Zoom in out To zoom in or out Click the center mouse button or roll the wheel If you do not have a three button mouse or a mouse with a wheel hold down the shift key and left click and drag to zoom in or out Macintosh For Macintosh operating systems use mouse and keyboard combinations to reposition and rotate the image e Rotate the image Click and drag to rotate the image e Reposition the image Ctrl gt click and drag to reposition the image Zoom in out Shift gt click and drag to zoom in or out 3 5 b Display Mode The 3D Viewer window allows you to view the image in three different modes wire frame lighting and textured e Wire frame shows the image in a transparent frame view e Lighting shows the image with different areas of light and shadow depending on the angle of view Use the slider bar to adjust the intensity of the lighting
303. or high capacity removable disk PC The following is the recommended system configuration for installing and running on a PC Operating system Windows 2000 Windows XP Processor Pentium 333 MHz or faster RAM 2 256 MB for all Bio Rad imaging systems 1 5 Quantity One User Guide Hard disk space Monitor USB port Printer Macintosh 23GB 17 monitor 1024 x 768 resolution required True color Required for the Hardware Security Key HSK Optional The following is the recommended system configuration for installing and running on a Macintosh Operating system Processor Model RAM Hard disk space Monitor USB port Printer MacOS 9 2 MacOS 10 2 and 10 3 PowerPC G3 processor or faster 2 256 MB for all Bio Rad imaging systems 23GB 17 monitor 1024 x 768 resolution required Millions of colors Required for the Hardware Security Key HSK Optional Note The default amount of memory assigned to this program on the Macintosh is 128 MB If the total RAM in your Macintosh is 128 MB or less you should reduce the amount of memory assigned to the program to 10 MB less than your total RAM With the application icon selected go to File Get Info in your Finder to reduce the memory requirements for the application See your Macintosh computer documentation for details 1 6 Installation Refer to the Installation Guide for detailed information regarding installation of The Disc
304. ortion across the gel You can also click on the Arrow button next to a standard value to apply that value to a particular band in a lane on the image Click on the button then click on the standard band The remaining bands in the lane will be numbered sequentially based on the initial assignment Chapter 6 Standards and Band Matching T Name KDa es57 00 2 Click here to apply this standard value 7 to a particular band on the image 4561 00 322 00 2027 00 Fig 6 9 Applying a single standard value k k h gt After you have applied the standard values to a lane the bands in that lane will change color to blue indicating that they are now standards You are now ready to select the standards regression curve to use for calculating the unknowns Removing Standard Values from Lanes The Clear from Lane command removes all the standard values from the lane s you select Click on the button then click on the lane or lanes from which you want to delete the standard values Showing the Modeling Lines Click on the Show Modeling Lines button in the Standards dialog to display lines across the gel connecting the same standard bands in different lanes Unknown bands that fall along these lines have the same values as the standards To redisplay only the band numbers with no modeling lines click on the Show Band Types button 6 1 d Standards Regression Curve After you have applied the s
305. ot matrix or film fogging Note Whole image background subtraction permanently changes the image You will be prompted to confirm the change Select Subtract Background from the Image menu 3 29 Quantity One User Guide amp Background for MicroSatellite 1D S Dark Contrast EI gt Background p 00 T Stripe Min n a Avg n a Max n a Background Box A Background Stripe k OK Auto scale Cancel Help Fig 3 19 Subtract Background dialog The Subtract Background dialog has a preview window which contains a smaller view of the image Changes in the subtract background controls are reflected in the preview window and are only applied to the main image when you click on OK 3 30 Chapter 3 Viewing and Editing Images Auto scale Click on the Auto scale button to automatically adjust the Dark Contrast and Background settings to optimal levels You can then manually adjust these settings using the other controls Dark Contrast Slider Use the Dark Contrast slider to reveal the level of background in the image before subtraction This slider is similar to the High slider in the Transform dialog Drag the slider handle to the left to make faint signals appear stronger Click on the slider bar to move the slider incrementally or type a value into the field next to the slider Note The Dark Contrast slider itself does not eliminate background intensity therefore the OK button will not activate if
306. our Create Contour Contour Information Contour to Band Draw Band b Fig 5 16 Contour tools Creating Contours Select Create Contour from the Band gt Contour submenu or toolbar and click on a pixel at the edge of the band This displays a contour that encloses pixels whose intensity is equal to or greater than that of the pixel at the cursor Contour encloses pixels with intensities greater than or equal to intensity of pixel at cursor Fig 5 17 Creating a Contour If the contour does not encircle the band reposition the cursor and click again A new contour will be drawn in place of the old 5 22 Chapter 5 Bands Contour Information To display information for the selected contour select Contour Information A pop up box will show the area total intensity and average intensity Converting a Contour into a Band When you are satisfied with the contour select Contour to Band from the menu or toolbar to redefine the contour as a band and assign it to the nearest lane Note Before you can convert a contour into a band you must define the lane containing the band The contour boundary will change color from yellow to red and a band line will appear on the nearest lane Note Gaussian modeling and the Plot Band command do not work on contoured bands To list areas and quantities of contoured bands in reports select the Contour Area and Contour Qty report formatting options Display this informati
307. ouse button the border changes to a dashed blue line indicating a selected area e To reposition the scanning area box you have created position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move Toredo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right As you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later E 7 Quantity One User Guide E 4 Step Ill Select Resolution To select from a list of possible scanning resolutions click on the Select button under Step III Select Resolution This will open the Select Scan Resolution dialog box 1 PDQUEST x A 36 3 x 36 3 423 x 423 v 63 5 x 63 5 35 3 x 95 3 Select Scan Resolution High For small gels with tiny features D Medium high For gels with small features _ Done Medium For most gels ES Medium low For gels with large features 127 0 x 127 0 Low For gels with very large features 2 Help Fig E 7 Select Scan Resolution dialog box Available resolutions are listed from highest
308. overy Series 1 6 Chapter 1 Introduction 1 6 a Hardware Security Key HSK The Discovery Series software is password protected using a Hardware Security Key HSK which is included in your software package You must attach the Hardware Security Key to your computer before you can run the software Fig 1 3 Hardware Security Key Before proceeding with installation plug the HSK into any available USB port on your computer The code for the HSK is EY YCY which is printed on the key itself Use this code to identify the HSK that belongs to The Discovery Series software Note Initial installation of a network server requires the Hardware Security Key included in the software package Installation of an additional Network Client User to a Network License Server System does not require an HSK Please refer to the Network License Installation Guide that ships with Network License 1 6 b Installing The Discovery Series for Windows Note You must be a member of the Administrators group to install Discovery Series software Insert The Discovery Series CD ROM The installation wizard will start automatically If the CD does not auto start click Start in your taskbar then click Run In the Open field type d setup exe where d is the letter of your CD ROM drive Select the software application you want to install On each panel click Next when you are ready to proceed You must accept the license agreement to continue with instal
309. ow and uses it to replace the value of the pixel being processed Out of Range Pixel This filter is useful for suppressing salt and pepper noise its effect on Gaussian noise is minimal This filter calculates the mean of the pixel values in the filtering window including the pixel being processed If the difference between the mean and the individual pixel value is above a certain threshold then the individual value is replaced by the mean Median Also useful for suppressing salt and pepper noise this filter calculates the median value of the pixels within the filtering window and uses it to replace the value of the pixel being processed The median filter produces very little blurring if a small sized window is selected Maximum This filter is useful for eliminating pepper noise in an image it worsens the effect of salt noise It replaces the value of the pixel being processed with the maximum value of the pixels within the filtering window Minimum This filter replaces the value of the pixel being processed with the minimum pixel value within the filtering window This filter is useful for eliminating salt noise in an image it worsens the effect of pepper MidValue This filter is useful for suppressing uniform noise within an image however it worsens the effect of pepper and salt This filter replaces the value of 3 37 Quantity One User Guide the pixel being processed with the mean of the maximum and minimum pixel values
