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FLUORCHEM Q USER MANUAL

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1. sssesssssesesenneeen nennen nnne nennen nenne nnn nennen nnn 65 Figure 3 2 File Pull Down Menwu lessen nnne nennen nnn nnne nnn nnn nnns nain n nnns na sanas nn annis 65 Figure 9 9 bile Oben Dialog BOX sid eto tee tator dan dex ote iduta dex E iui ided x cau dE 66 Figure 3 4 Save Load analysis feature ccccccccccccsseseeeceeeeeeeeeeeeeeeeeeeseeaeseeeeeeessuaaseeeeeeeeesseaeseeeeeeeesesaaageses 67 Figure 3 5 File Save As Dialog BOX cae eene nec edant Ln Ua an YR E pE sed Ungu WE RE UD aS 67 Figure 9 5 Printer Set DialOG BOX iiio fua ao mma katu a bun ineo ani cda deded ora usd 70 gie Irc 7 Pine xcs Boss ART E 70 Figure S S Edit Be uum 71 Figure 329 Ready tO Grop OF CODY sie eo et Caesia ote ere een veh oe een sad ene hoenedeed menial 71 Figure 3 10 AlphaView interface after CROP has been selected ccccccecsseeeeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeenas 72 Figure 1a Image Pull DOWN MENU xcci oskin astra car boe dean vb a wed Guus dusk uev bdeiniinleseet use Rodas Nds 73 Figure 3 12 Image Arithmetic dialog BOX 2 9 1 cron efe reed wenn DL oett Siesta eee eae eae 74 Figure 3 T3 Image Gonversion dialog BOX i e thoai eei eee t ated eos etnies Mahe ERO eae 75 Figure 3 14 Image Resize dialog DOX ve ha eee eb re uf d esee abesset fedus 77 Figure 3 T5 Image Iritfo dialod DOX x39 nics oe eod a Dueh og n aco otal a neces 78 Figure 3 16 Setup Pull Down MOD ei siae onse co bad e diuo t Pe sri dede e
2. Q C BHEHENMHBENEHENEHEBEHENBENBHN C EE EE ge SE Fe Sp Q EEE gee EESE EEE Ej Figure 4 6 Pen Style Selection Tools 97 Arrows and Straight Lines The LINE ENDS menu specifies the style of the ends of straight lines no arrow single arrow or double arrow Click on the appropriate checkbox for the desired style Note these line ends work with any line thickness Annotate a Color Line Ends Pen Width CO Text Style O Pen Style CO Text Orient o Q O Figure 4 7 Line Ends Selection Tools Text Background and Font The TEXT STYLE menu specifies the style of text Click on the appropriate checkbox to show text with or without a background An opaque background is useful if annotations will be made on an image that has wide variations in gray scale By using an opaque background text will not be lost in the background of the image SANDO XE C2 Color C3 Line Ends Pen width 9 Test Style Q Pen Style Q Text Orient Transparent Back Set Fort C3 Opaque Back Figure 4 8 Text Style Selection Tools This is also the window in which specific font is chosen When the Set Font button is depressed a selection box appears from which text style can be chosen 98 Italic O Arial M arrow Bold i Arial Unicode MS Bold Italic F Batang Book Antiqua Bookman Old Style Effects Strikeout Underline Script Westen Figure 4 9 Font
3. Symbols Figure 1 5 Ultra Violet Light WARNING face shields long sleeve lab coats can be obtained from major scientific distribution catalogs e g Fisher Scientific 19 gt Figure 1 6 CAUTION Risk of electric shock gt Figure 1 7 HAZARD please take appropriate precautions I Figure 1 8 Earth ground Terminal M pent Fuse symbol Power Ratings DE 500FCQ Multilmage III cabinet is rated at 250V 50 60 Hz 3A Fuse 250V 3 15A 20 Camera Components and Installation 1 Connect the Camera cable camera power supply and miniDIN connectors to the back side of the camera Cabinet Connection Camera Connections Figure 1 9 Camera Setup Step 1 2 Connect the power cable standard three prong end to the power strip Figure 1 10 Camera Setup Step 2 21 3 Complete the installation by connecting the camera cable to the USB port Figure 1 11 Camera Setup Step 3 Connecting the Printer The Mitsubishi P93DW UB printer connections are color coded for convenience see figure in section 1 3 2 Plug the USB cable into the back of the printer and into the proper USB connector on the back of the computer Plug in the standard three prong power cable to the back of the printer and to the power strip then turn the power on NOTE The Mitsubishi P93D UB printer may be connected to any USB port on the computer however the driver may need to be manually re installed depending on which
4. The Horizontal Alignment tool aligns annotations in a straight horizontal line This is pecially useful for labeling lanes etc To use this tool draw text on the image select it click the Horizontal Alignment tool then deselect the text The text will now be aligned in a straight line across the image Text Prior to Horizontal Alignment Text After Horizontal Alignment The Vertical Alignment tool aligns annotations in a straight vertical line This is especially usatu for labeling markers etc To use this tool draw text on the image select it click the Vertical Alignment tool then deselect the text The text will now be aligned in a straight line down the image 102 False Color These tools consist of eleven pre defined color palettes that can be applied to an image To select a palette simply click on one of the four buttons labeled GRAY PALETTE HIGH LOW NEXT or PREVIOUS Figure 4 13 False Color Selection Box When a palette is selected its range of colors is displayed to the left of the palette buttons and automatically applied to the image To apply a different palette to the image click the NEXT or PREVIOUS buttons Note changes in Black level White level and Gamma setting can alter the effect of each of the palettes and can enhance the results produced Gray Scale Palette 0 This is the default or standard gray scale consisting of different gray levels ranging from black to white High