310. p If this checkbox is selected Quick Guides will always appear on top of images and never be obscured by them Quick Guide Placement and Toolbar Placement These checkboxes determine on which side of the screen the Quick Guides and toolbars will first open Placement Behavior This setting determines whether a Quick Guide or toolbar will always pop up in the same place and format Always Auto or whether they will pop up in the last location they were moved to and the last format selected Save Prior Toolbar Orientation These option buttons specify whether toolbars will first appear in a vertical horizontal or expanded format when you open the program 2 19 Quantity One User Guide Tool Help Delay and Persistence Specify the amount of time the cursor must remain over a toolbar icon before the Tool Help appears by entering a value in seconds in the Tool Help Delay field Specify the amount of time that the Tool Help will remain on the screen after you move the cursor off a button by entering a value in seconds in the Tool Help Persistence field 2 5 e Application Click on the Application tab to access the following preferences Relative Quantity Calculation The Relative Quantity Calculation option allows you to define how the relative quantities of defined bands in lanes will be determined for all reports histograms and band information functions either as a percentage of the signal intensity of an entire lane or a
311. pecify the amount of virtual memory allocated for the application at start up enter a value in megabytes in the Memory Allowance field The default value of 512 megabytes is recommended If you receive a warning message that the amount of virtual memory is set too high you can enter a smaller value in this field However this should be considered a temporary fix and you should consider expanding your hard drive Enable DOS Filename Parsing If this checkbox is selected for 8 character file names ending in two digits the final two digits are interpreted as version and exposure numbers For example the file name IMAGE 11 1sc would be parsed as IMAGE ver 1 xpo 1 1sc This is designed to 2 16 Chapter 2 General Information enable backwards compatibility for users with DOS image files You should only check this box if you are using these image files Enable UNIX Filename Parsing This is similar to DOS file name parsing Windows and Macintosh users are unlikely to run into difficulties with UNIX parsing therefore this setting is checked by default Maximize Application Window In the Windows version select the Maximize Application Window checkbox to automatically maximize the application window when Quantity One first opens If this is unchecked the menu and status bars will appear across the top of the screen and any toolbars will appear floating on the screen 2 b b Paths Click on the Paths tab to access the following pre
312. percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less than 1 percent of the pixels in your image saturated Consequently the default value for this field is 0 15 percent Reminder When this check box is selected the software will warn you to turn off your transilluminator light when you exit the Gel Doc XR acquisition window or when your system is idle for more than 5 minutes Print Footer Information The check boxes in this group allow you to specify the information that will appear at the bottom of your printouts Simple Acquisition Mode The Simple Acquisition mode option allows you to reconfigure the Gel Doc XR acquisition window to simple mode In simple mode the acquisition window contains the same steps as the advanced acquisition window with the exception of Step IV B 13 Quantity One User Guide Optimize display and Step V Analysis Manual exposure is also disabled in simple mode E Gel Doc XR Step Position Gel Live Focus IRIS ZOOM FOCUS 4 Open P Tele 4 Near Y Close BWwide Y Far Step Il Image Mode GUY C white Step IIl Acquire Image Auto o Expose Q Freeze Step IV Select Output Pin e Save Options 2 Help Fig B 10 Simple Acquisition Window To change the acquisition window to Simple Acquisition Mode check the box marked Simple Acquisition Mode The change will tak
313. ping Gaussian modeling can provide more accurate quantitation than regular band detection Gaussian modeling fits a Gaussian curve to each band profile and calculates band quantity from the area under the curve Since the profile of a well resolved distinct band conforms to the shape of a Gaussian curve this creates a band profile that is as close to ideal as possible Before After Fig 5 13 Profiles of two overlapping bands before top and after bottom Gaussian mod eling Modeling better resolves the band quantities Chapter 5 Bands For a band that overlaps with an adjacent band Gaussian fitting provides the best way to resolve the area that overlaps This quantity would be lost with conventional band detection Note Gaussian modeling requires little or no background in lanes Subtract lane background using the Lane Background command see section 4 2 Lane Based Background Subtraction prior to modeling Also high resolution images require significantly more time to model To reduce image resolution use the Reduce File Size command see section 2 2 f Reduce File Size To model bands using Gaussian fitting first detect the bands then select Gauss model Bands from the Band menu Note Gaussian modeling will not create bands or eliminate detected bands It will simply apply a Gaussian curve to the profiles of detected bands In the pop up box select All to model all lanes or One to model a sing
314. play window will open in live mode giving you a live video display of your sample If no image is visible make sure the camera is on check the cable connections make sure the iris on the camera is not closed and make sure that the protective cap is off the camera lens Also check to see that the transilluminator is on and working The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are six basic steps to acquiring an image using the ChemiDoc EQ 1 Position and focus the gel or other object to be imaged 2 Select Mode 3 Acquire the image C 3 Quantity One User Guide 4 Optimize the display 5 Analysis 6 Select the output C 2 Step Position Gel The ChemiDoc EQ window will open in live mode giving you a live video display of your sample In this mode the Live Focus button will appear selected and frames will be captured and displayed at about 10 frames per second depending on the speed of your computer You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note Newer versions of the ChemiDoc EQ feature a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens These buttons will not be visible if you are connected to
315. pop up buttons on the left side of the form offer a number of choices pertaining to the individual lanes on your gel Clicking on one will open the Lane Choices list Band set type standard or experimental Lane number Band set name Sample name Category attributes information see above for individual lanes Lane Report will open a customizable lane report form that can be printed or exported Assign Band Set allows you to select the specific band set that is to be applied to that lane Unassign Band Set allows you to remove the band set that is currently applied to that lane View Band Set displays the band set form for the band set that is currently applied to that lane Appendix K 21 CFR Part 11 K 1 Introduction Effective August 20 1997 the United States Food and Drug Administration FDA released Part 11 Electronic Records Electronic Signatures of title 21 of the Code of Federal Regulations This rule states the conditions under which the FDA considers electronic signatures and electronic records to be trustworthy reliable and equivalent to traditional handwritten signatures In this manner it defines the conditions under which an organization must operate to meet its record keeping and record submission requirements when using electronic signatures and records rather than handwritten records and signatures K 1 a CFR and The Discovery Series The purchase of a separate license allows the use of the 21
316. r arrays Note You cannot create a volume array in an image with asymmetric pixels i e different dimensions in x and y If you want to create a volume array in such an image select Reduce File Size from the File menu to change the pixel dimensions of the image see section 2 2 f Reduce File Size What Is a Volume Array A volume array is a matrix of volume circles or rectangles that can be sized positioned as a group and overlaid on images of blots wells or cells for easy quantitation The individual cells in the array have the same functionality as standard volumes You can define cells as background volumes standards and or unknowns as described in the sections above 7 12 Chapter 7 Volume Tools You report the array data as you would standard volumes using the Volume Analysis Report Creating a Volume Array On the Volume menu or toolbar select the Volume Array Tool This will open the Build Volume Array dialog Build Volume Array Type 96 wells 384 wells 1536 wells rows x cols TOWS cols Well Shape circular rectangular Cancel Fig 7 10 Build Volume Array dialog In the dialog you can select a standard microtiter plate dimension 96 wells 384 wells or 1536 wells or select Rows x Cols and enter the number of rows and columns in the array in the appropriate fields Select the shape of the wells cells Circular or Rectangular and click on OK The arr
317. r delete lanes using the single lane commands see section 4 1 c Single Lanes If Auto Frame Lanes does not work on the image you will be prompted to create a lane frame manually To delete the lane frame select Clear Analysis from the Edit menu Manual Frame Lanes If Auto Frame Lanes does not work with your images you can frame the lanes manually Select Frame Lanes from the Lane menu or toolbar In the dialog enter the number of lanes in the gel and click on OK E Quantity One x Number of lanes to create Cancel Fig 4 3 Frame Lanes dialog 4 3 Quantity One User Guide Corner anchor points Fig 4 4 Lane frame created using the Frame Lanes command The lane frame will be marked by corner anchor points with no interior anchors You can edit the frame as described below 4 1 b Editing the Frame If the frame is too large or small or does not follow the lanes on the image you can adjust it using the frame editing commands HOF Og puto eg Edit Frame Id Add Adjust Anchors Sind x Remove Anchors nge iam Move Frame Lane Width Rotate Frame Lane Background Resize Frame Stretch Frame Plot ane Fig 4 5 Edit Frame tools 4 4 Chapter 4 Lanes Adjusting the Entire Frame The following commands are located on the Lane Edit Frame submenu e To stretch the frame in one direction e g to encompass additional bands at the top or bottom of the image
318. r full network license If your network license is not activated when you click on Check License notify your network administrator HSK users have 30 days to purchase the software and obtain a purchase order number and software serial number from Bio Rad When you have this information click on the Check License or Registration Form button in the Software License screen to register your software 1 10 Chapter 1 Introduction Software License Registration Form p xj Please fill out the form below to authorize full use of your software If you have Internet access please send the form directly to Bio Rad by clicking on the Submit via Internet button This will immediately update your registration If you do not have Internet access you can print out this form and send it to Bio Rad NOTE There will be a delay while we update your registration You can either 1 Fax the form to Bio Rad at 1 510 741 5885 We will then e mail or fax you your passsword 2 Call 1 800 424 5723 x2601 inside U S or 1 510 741 2601 Intl and ask for Software Registration 3 E mail the contents of this form to LSG Software Registration Bio Rad com For network license registrations or software upgrade registrations please submit your request for a new password by email only Include your system ID purchase order number and the software catalogue number purchased along with the standard registration info in your message text Software U
319. r of exposures will be taken at regular intervals between the starting exposure time and the total exposure time Appendix I VersaDoc Optimize Exposure x Settings Total Exposure Time sec 20 0 Starting Exposure Time sec 1 0 Number of Exposures 5 O Save Images Cancel OK Fig I 7 Optimize Exposure dialog Note You should specify no more than 10 exposures in the Optimize Exposure dialog to avoid excessive build up of image background in later exposures The fewer the exposures the less background will be added to the image See the Release Notes for additional instructions on reducing background in images captured using Optimize Exposure Select the Save Images checkbox if you want to automatically save each exposure as it is taken Click on OK in the settings dialog to begin taking exposures If you selected Save Images a Save dialog box will open in which you can specify the base file name and location of the exposure files When you click on Save the exposures will be taken The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time The exposure status bar will show the progress of each exposure Depending on which dark subtraction type you have selected under Options a dark image may be acquired immediately following each exposure When each exposure is complete an image window containing that exposure will open in fr
320. r take multiple exposures using the Optimize Exposure feature described below Flat Fielding will be disabled UV Illumination Flat Fielding When you first select the Flat fielding checkbox and then acquire an image using the UV transilluminator you will be prompted to remove your sample and place the fluorescent reference plate on the VersaDoc sample stage see the VersaDoc User Manual A reference image of the plate will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a Flat Field corrected image White Illumination Flat Fielding When you first select the checkbox and then acquire an image using the white light conversion screen you will be prompted to remove your sample and collect an exposure of the conversion screen A reference image of the screen will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a Flat Field corrected image For subsequent UV or white light trans exposures you will be prompted to either use the appropriate saved Flat Fielding image or acquire a new one Any changes in light source filter or lens setting will require the acquisition of a new Flat Fielding reference image Acquire Click on the Acquire button to capture a single image for each enabled channel An exposure will be taken for each enabled channel based on the time selected for that channel in Step III The
321. redraw the image yourself In white light mode dragging the High slider handle to the left will make weak signals appear darker In UV mode dragging the High slider handle to the left will make weak signals appear brighter Dragging the Low slider handle to the right will reduce background noise You can also type specific High and Low values in the text boxes next to the sliders Clicking anywhere on the slider bars will move the sliders incrementally B 8 Appendix B Gel Doc XR Gamma Slider Some images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashion Adjusting the Gamma slider handle changes the light and dark contrast nonlinearly Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background and visa versa This will only affect how the image is displayed on the screen not the actual image data Auto scale Clicking on Auto scale will adjust your displayed image automatically The lightest part of the image will be set to the minimum intensity e g white and the darkest will be set to the maximum intensity e g black You can then fine tune the displ
322. rlay toolbar will also pop up to allow you to annotate your image The image will not be saved until you select Save or Save As from the File menu Analyze Clicking on Analyze will open a separate image window displaying the captured image The default name for the image will include the date time and user if known You can then analyze the image using the other features in the main application The image will not be saved until you select Save or Save As from the File menu A 7 Step VI Select Output In Select Output you can select Video Print and Save as your output options Step VI Select Output amp Video Print Save Fig A 8 Output options Video Print Clicking on Video Print will automatically send the currently displayed frame either live or integrated to a video printer You can add information about your image to the A 10 Appendix A Gel Doc bottom of the printout by selecting the appropriate checkboxes in the Options dialog box See section A 9 Options Save Clicking on Save will open a separate image window displaying the captured image A Save As dialog box will automatically open displaying the default file name for the image which will include the date time and user if known You can then change the file name and storage directory You can also export your image as a TIFF image for viewing with other applications A 8 Exposure Status The Exposure Status bar shows the progres
323. rm command Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox Warn on Saturated Pixels The Warn on Saturated Pixels option is checked by default Uncheck this box to disable this warning Filters The PharosFX family of imagers allows you to reposition filters Click Filters to open the PharosFX Filter Placement dialog box E PharosFX Filter Placement Filter wheel Filter wheel B Filter position 1 Blank Filter position 1 605nm BP Filter position 2 380nm BP Change Filter position 2 Blank Change Filter position 3 640nm BP Change Filter position 3 Blank Change Filter position 4 Blank Change Filter position 4 Blank Cancel OK Restore Defaults Fig 8 12 PharosFX Filter Placement H 16 Appendix H FX Use the drop down boxes to change the filters in each position in Filter wheel A and Filter Wheel B Note that Position 1 in Filter wheel A and Positions 1 and 4 in Filter wheel B cannot be changed To create a custom filter click Custom in the filter type list Enter a name for the filter then click OK The name of the custom filter will appear in the chosen filter position Blank 390nm BP 530nm BP l 555nm LP Filter wheel 4 605nm BP Filter position 1 Blank 640nm BP Filter position 2 530nm BP 4 695nm BP Filter position 3 B40nm BP Filter position 4 Blank E PharosFX Filter Placement Cancel OK Cancel OK
324. rols will appear active but instead of capturing real images the window will create simulated grayscale images You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated H 1 PharosFX Acquisition Window To acquire images using the PharosFX go to the File menu and select PharosFX The acquisition window for the imager will open displaying the control panel for the imager and the scanning area window H 2 Appendix H FX Molecular Imager PharosFX Step Select Application y Channel 1 Channel 2 y Channel 3 y Channel 4 3 Select Radioisotopes K Screen Kodak Lowest 532nm EX Blank 390nm BP Step Il Select Scan Area Click and drag in diagram to set scan area Top B Left 1 Bottom a Right i xj Quadrant Mode Step Ill Select Resolution 50 micrometer 100 micrometer 200 micrometer 800 micrometer Image file size 0 00 Mb Q Acquire 9 Options Auto Save After Scan Highlight Saturated Pixels Hide Grid Filters x Wam On Saturated Pixels Info Help iy ahh deb tke En Gee ais Fig H 2 PharosFX acquisition window The default scanning window is marked by grid lines that divid
325. rom an image including any lanes bands volumes standards text overlays etc select Clear Analysis from the Edit menu Since this process is irreversible you will be prompted to confirm the selection 3 15 Sort and Recalculate To update renumber and recalculate all lane and band information select Sort and Recalculate from the Edit menu 3 16 Automation Manager The Automation Manager allows you to save objects such as lanes automated band detection standard bandsets volume overlays and text and line overlays in a template file These files can then be automatically applied to images either individually or in batches To open the Automation Manager select Analysis Automation Manager 3 42 Chapter 3 Viewing and Editing Images Automation Manager 5 pl Een dg MM i Protein scr Colony scr L New Open 55 Close Colony scr x Use Single Image For Template r2 Edit Selected Template Proteins test Raw 1 D Image Modified Options JLanes uto Detect Bands x Apply Standards x Text amp Line Overlays Volume Overlays Template Saved r3 Select Images Proteins test Raw l D Ima KE n Select All Save Save s wf Default Apply r4 Apply Selected Template Close Window 4l Help Fig 3 26 Automation Manager dialog 3 16 a Step 1 Select Templat
326. ropriate reference plate to ensure a uniform intensity in the image This will compensate for normal variations in image pixel intensity that occur with a transilluminating light source D 9 Quantity One User Guide To enable this feature select the Flat Fielding checkbox UV Illumination Flat Fielding When you first select the Flat fielding checkbox and then acquire an image using the UV transilluminator you will be prompted to remove your sample and place the fluorescent reference plate on the ChemiDoc XRS sample stage and turn on the UV transilluminator see the ChemiDoc XRS User Manual x AN Illumination Flat Fielding Remove your sample from the ChemiDoc XRS Continue X Cancel Fig D 9 Illumination flat fielding reminder A reference image of the plate will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a flat field corrected exposure White Light Illumination Flat Fielding When you first select the checkbox and then acquire an image using the white light conversion screen or the white light transilluminator you will be prompted to remove your sample turn on the white light source and collect an exposure of the white light screen transilluminator see the ChemiDoc XRS User Manual A reference image of the screen transilluminator will be acquired and saved on your computer hard drive The reference image will be applied to the sample imag