5. du 783 0428 1 A e an 53b 10 31 0 4571 1 n 174 13 11k E41 1235 n Agno hw based on Standard Sample Value Least Square Fit D UELLE Em EUN CE CEP Ba A o ae o e O M X Deselecting Show All Lanes The Active Graph The active graph is shown in the upper quadrant The x axis represents the distance in pixels from the top of the template The y axis represents the average pixel intensity across the width of the scan Note Lane scans are shown without axes in order to show as much detail as possible in a small window To show the graph s axes choose Axes from the Options pull down menu To change the background color of the graph choose Background Color from the Options pull down menu AlphaView automatically detects the peaks and integrates the area under each peak Adjustments that can be made manually are described below 156 The Data Window Once a peak is defined its integration data and associated information are displayed in a table located in the lower right quadrant of the screen The data table is updated any time a peak is deleted or added peak boundaries are redefined or the background value is re set Lane Profile Result Data BaseLine Options Lane Peak Distance Width Height Area a b T 30 34 2 DE 2 12 T 18 B3 1 40 3 18 T 23 69 1 52 4 25 g 35 121 2B x 34 g AB 208 450 b 44 10 Bt 333 nir f AB 10 1
6. Despeckle Filters The despeckle filter is a type of smoothing filter based on data rejection Pixels in the neighborhood usually the adjacent pixels are used as a data set upon which the average and standard deviation of the set are calculated If the pixel of interest the center of the neighborhood is different from the neighborhood average either greater or lesser by a threshold a multiple of the standard deviation it is replaced by the average value The effect of the filter is to smooth pixels that are much different from their neighbors Artifacts such as hot pixels cosmic rays etc are commonly rejected by this type of filter The filter strength is controlled by the threshold factor For large factors very little data is rejected as only very large deviations are required for rejection where as low thresholds result in more smoothing For example setting 1 results in outliers of 1 standard deviation greater or lesser than the neighborhood average to be corrected for setting 2 results in outliers of 2 5 standard deviations to be corrected for and setting 3 results in outliers of 5 standard deviations to be corrected for These filters are particularly effective in eliminating random noise contained in an image and produce less blurring than the Noise filter described above 3 D Contour Filters These filters are particularly useful for visualizing faint bands They produce a 3 D effect defining edges and making det
7. Sample Annotations Figure 4 11 Sample Annotations Annotated image showing freehand drawings lines with various characteristics circles squares text with various characteristics The Editing Tools When the cursor is in edit mode it can be clicked on an object to select it Note the cursor can be toggled between edit and drawing modes by clicking the right mouse button When an object is selected small square boxes appear at the corners Selected objects can be resized copied deleted or moved e To resize an object click on one of the gray boxes at the corners of its perimeter and drag the box until the object reaches the desired size e To copy an object use the Copy tool e To delete an object use the Cut tool or the Eraser e o move an object click within its boundary and drag it into the desired location To select more than one object outline them with the mouse any objects that fall completely within the outline drawn will be selected Note The entire object must be enclosed by the cursor s movement in order to be selected 101 Figure 4 12 A Selected Object x Once an object or group of objects has been selected clicking on the Cut tool deletes it from the image The Copy tool makes an exact copy of the selected object The new object becomes the selected object and can be repositioned by placing the cursor within the objects boundary and moving it to the desired location