327. rpolation in the dialog Next indicate whether the curve should be extrapolated beyond the highest and lowest known values by selecting Yes or No next to the Extrapolation prompt Note that values extrapolated from the Point to Point curve may be unreliable 6 28 Chapter 6 Standards and Band Matching Displaying the Calibration Curve To display the calibration curve click on the Show Curve button at the bottom of the Quantity Standards dialog A graph of the known quantities versus measured intensities will be displayed in a separate window Calibration Curve Cal 1 3 00 Quantity 0 000 0 500 100 150 200 250 300 350 400 450 Trace Density OD x mm G Known Lin 7 Check Titles Print J Done J Fig 6 23 Calibration curve with linear regression and extrapolation Known values used to calculate the curve are marked by circles Values identified as outliers are marked by squares e To change the status of a point on the graph click on it The status will toggle between Known and Outlier e To display a legend at the bottom of the graph click on Titles e To print the graph click on Print e To close the graph window click on Done 6 29 Quantity One User Guide 6 3 c Applying the Calibration Curve After you have generated a calibration curve you are ready to calculate the quantities of the unknown bands Select a button next to Calibrate to calculate the quantity of an individual band match lane
328. rs and number of pixels and the file size of the image inside the crop area are listed at the bottom of the crop box 1 To reposition the crop box position the cursor at the center of the box The cursor will change to a multidirectional arrow Then drag the box to a new position 3 24 Chapter 3 Viewing and Editing Images 2 To resize the box position the cursor on a box border line or corner The cursor will change to a bidirectional arrow You can then drag the border or corner in or out resizing the box 3 To redraw the box position the cursor outside the box The cursor will change back to the Crop tool and you can redraw the box After you are satisfied with the size of crop box position the cursor inside the box slightly off center The cursor will change to a scissors symbol Then click to perform the crop A pop up box will prompt you to 1 crop the original image 2 crop a copy of the original image or 3 cancel the operation Quantity One x Ready to crop Image 3 1D Scan Crop current image or crop copy of image If vou want to crop multiple pieces from the image or there is some analysis pou want to preserve create a copy of the image then crop Crop Cancel Fig 3 17 Crop box and pop up Crop dialog If you click on the Copy and Crop button you will be prompted to enter the name and version number of the image copy before cropping 3 25 Quantity One User Guide
329. s deassigned The second status box is designed to supplement Tool Help see above It provides additional information about the toolbar buttons If you hold your cursor over a 2 2 Chapter 2 General Information button a short explanation about that command will be displayed in this second status box 2 1 d Secondary Toolbars Secondary toolbars contain groups of related functions You can open these toolbars from the main toolbar or from the View Toolbars submenu The secondary toolbars can be toggled between vertical horizontal and expanded formats by clicking on the resize button on the toolbar itself Expanded format Image Tools 2 2 Horizontal Flip 2 Rotate 90 Left Rotate 90 Right 2 Custom Rotation 2 Filter wizard 2 Crop Vertical Flip Rotate 180 Li LB P es 80 les st Let Invert Data Horizontal format Image Tools x Click on resize button gg to toggle format Vertical format Hold cursor over icon to reveal the tool tip Click on question marks for on line help Fig 2 3 Secondary toolbar formats and features The expanded toolbar format shows the name of each of the commands Click on the icon next to the name to display on line Help for that command 2 3 Quantity One User Guide 2 1 e Quick Guides The Quick Guides are designed to guide you through the major applications of the software They are
330. s a percentage of the signal intensity of the defined bands in a lane Selecting of Lane means that the total intensity in the lane including bands and the intensity between bands will equal 100 percent and the intensity of a band in that lane will be reported as a fraction thereof Selecting of Bands in Lane means that the sum of the intensity of the defined bands in a lane will equal 100 percent and the intensity of an individual band will be reported as a fraction of that sum If you create adjust or remove bands in a lane with Relative Quantity defined as of Bands in Lane the relative quantities of the remaining bands will be updated Relative Front Calculation The Relative Front Calculation option lets you select the method for calculating the relative positions of bands in lanes This affects the calculation of both Relative Front and Normalized Rf values Relative front is calculated by either 2 20 Chapter 2 General Information 1 Dividing the distance a band has traveled down a lane by the length of the lane Follow Lane This is useful if your gel image is curved or slanted 2 Dividing the vertical distance a band has traveled from the top of a lane by the vertical distance from the top of the lane to the bottom Vertical Note Lane and band refer here to lanes and bands as defined by overlays on the gel image For example the top of a lane refers to the beginning of the lane line created in Qu
331. s been Gauss modeled the modeling will be automatically removed from that lane The Gaussian models in a lane are interdependent so changing a single band invalidates the modeling After you have adjusted the bands you can remodel the lane Incorrect Modeling The Gauss model Bands command will try to model all the bands in the selected lanes Carefully review the results of modeling using the Plot Band Bands in Lane and Band Information commands as described above If the Gaussian curve does not adequately conform to the profile of a band or if the Gaussian peak and trace quantities differ greatly from the normal peak and trace quantities in the Band Information dialog it may be because there is too much lane background Use the Lane Background command with a smaller rolling disk size to remove more background then remodel the lane If Gaussian modeling does not work well with the bands in the image simply remove the modeling To remove the modeling from a particular lane select Remove One Gauss model from the Band menu and click on the lane To remove the modeling from all lanes select Remove All Gauss models Removing Gaussian modeling will not affect band detection 5 20 Chapter 5 Bands 5 8 Irregularly Shaped Bands in Lanes If some bands in lanes are irregularly shaped you can use the contour or drawing features to define them These functions give you more control over defining bands than either Detect Bands or Create
332. s faint colonies e g O D 0 05 counts 2 000 you may want to increase this value to 20 00 Quantity One User Guide Averaging Averaging is designed to prevent random signal noise such as salt or pepper in the image from being detected as colonies If the image is noisy you should select the highest value that still results in good separation of colonies default 3 A low averaging value may result in noise being detected as colonies A high averaging value may result in two closely spaced colonies being counted as one 8 3 Displaying the Results When you click on Count the number of detected colonies will appear in the Results section of the dialog in the White column rStep 2 Adjust Results White Blue Colony count 1186 Adjusted count 1186 o Count vs Peak density Cutoff ju White Blue zz Fig 8 3 Example of a dish with white colonies The colonies will also appear marked as gold triangles on the image itself Note Ifthe colonies are not marked on the image make sure that the Mark White Colonies checkbox at the bottom of the dialog is checked A text box on the image will indicate how many colonies were detected on the image Chapter 8 Colony Counting Doing a Recount To recount using different parameters change the Sensitivity and or Averaging settings This will erase the old count Then click on Count again to recount Redrawing the counting region circle or clicking on
333. s of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical representation of the remaining time before exposure is complete If the exposure time is less than 1 second the status bar will not refresh itself for each exposure it will remain at 100 percent A 9 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your Gel Doc EQ system Quantity One User Guide DAC Settings Min so Max 130 Imaging rea cm 4 Width 20 00 Height 15 00 r Auto Exposure Threshold Saturated Pixels o 15 r Reminder xj Show Light Warning Show Filter Warning r Video Printing Footer Information x File Name x Date Time X Exposure Time x Contrast Settings r Save Options Make Backup Copy r Misc Options J Simple Acquisition Mode Defaults Cancel OK Fig A 9 Available options in the Gel Doc EQ acquisition window Click on OK to implement any changes you make in this box Clicking on Defaults restores the settings to the factory defaults DAC Settings Note The default DAC settings are highly recommended and should be changed with caution These sliders may be used to adjust the minimum and maximum voltage settings of your video capture board The minimum slider defines the pixel value that will appear A 1
334. se in the image Next select the size of the filter to use on that noise Finally filter the image Select Filter Wizard from the mage menu or toolbar to open the dialog 3 34 Chapter 3 Viewing and Editing Images Filter Wizard for Proteins 1D Scan Step 1 Identify Noise Characteristics Study the density distribution histogram below and select the shape you see x Pepper us x Salt Look at the density distribution plot for a background region of the image and select the shape of the plot you see Gaussian Gaussian outlier Uniform Outlier LU Pul Density Distribution Histogram Step 2 Select Filter Size Select a size in pixels that is larger than the average noise feature 0383 5435 C 74 939 Filter selected Median 3X3 OK Cancel Help Fig 3 22 Filter Wizard dialog The dialog contains settings for identifying the different types of noise in the image It also includes a density distribution histogram of the noise in the image to aid in filter selection Step Identify Noise Characteristics The first step is to identify the type of noise in the image Examine both the image and the density distribution histogram then select one both or neither of the following checkboxes e Salt This type of noise appears as specks that are lighter than the surrounding background The density distribution histogram of this type of noise displays noise p