8. Adjust regions after loading a protocol by Export View OE selecting moving and resizing regions Band F C Help on these tools CO CO 7 CD Cl CO n2 RGB 4751 4806 4013 Zoom 113 X 246 Y 493 fi C Program Files 4lph 2 Gl Appendix X doc Micr AlphaInnotech Alph n U 4 14PM a In the figure above bands 1 6 are the Color Control bands serving as loading controls Any color channel can be used to perform the Loading Control Normalization Bands 7 12 of green channel data represent the signal from the phospho specific antibody with band 7 being the reference sample not subject to treatment Bands 13 18 of red channel data represent the dilution series with the following amounts of protein from left to right 0 0 4 1 1 3 3 10 and 30 nanograms Select the Background tab and select Local Background Select the Control Tab and Identify Loading Controls by choosing the blue channel and selecting bands 1 6 De activate the Loading Control tool by selecting the Identify Loading Controls button or right mouse click Select Identify Experimental Bands and select bands 7 12 The Identify Experimental Bands feature is automatically de activated if the number of experimental bands matches the number of control bands Press the Identify Experimental Bands button again and select bands 13 18 G9 Y Alphalnnotech AlphaViewQ
9. Blue Bkgd SD Standard deviation of Blue pixel gray levels in background region Blue Signal Noise Signal to Noise ratio of the Blue band Blue Average Blue Bkgd Average Blue Bkgd SD Grenng Results of Std Curve quantitative analysis for green channel data Background Corrected Sum Green BC Average Area Green BC Average Background Corrected Average Green Average Green Bkgd Average Green Bkgd Average pixel level of Green Background region Green Bkgd Average Sum Bkgd area Green Bkgd SD Standard deviation of Green pixel gray levels in background region Green Signal Noise Signal to Noise ratio of the Green band Green Average Green Bkgd Average Green Bkgd SD Red BC Sum Background Corrected Sum Red BC Average Area Red BC Average Background Corrected Average Red Average Red Bkgd Average Red Bkgd Average Average pixel level of Red Background region Red Bkgd Sum Bkgd area Red Bkgd SD Standard deviation of Red pixel gray levels in background region Red Signal Noise Signal to Noise ratio of the Red band Red Average Red Bkgd Average Red Bkgd SD Area of Background Region ControlTab 2 0 O Loading Control Normalized Average BC Average of C2 Experimental Band BC Average of corresponding Loading control mean of all loading controls LC Regions Specifies the channel and region number used for each Experimental Band and corresponding Loading
10. Low Palette 1 This is a modified gray scale palette in which black is replaced with green and white is replaced with red Over and under exposed areas of the image are thus shown as green or red while areas within the linear range of the CCD chip are shown in gray scale The Saturation Palette is especially useful during quantitation as areas outside the linear range of the instrument do not give accurate quantitative information This palette allows the user to avoid those areas during quantitative analysis This palette can also be accessed by clicking the Show Saturation checkbox in the Camera Setup and Preview function accessible by clicking on the Camera icon in the ToolBar window 103 Other Palettes Palettes 2 through 11 These are color substitution palettes in which the gray levels are translated into different color ranges These palettes can be useful to help distinguish features and highlight details on an image Palette 2 maps the gray scale levels to a red green blue palette Values of 0 are mapped to red saturated to blue and values in between to green Palette 3 maps the gray scale levels to a red green blue palette Values of 0 are mapped to blue saturated to green and values in between to red Palette 4 maps the gray scale levels to a red green blue palette Values of O are mapped to green saturated to red and values in between to blue Palette 5 maps the gray scale levels to a cyan magenta yellow palette
11. Once the protocol is selected click on the Auto Image Capture button to begin the acquisition process Acquiring protocol FtBr Gel Channel EtBr Total channel time Time remaining 108m Acquire stage Filtering image Camera exposure time ome Cancel Figure 2 11 AIC Status Window Once the Auto Image Capture process is initiated a pop up window labeled Acquiring protocol protocol name is displayed to indicate the status of the acquisition process This progress window shows the current operation status and overall imaging progress NOTE The images displayed in the Acquiring Protocol windows are animations to display status and do not reflect upon the image being acquired 46 Compare View Two or more images can be opened in compare view To open compare view click on the Compare View icon Compare View toolbar button If only two images are opened in the tab view images will directly open in compare view If more than two images are opened in tab view then a dialog box pops up through which image can be selected to open in compare view Select Images Please select two or more images to view in comparer view Title File Path Samples tit C Images S amplez tif Sample tif C Images S ample tif caomassie l 6 tit Images coamassieT 6 tit If two images of same size are opened in compare view then synchronize checkbox in status bar will be available to use By activ