335. select Stretch Frame from the submenu or Lane toolbar and drag an anchor point in or out The opposite anchor point will remain fixed while the frame expands or contracts e To move the entire frame to a new position select Move Frame from the submenu and drag an anchor point The entire frame will move e To rotate the frame select Rotate Frame from the submenu and drag an anchor point The entire frame will rotate e To resize the entire frame select Resize Frame from the submenu and drag an anchor point in or out The frame will expand or contract from the center Adding and Adjusting Frame Anchors To adjust a corner anchor point select Add Adjust Anchors from the Edit Frame submenu or toolbar and drag the anchor This will move the anchor point and attached frame lines If the gel lanes are not straight you can create additional anchor points along the frame to change the shape of individual lines Still using Add Adjust Anchors click anywhere on the frame lines This creates interior anchor points both where you clicked and on the other side of the frame connected by a frame line Then drag the anchor points to bend the frame Add and adjust as many anchor points as you need to bend the lane lines to follow the lanes in the gel 4 5 Quantity One User Guide New anchor points New anchor lines E Fig 4 6 Adjusting the anchor points of the lane frame Removing Unadjusti
336. ser Company or Institution Purchase Information Software and System Dr Mr Ms First Name Last Name Phone Fax E mail Submit via Internet amp Print ET Close Fig 1 6 Software License Registration Form Fill out the information in the Software License Registration Form Be sure to enter your purchase order number and software serial number under the Purchase Information tab when registering 1 7 a Registering by Internet If you have Internet access from your computer click on the Submit via Internet button to send the Software Registration Form directly to Bio Rad Your information will be submitted and a temporary password will be generated automatically and sent back to your computer Simply continue to run the application as before Bio Rad will confirm your purchase information and generate a permanent license After 2 3 days click on Check License in the Software License screen again to update to a permanent password The Software License screen will not appear automatically after the temporary password has been generated the software will 1 11 Quantity One User Guide simply open normally Go to the Help menu and select Register to open the Software License screen 1 7 b Registering by Fax or E mail If you do not have Internet access click on the Print button in the Software License Registration Form and fax the form to Bio Rad at
337. sition mode Reset Referenced Dark Auto Exposure Threshold Saturated Pixels 0 15 Cancel OK Fig B 9 Available options in the Gel Doc XR acquisition window Click OK to implement any changes you make in this box Information This field describes the instrument model and the camera serial number Imaging Area These fields are used to specify the size of your imaging area in centimeters which in turns determines the size of the pixels in your image i e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images B 12 Appendix B Gel Doc XR Save Options To automatically create a backup copy of any scan you create select the Make Backup Copy check box With this check box selected when you save a scan a backup copy will be placed in the same directory as the scanned image Backup images are identified by Backup of followed by the original image file name Note Backup files are read only files If you attempt to save a backup file you will be prompted to give it a new name This protects the backup file and preserves it from any changes Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the
338. ss than the number of screen pages listed in the report window This is because the print command reformats the data to make maximum use of the paper size Exporting Report Data You can export the report data to a spreadsheet application for further computation and analysis Click on the Export button to open the Export Report dialog Export Lane Report Field Separators Commas 1 2 3 format Tabs Excel format Export Destination File Clipboard Cancel Help Fig 12 4 Export Report dialog Exported data can be separated by commas or tabs depending on the requirements of your spreadsheet application You can save the data to a text file or to the clipboard by selecting the appropriate option button If you select File when you click on OK a dialog will open in which you can save the file Reformatting Report Data Click on the Reformat button to open the options dialog for the report and select different display options This button is not available in all reports 12 4 Chapter 12 Reports 12 2 Lane and Match Reports There are four different lane and match reports Lane Report All Lanes Report Match Report and All Matches Report Lane Report generates a report on any you lane you select First select the report from the Reports menu then click on the particular lane All Lanes Report generates a report on all the lanes in the current gel image Match Report generates a report on a matched
339. ssing the light lane improving the detection of faint bands Quantity One User Guide Select No next to the Normalize prompt to apply the band detection parameters in the same way to every lane in the gel image Select Yes to normalize for the intensity of the lane Shadow Rejection Shadow bands are common gel artifacts Shadow bands are spaced at tandem repeat intervals and decrease in intensity as they progress further from a real band The Shadows parameter is designed to limit the detection of shadow bands see also Band Limit Select Reject to turn on the shadows filter A band will detected only if it is darker than the one above it or spaced further than one tandem repeat unit from the previous band This greatly reduces the number of shadows identified as real bands Select Accept to turn off the shadows filter Band Limit If you know that all the lanes in a gel contain a specific number of bands you can click on the Limit button next to the Bands prompt and type in the number of bands that you know are present Only that number of bands will be detected in each lane reducing the need for later editing Sensitivity The Sensitivity setting determines the minimum signal intensity in the image that will be defined as a band The higher the sensitivity value the more bands will be detected If the sensitivity is set too high background noise may be detected as bands If the setting is too low real bands may be missed
340. t favors the most recent member clusters in forming new clusters Centroid Median dy 05 d j 05 d 025 d Centroid and Median are similar to UPGAMA and WPGAMA respectively but the distance formula contains an additional third term Centroid and Median methods are not monatomic hierarchical clustering algorithms In other words the similarity value between cluster k and any other cluster may be greater than the similarity between cluster p and cluster q This condition occurs if the centroids or medians of the different clusters have approximately the same distance as the distances between the samples that make up the cluster Ward s This method attempts to minimize the information by describing a set of N samples using a fewer number of clusters FEES d E M nj ki n n P nen nen P4 12 17 Quantity One User Guide Options Click on the Options button in the PAylogenetic Tree window to open the Options dialog Align Right Proportional Align Right Proportional xj Fit Tree in Window x Fit Tree in Window Restore Original Tree Restore Original Tree _ Designate New Root Clusters fo Min E AII Other Methods Max 17 8 Neighbor Joining Only Fig 12 13 Two forms of the Options dialog The box on the left has controls that are specific for the Neighbor Joining method The box on the right is applicable to the other tree methods Both boxes share certain features
341. t removes small noise features on an image while leaving larger features e g bands relatively unaffected A wide range of filters are available for removing different types of noise from images Depending on the nature of your data you will probably need to use only one or two of the available filters However you should experiment with several different filters before selecting the ones that work best for your images The filtering commands are located on the Image menu and toolbar 3 33 Quantity One User Guide Image Lane Band Match Volume Image Tools x Holme a Transform Ctrl B o 3B q Crop Advanced Crop Vertical Flip Horizontal Flip Rotate Subtract Background Filter Wizard L Filter List Weighted Mean Out of Range Pixel Median Maximum Minimum MidValue PowerMean ContraMean Adaptive Fig 3 21 Filtering commands Invert Data Note Since filtering is an irreversible process you will be asked if you want to create a copy of the original image before you filter If you are experimenting with various filters you should create copies of the image and compare them side by side If you filter the original image and save it you cannot return to the original unfiltered state 3 11 a Filter Wizard The Filter Wizard is designed to guide you through the filter selection process First you identify the type of noi
342. tandard values to the image you are ready to select the regression model to use to calculate the values of the unknown bands Note You must apply the values to a lane before you can view and adjust the regression curve Quantity One User Guide Click on the Standard Curve button in the Standards dialog then click on a lane A graph of the standards regression curve is displayed on the image and the Standard Curve Options dialog is displayed as well Regression Method Elder Southem Std Curve Options for Image 1 Correlation Coefficient N A a DE SREE ne Log Mol Wt r Regression Model r Display Options interpo le 3 40 3 60 f Point to Point Semi Log x Show Standard Points Elder Southem X Show Calculated Points lt Logistic x Perform Log Transform Linear x Show Numerical Data of Points Quadratic x Display Correlation Coefficient Cubic Cubic Spline Close Help Fig 6 10 Displaying the standard curve and options dialog As you click on different lanes the curve is displayed for each lane in turn The X axis is the standard value and the Y axis is the Rf value Use the Std Curve Options dialog to change the regression model for the curve as well as various display options Standards Regression Models Select different regression models in the Std Curve Options dialog while you study the standard curve with the standard points displayed Then choose a curve that
343. tc Values can be displayed and printed in report format using the lane and match reports on the Reports menu 6 1 f Saving Opening and Deleting Standards Standards are saved with the image you can also save copies of them in a separate archive that is available to all images To save the standard values with the image click on the Close button to close the Standards dialog then save the image When you open the image again the standards will be available when you select Standards To save these standards for use on other images click on the Archive button The standards will be saved in an archive file separately from the image To use a set of archived standards open any image then choose Standards from the menu or toolbar and select the archived standards They will be imported into the image 6 12 Chapter 6 Standards and Band Matching To delete a set of standards you have created open them then click on the Delete button at the bottom of the dialog A pop up box will ask you to confirm that you want to proceed with the deletion before completing the action This will delete the standards from both the image and the archive To modify a set of standards you have created open them make your changes then save the image and if desired archive the new standards Read Only Standards You can make archived standards read only Read only standards cannot be deleted from or modified in the archive using the methods des
344. ted volume labels However if you change the type for a particular volume this will renumber the subsequent volumes without changing the text or style of the User label If you want to remove the User label and return the Auto label for the selected volume click Use Auto Label Note that changes do not take effect until you click Done So if you find you want to keep the User label click Cancel instead Editing Auto Labels To modify the style of the Auto labels click Edit Auto Labels Like the Edit User Label tool this opens the Text Overlays Properties dialog box Use the options in 7 6 Chapter 7 Volume Tools the Text Overlays Properties dialog box to modify the style of the Auto labels Note that you must first select the text in the text field before making any changes Note X Editing the Auto labels affects only the style of the labels not the text or numbering of the Auto labels Modifying the style of your Auto labels does not affect the style of any existing User labels To modify the styles for your User Labels you must modify each individually See Editing User Labels for further information When you first create volumes they appear labeled Click Show Hide Volume Labels on the Volume menu or toolbar to hide the labels 7 3 Volume Features Each new volume you create initially has a green border which indicates that the volume is selected If you click elsewhere on the image the border will change to blue
345. the band s in one gel and the same area will be magnified in all the other gel images Note that the images must be approximately the same size AutoScale Annotations Select AutoScale Annotations if you want to display annotations in the image as a percentage of the image window not the image This allows you to zoom in on an area of the image but have displayed annotations remain the same size in the image window Disable hardware Acceleration for 3D Viewer If you are having problems using the 3D Viewer install the latest drivers for your graphics card If after updating your drivers you are still having problems select Disable hardware acceleration for 3D viewer This allows Quantity One to emulate a graphics driver to render a 3D image Note Disabling hardware acceleration reduces performance for the 3D Viewer 2 18 Chapter 2 General Information 2 5 d Toolbar Click on the Toolbar tab to access the following preferences which determine the behavior and positioning of the secondary toolbars and Quick Guides Show Default Quick Guide at Startup If this checkbox is selected the Volumes Quick Guide will open automatically when you open the program If you are in Basic mode the Basic Quick Guide opens instead Align Quick Guide with Document If this checkbox is selected the Quick Guides will open flush with the edge of your documents Otherwise they will appear flush with the edge of the screen Guides Always on To
346. the histogram and the Cutoff slider to correct this Drag the Cutoff slider to the right until it is centered on the right edge of the background peak Background peak Count vs Peak density Cutoff mark Cutoff Fig 8 5 Using the Cutoff slider The yellow portion of the bar beneath the histogram marks the range of image data has been designated as background noise and is not being considered for colony counting purposes The gold portion of the bar marks white colony data range Chapter 8 Colony Counting The colony count displayed in the dialog and on the image should decrease On the image you should also see the incorrectly identified colonies disappear as you drag the slider White and Blue Colonies If you know you have white and blue colonies in the image and there are two clear peaks on the histogram to the right of the background peak you can use the histogram to distinguish between these types of colonies Results White Colony count a 3 Adjusted count 93 White peak Blue peak Count vs Peak density Cutoff White blue division mark Fig 8 6 Using the White Blue slider Drag the White Blue slider to the left until it is positioned between the two peaks The white colony data range is indicated by gold on the bar beneath the histogram and the blue colony data range is marked with blue As you drag the slider the numbers of white and blue colonies will change in the dialog and
347. to 1 0 4 13 Quantity One User Guide Adding and Removing Lanes from the Graph To add a lane to the graph click directly on the lane in the image The plot of the lane will appear in the graph Each lane you add will be displayed in one of eight colors and will be identified by color in a legend underneath the graph If you add more than eight lanes the colors will repeat but each lane will still be identified underneath the trace display There is no limit to the number of lanes that can be displayed simultaneously To remove a lane profile click on the Remove Lane button A pop up box will prompt you to select the lane to remove If you remove a lane the colors of the remaining lanes will change Check the lane legend for an updated color code Magnifying the Graph Use the Zoom In and Zoom Out buttons in the dialog to magnify regions of interest in the profiles Alternatively drag the cursor horizontally across the graph and release the mouse button to magnify the defined range Note The magnifying functions in Compare Lanes only magnify the profile in the direction of the X axis Therefore the profile will appear to stretch without increasing in height The Full View button returns the graph to its default display If you have magnified part of the graph the Left arrow and Right arrow scroll buttons can be used to pan left or right along the graph Show Gaussian Modeling The Show Gauss Model checkbox is active i
348. ty arrays samples with very closely spaced bands Files scanned at 50 micrometers can be very large e 100 micrometer resolution should be used for typical gels and arrays e 200 micrometer resolution is useful for gels with large bands and dot blots e 800 micrometer resolution should be reserved for very large objects such as CAT assays F 5 Quantity One User Guide File Size of Images Image File Size below Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance F 4 Acquire the Image Once you have selected your scan area and resolution you ready to acquire an image Click on the Acquire button There may be a short delay while the image laser warms up then the scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected Note Ifthe image you are scanning has more than 10 saturated pixels you will receive a warning message
349. ty of the pixel Signal intensity of a single pixel Intensity r 3 D View 2 D View Fig 1 1 Representation of the pixels in two digitally imaged bands in a gel For a data object to be visible and quantifiable the intensity of its clustered pixels must be higher than the intensity of the pixels that make up the background of the image The total intensity of a data object is the sum of the intensities of all the pixels that make up the object The mean intensity of a data object is the total intensity divided by the number of pixels in the object The units of signal intensity are Optical Density O D in the case of the GS 700 imaging densitometer GS 710 calibrated imaging densitometer and GS 800 calibrated densitometer the Gel Doc EQ system ChemiDoc EQ system ChemiDoc XRS system with a white light source or the Fluor S Multilmager system Fluor S MAX MultiImager system Fluor S MAX2 Multilmager system and VersaDoc imaging systems with white light illumination Signal intensity is expressed in counts when using the Personal Molecular Imager system or the Molecular Imager FX system Molecular Imager FX Pro fluorescent imager 1 2 Chapter 1 Introduction Molecular Imager FX Pro Plus multiimager system or in the case of the Gel Doc EQ ChemiDoc EQ ChemiDoc XRS Fluor S Fluor S MAX or VersaDoc when using the UV light source 1 3 Gel Quality Quant
350. u and click Export to JPEG Image This opens the Export to JPEG Image dialog box BITUITTTITIDS r Export Mode ER Quality p 50 X Cancel 2 Help Fig 13 4 Exporting to JPEG In the Export to JPEG dialog box under Export mode use the slider to adjust the image quality The higher the quality the larger the file size Click Export to complete the operation The default file name and location are based on the original image You can change the file name or select a different folder to save in Click Save to complete the export 13 8 Appendix A Gel Doc EQ Fig A 1 Gel Doc EQ Before you can begin acquiring images the Gel Doc EQ system must be properly installed and connected with the host computer See the Gel Doc EQ hardware manual for installation startup and operating instructions A 1 Quantity One User Guide To use the Gel Doc EQ you will need to have the Bio Rad supplied acquisition board installed in your PC or Macintosh The drivers for this board will be installed when you install the main software application Make sure that your Gel Doc EQ camera is turned on If the camera is not turned on the Gel Doc EQ acquisition window will open and an image capture error will be displayed Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real
351. uce File Size pop up box 2 3 Imaging Device Acquisition Windows The File menu contains a list of Bio Rad imaging devices supported by Quantity One These are 1 Gel Doc EQ 2 ChemiDoc EQ 3 ChemiDoc XRS 4 GS 800 Calibrated Densitometer 2 14 Chapter 2 General Information SU vow 8 VersaDoc Personal Molecular Imager FX Molecular Imager FX PharosFX To open the acquisition window for an imaging device select the name of that device from the File menu See the individual chapters on the imaging devices for more details 2 4 Exit To close Quantity One select Exit from the File menu You will be prompted to save your changes to any open files 2 5 Preferences You can customize basic features of Quantity One such as menu options display settings and toolbars using the Preferences dialog box Select Preferences from the Edit menu to open this dialog 2 15 Quantity One User Guide E Misc Paths Display Toolbars Application Imagers Security Memory Allowance ERT RET O Enable DOS filename parsing Maximize application window x Enable Unix filename parsing Y OK X Cancel 2 Help Fig 2 13 Preferences dialog box Click on the appropriate tab to access groups of related preferences After you have selected your preferences click on OK to implement them 2 5 a Misc Click on the Misc tab to access the following preferences Memory Allowance To s
352. uisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated D 1 ChemiDoc XRS Acquisition Window To acquire images using the ChemiDoc XRS go to the File menu and select ChemiDoc XRS The acquisition window for the instrument will open displaying a control panel and a video display window D 2 Appendix D ChemiDoc XRS E chemioe xes 9 xi Step Select Application Select White Transilluminati White TRANS 2x Gain 1x1 Bin r Step Il Position Gel Live Focus IRIS ZOOM FOCUS 0000 t Tee res 3 Show Alignment Grid r Step III Acquire Image Auto Manual Live OE nose OE spose Q acquire pe a X Flat Fielding Q Freeze Display Options Highlight Saturated Pixels Image file size 255 Mb Options Z Help r Exposure Status Fig D 2 ChemiDoc XRS acquisition window When the ChemiDoc XRS window first opens no image will be displayed The control panel has been arranged from top to bottom to guide you through the acquisiti
353. ular application To select from the list of applications click on the Select button under Step I Select Application E 4 Appendix E GS 800 X ray film Gel Photograph Coomassie blue Silver stain Copper stain Zinc stain Stain all Blot Fig E 3 Example of the application tree in the GS 800 dialog box The applications are listed in a tree that expands from left to right First you select the category of your application then you select your particular application To exit the tree without selecting press the Esc key Gray file X ray film Blue film Linear r Coomassie Blue Silver stain Gel L Copper stain Zinc stain Stain all r Positive dark bands light background Photograph _4 L Negative light bands dark background Coomassie stain Colloidal gold stain Amido black stain AP Substrate BCIP NBT HRP Substrate 4CN HRP Substrate DAB Blot Fig E 4 Applications available in the GS 800 acquisition window E 5 Quantity One User Guide Choosing Your Own Settings If you know the filter and light source settings you want or want to experiment with different settings you can choose them yourself Filter Red x Green Blue White Light Reflective Transmissive Fig E 5 GS 800 Custom Application controls Next to Filter click on either the Red Green Blue or White che
354. urve for quantitating unknown bands These tools are found on the Match menu and Match Tools toolbar Match Volume Analysis Reports Match Tools Lx Standards Standard Curve Match Unmatch Outlier Normalize Matched Band Set Show Band Types Shift F5 Show Band Models Ctrl F5 Match Graphs gt Fig 6 1 Standards and matching tools 6 1 Standards If your gel includes lanes of standards you can enter the values of the standards and automatically calculate the values of the unknown bands in the gel Quantity One User Guide Note In general the more standard lanes in a gel the greater the accuracy of the calculated band values We recommend a minimum of two standard lanes per gel Also the modeling algorithm works best with standard lanes spaced evenly across the gel as shown in the following figure Multiple standard lanes also facilitate comparisons of sample similarity Standard Lane Standard Lane Standard Lane 2 Image 2 1D Scan t Fig 6 2 Standard lanes as defined on a gel image With the image open and lanes and bands defined select Standards from the Match menu or toolbar A dialog will open listing any saved standards as well as predefined Bio Rad standards Chapter 6 Standards and Band Matching Quantity One Ea 2 Select Standards New Standard S Protein S Bio Rad DNA 1 kb Molecular Ruler S Bio Rad
355. usible clusters and are affected the least by samples that are outliers 12 14 Chapter 12 Reports The report window for a phylogenetic tree is slightly different than the standard report window The tree appears in a window with scroll bars for moving up and down and left and right To print the tree click on the Print button Click on the Cluster Method button to select a different clustering method for displaying the tree This will automatically recalculate and redisplay the tree Neighbor Joining This type of phylogenetic tree is computed is based on minimizing the total branch length at each stage of clustering The method also finds branch lengths between nodes The approximate distance between any two samples in this tree can be found by adding the branch lengths that connect the samples Other Methods 2 The following methods are based on the algorithm below 1 Begin with n clusters one cluster for each sample 2 Compute the similarity matrix for the samples 3 Convert the similarity matrix into a distance matrix d using the appropriate distance formula 4 Join the two clusters with the minimum distance into one cluster Compute the similarity value for this cluster 5 Recompute the distance matrix d using the cluster that was formed in Step 4 Steps 4 and 5 are repeated until there is only one cluster The difference between the methods below is based on the definition of minimum distance in Step 4 and on t
356. ut instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated C 1 ChemiDoc EQ Acquisition Window To acquire images using the ChemiDoc EQ go to the File menu and select ChemiDoc EQ The acquisition window for the instrument will open displaying a control panel and a video display window Appendix C ChemiDoc Chemi Doc r Step Position Gel Live Focus IRIS Z00M FOCUS 4 Open 4 Tele 4 Near Close Y Wide Y Far C Show Alignment Grid r Step Il Image Mode UV White Chemi r Step III Acquire Image Auto Manual Live Qo Qi Q cure nr Time sec 34 Fa ee O Freeze r Step IV Optimize Display High 255 o Low fo A Gamma 1 00 X Highight Saturated Pixels Step V Analysis y Step VI Select Output Exposure Status Llnvert Display 53 Annotate Video Print Auto scale Reset z Autoscale Ree Analyze Save Options 2 Hep Fig C 2 ChemiDoc EQ acquisition window The ChemiDoc EQ video dis
357. utton in the Densitometer Options dialog box You can also specify how often you want the GS 800 to automatically recalibrate Either click on the Calibrate Before Every Scan checkbox or specify a period between automatic recalibrations in minutes in the Recalibration Interval field Note The scanner will automatically recalibrate each time you change your filter or your reflective transmissive setting If you select a different application with the same filter and light settings it will not auto recalibrate To print out a calibration report each time the densitometer calibrates click on the Calibration Report checkbox E 6 Acquire the Image Note Before scanning in transmissive mode make sure the white balance region of the scanning area is not covered or obstructed in any way To begin to scan click on the Acquire button The scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected After the scan is complete a window will open displaying the scan image at which point you can analyze and save it Note The image will open with a default file name that includes the date time and if applicable user name However unless you have selected Auto Save After Scan the file w
358. ve again and you can add the adjusted count to the spreadsheet Loading Another Image After you have saved the count s for the current image you can open another dish image by clicking on the Load Next button This will open a standard Open dialog from which you can select the image The new image will be loaded into the Colony Counting dialog Note The image will only be loaded into the Colony Counting dialog it will not open in a separate image window in Quantity One After you have saved the count s for the new image to the batch file you can either load another image using the Load Next command or click on Close to close the Colony Counting dialog 8 11 Quantity One User Guide 8 12 9 ReadyAgarose 96 Plus Ready Agarose 96 plus gels are pre cast agarose gels that accommodate 96 samples and standards These high throughput gels allow loading samples from 96 well microtiter plates The gels are 4 and 12 multi channel pipet compatible and the tip to tip spacing of the multi channel pipetors results in adjacent samples from microtiter plates being loaded in alternating wells on the Ready Agarose 96 plus gel The Ready Agarose 96 plus feature of Quantity One allows you to manipulate the gel image to rearrange the lanes from samples run on the ReadyA garose 96 plus gels and put them in a 96 well microtiter plate format thus simplifying sample tracking for analysis This allows easy comparison and analysis of gel data based
359. ween the end of the primer region and the beginning of the stretch of repeats Chapter 11 Differential Displays and VNTRs PCR Primer Sequence between primer and VNTR VNTR repeat unit Fig 11 3 Diagram of components of a DNA fragment In the Repeated Unit Size field enter the size in base pairs of the repeated element e g if you are working with CA repeats enter 2 Testing for Ambiguity The number of repeats calculated by the software may not be a whole number due to the limitations of exact band size determination Since the number of times an element is repeated must be a whole number the calculated value is rounded to the nearest whole number In the dialog box you have the option to flag those numbers Select Yes after Test for ambiguity to flag values that deviate from the nearest whole number by a certain fraction These values may warrant further review In the Ambiguous if rounded field specify how much the calculated value must deviate from the nearest whole number for it to be flagged Finally specify how to identify the ambiguous values using the Flag with button or the Leave Blank button When you have entered all the information in the dialog box click on the Done button The number of tandem repeats will be displayed next to the bands Note that if no standards have been defined on the gel no numbers will be displayed If the
360. well microplates high density arrays samples with very closely spaced bands Files scanned at 50 micrometers can be very large 100 micrometer resolution is useful for typical gels and arrays 200 micrometer resolution is useful for gels with large bands and dot blots 800 micrometer resolution should be reserved for very large objects such as CAT assays G 12 Appendix G FX File Size of Images Image File Size below Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance G 5 Acquire the Image Once you have selected your application scan area and resolution you are ready to acquire an image Click on the Acquire button There may be a short delay while the image laser warms up then the scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected Note Ifthe image you are scanning has more than 10 saturated pixels you wi
361. when possible Maximum cuts object 3 Cutting tip size 1 0mm 1 5 mm Material Glass backed gel v Fig 10 17 Cut options If you want to make multiple cuts check the box labelled Make multiple cuts when possible Then enter the maximum number of cuts per object Note Make multiple cuts when possible is inactive when using Lane Excision Next select the cutting tip size you are using Quantity One uses this to determine when you have reached the maximum capacity for wells on the plate Finally select the material you are cutting from the drop down list Plate On the plate tab select the plate type you are using for this cut run then select the volume of each well Quantity One uses this setting in conjunction with your cut settings to determine whether you will exceed the well capacity 10 21 Quantity One User Guide Specify Options Cut Plate Hydration Wash 96 well MTP 2 00 ml per well 384 well MTP 0 05 ml per well 96 Tubes 1 00 ml per tube Loading order 41 2 C amp 1 B1 Fig 10 18 Plate options Load order determines the order in which wells will be filled on the plate Hydrate To keep gels from drying out during long cut runs you can have the EXQuest spot cutter automatically hydrate your gel s during the cut run Check the hydrate box then enter the time in minutes between hydration runs Specify Options Cut Plate Hydration Wash x
362. which it was created Type Unknown standard or background Volume Sum of the intensities of the pixels inside the volume boundary x area of a single pixel in mm 2 Adj Vol Volume minus the background volume if there is no background volume this is simply the volume 12 21 Quantity One User Guide Volume The volume expressed as a percentage of all the volumes in the image Concentration The quantity as calculated from the standards and the regression method If you have not defined standards this is not calculated Area The total area of the volume box you have drawn in mm 2 Mean Value The mean intensity of the pixels inside the volume boundary Std Deviation The standard deviation from the mean intensity Min Value The value of the lowest intensity pixel in the volume Max Value The value of the highest intensity pixel in the volume Density The total intensity of all the pixels in the volume divided by the area of the volume Mean Background The mean intensity of the pixels in the background volume Num Pixels The number of pixels inside the volume X location The distance in mm from the left edge of the image to the center of the volume Y location The distance in mm from the top edge of the image to the center of the volume Width The width of the volume in mm Height The height of the volume in mm Specify the preferred Background Subtraction Meth
363. window displaying the captured image A Save As dialog box will automatically open displaying the default file name for the image which will include the date time and user if known You can then change the file name and storage directory You can also export your image as a TIFF image for viewing with other applications C 8 Exposure Status The Exposure Status bar shows the progress of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical representation of the remaining time before exposure is complete If the exposure time is less than 1 second the status bar will not refresh itself for each exposure it will remain at 100 percent C 9 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your ChemiDoc EQ system Quantity One User Guide DAC Settings Min eo Max 130 Imaging Area cm 4 Width 20 00 Height 15 00 Auto Exposure Threshold Saturated Pixels o 15 r Reminder x Show Light Waring Show Filter Warning r Video Printing Footer Information xj File Name xj Date Time x E posure Time X Contrast Settings Save Options Make Backup Copy r Misc Options O Simple Acquisition Mode Defaults Cancel OK Fig C 10 Available options in the ChemiDoc EQ acquisition window Click on OK to impleme
364. within the filtering window e PowerMean This filter is useful for suppressing salt and Gaussian noise within an image it worsens the effect of pepper noise It replaces the value of the pixel being processed with the power mean of the pixel values within the filtering window e ContraMean This filter is useful for suppressing pepper and Gaussian noise within an image it worsens the effect of salt It replaces the value of the pixel being processed with the contra harmonic mean of the pixel values within the filtering window Adaptive This filter is useful for suppressing Gaussian noise and salt and or pepper within an image If the image contains a mix of salt and pepper select this filter To begin filtering select a filter type from the pull down list A pop up box will prompt you to select a filter size Quantity One x A Select filter size Select a size that is larger than the average noise feature 5x5 77 9x9 Cancel Fig 3 23 Selecting a filter size Click on a button to select a size See Step 2 Select Filter Size for guidance Because filtering is an irreversible process you will be prompted to filter the original image filter a copy of the image or cancel the operation If you choose Copy and Filter enter a name and or version number for the new copy in the pop up box and click on OK 3 38 Chapter 3 Viewing and Editing Images 3 12 Invert Data The Invert checkbox in
365. ws 13 1 Print Image Print Image prints a copy of the active image window and any image overlays that are displayed The Print Image command opens the standard print dialog box for your operating system When you are satisfied with all the print parameters click Print to send the image to the printer 13 2 Page Setup Specify the printer settings using the Page Setup command on the File gt Print submenu This will open the standard Page Setup dialog box for Windows or Macintosh in which you can select the paper size page orientation printer etc 13 3 Print Settings Use the Print Settings command to configure what information to include and how the image appears each time you print an image To open Print Settings click Print Settings in the File gt Print submenu Any changes made to the Print Settings take effect immediately You can also leave the Print Settings dialog box open to quickly change the settings for images as you print Note You can have multiple Print Settings dialog boxes open one for each open image with different settings However If you use the print command file gt Print gt Print Image or CTRL P for an image that does not have the Print Settings dialog box open the print format will be based on the last Print Settings dialog box where changes were made 13 1 Quantity One User Guide Print Settings For Criterion Raw 1 D Image Crta R v 0 moi dad ddom je a Headers And Footers Pagin
366. y New Lane Created Initial Lane Number 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Number 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Nurnber 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Number New Lane Created Initial Lane Number 2003 08 04 09 27 53 Matthew Halsey 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Number 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Number 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Nurnber 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Number 2003 08 04 09 27 53 Matthew Halsey New Lane Created Initial Lane Number al l EE Page of amp eje ajaja Fig K 2 Audit Report Each entry in the Audit report contains the date and time of the event as well as the name of the user who performed the event The Audit Report also contains a description of the event with details the file name and the version of Quantity One used when the event occurred The comment column allows you to enter comments about particular events To enter a comment click the cell under the comments heading for the event to which you want to add the comment This opens the comment editor dialog box Enter your comment and click Done K 6 Appe
367. you can select it from the application tree by selecting Custom and the name you created You can delete the application by selecting Custom Delete and the name of the application Radioisotopes Chemiluminescence Chemifluorescence DNA Stain Gel Protein Stain Gel Fluorophores Microtiter Plate E Densitometry d Custom Create Delete High Sample Intensity Medium Sample Intensity Low Sample Intensity Fig G 7 Selecting a custom application You can edit a custom application by selecting Custom Edit and the name of the application You can also use this feature to create a new custom application from an existing one Step Select Application J Channel 1 Channel 2 Channel 3 Channel 4 Densitometry Coomassie Blue Gel Blot Medium 532 1064nm EX Blank 555nm LP 1064nm BL Fig G 8 Application selection and settings G 10 Appendix G FX Once you select an application the application name and settings appear below the Select button G3 Step Il Select Scan Area To select a scan area drag your mouse within the scanning window In the scanning window your cursor appearance will change to a cross The border of the scan area you are selecting is marked by a frame Drag cursor to define scan area Fig G 9 Selecting a scan area If you are in quadrant mode note that the frame locks onto the next quadrant as you drag
368. your densitometer have specific density values Before scanning for the first time you must enter the values for your transmissive step tablet into the software You can use the default values in the software for the reflective step tablet Transmissive Step Tablet Before you scan in transmissive mode for the first time you must specify the values for your transmissive step tablet First make sure that the Transmissive checkbox is selected in the main acquisition window then click on the Options button In the Densitometer Options dialog click on the Edit Step Tablet button The Step Tablet Values for GS 800 Transmissive dialog box will open Appendix E GS 800 E Quantity One 4 4 0 BETA VERSION 020 File Edit View Image Lane Band Match Volume Analysis Reports Window Help ONE WI E La Densitometer GS 800 Calibrated Transparency rStep Select Application 1 HH Seen Step Tablet Yalues for GS 800 Transmissive X ray film Tablet serial number TJE Gray film Quantity Offset o ooo Filter Red Step Diffuse Specular Step Diffuse Sp 1 o os 0 07 12 1 75 2 4 Light Reflective 1 14 1 61 19 2 83 fac 1 29 1 82 20 2 98 laz Fig E 10 Step Tablet dialog box 2 o z1 0 30 13 1 91 E rStep Il Select Scan Are 3 0 37 0 52 14 2 06 e Preview scan 4 o 53 0 75 15 2 21 5 1 Click and drag in diagar 5 o 68 0 96 16 2 36 3 3 Top o o 6

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