12. c D an A T ne REI N j n ERE w LL C mE E pem e LET _ ss e C E S Em SS M EE REN e m mM Tt am Sum E c EG A c PRo p T M Ss a SS sere C ART E CE p a a TRUE s T M A REDE _ C AES PERDITIS T uL NW 3D Scan hie If a lane has been scanned with a large width it may take a long time to display i gr aph n 3D mode If this is the case select 3D View Show Hidden Lines from the utility menu in the i raph window This will result in a graph that is not as clean but which displays on she screen very quickly Smooth Dat i omooth Dau nction found in the Data menu minimizes single pixel background noise in a original graph smoothed graph When this function is selected the menu option changes to Unsmooth Data Selecting this undoes the smoothing operation The Invert Button The INVERT function reverses the gray scale assignments so that zero corresponds to white and 4 096 corresponds to black This function can be selected by placing an X in the INVERT checkbox or by selecting Invert Data from a graph s Data pull down menu If the image has dark bands and light background then INVERT should be selected If the image has light bands and dark background the INVERT option should not be activated On the
13. features In the Camera Setup amp Preview window click the Live button Open the door to the cabinet and position your sample on the preferred illumination Source that require epi or transillumination of UV energy should be placed on the purple UV filter glass For colorimetric samples such as protein gels films or blots use the told dow GR or your sample For chemiluminescence use the fold down white light table or the adjustable tray if used in conjunction with the fast lens Manuall With the door still open to allow light to enter into the cabinet use the monitor s real time LIVE readout display to position and focus your sample in the middle of the preview image Focus on the object by adjusting the top knurled knob on the lens For fluorescence and CE he door to the cabinet can be used to adjust the amount of light that enters into the cabinet if there is too much light to obtain a good focus setting For colorimetric samples it is necessary to decrease the aperture to acquire the image NOTE Business cards and other pieces of paper with small text often are the easiest objects to obtain optimal focus settings 3 Capturing a bright sample like fluorescently labeled gels colorimetric samples and film a b e 2o 7o2 Close the cabinet door Choose the appropriate optical filter for your sample type e Position 1 for colorimetric gels and film no filter e Position 2 for Ethidium B
14. 1 10 220 13 117 719 1384 06094 0 6094 E 11 255 13 111 E77 1304 O7064 0 7064 1 12 274 14 110 703 1354 07590 0 7590 1 13 313 10 45 254 4 27 0 8670 0 8670 2 1 128 12 15 138 5 B4 0 3546 03546 2 7 87 225 3 42 Example of an Auto Lane Data Table PEAK is the number assigned to each peak on the graph beginning on the left and moving right DIST is the distance in pixels that each peak starts from the beginning of the line scan WIDTH and HEIGHT refer to the size of each peak AREA is the integrated area under each peak This number reflects the intensity of each peak is the percentage each peak contributes to the total density measured on the graph The sum of this column will be 10096 for each lane Auto Lane Editing Features After the image has been evaluated for lanes and bands the following editing options will appear in the Lane Profile tab m EU m Tl 3coring Tools Result Gel Smiling Mol wt Mass Std Display Options Show Result v Line Auto Peak Adjust 3D View Min Area a Min Height 0 Min width D I Help on these tools Figure 5 47 Auto Lane Editing Features N N Add Lane This feature allows an additional lane to be placed on the image Once the button is selected the additional lane can be viewed and positioned on the image A left mouse click will place the lane on the image The data tables will readjust accordingly and the ba
15. 3 90 259 Gamma 2 3 t r9 Auto contrast Auto Enhance Level phng UDOocgagOg Gemma Linear Log H Show Grid Minmm 9J a 9 Maximum 3 4 J Auto contrast Auto Enhance Level nphnpDmeicus s 55 Gamma Linear Log Show Grid Image of film with log Contrast Adjustments selected Minimum JY 9 log provides minimum and oa Maximum 3 Ee maximum adjustment tools from 0 to 100 adjustment to the Auto contrast Auto Enhance Level phn DOOEDOEO displayed image Gamma Linear Loa C Show Grid Image of film with equal Contrast Adjustment Minimum a o selected Equal Maximum 4 JE automatically adjusts the image display for maximum contrast which is beneficia Auto contrast Auto Enhance Level nspng DOXEBDE 56 Multicolor Image Display Channel Viewer Channel Viewer performs a preliminary review of a composite display of a multichannel image to view each channel separately in the context of the composite image While each channel may also be displayed as a single channel using the Contrast adjustments window the Channel viewer provides a convenient tool to explore the image in the composite display mode Note Channel viewer is only active when selecting a multichannel image Select Channel Viewer to open a display window capable of showing each channel independently Hold d
16. 3 3 165 z O4 5 125 123 151 T2 7 B 287 167 1 2 0 02 f 128 202 143 z 0 04 8 158 205 152 0 02 9 492 205 215 3 0 05 10 30 209 17 f 0 12 11 128 212 187 11 0 15 Figure 5 13 Selecting the Colony Count Data Window to Show Individual Spot Details The data table gives the individual spot number the AOI in which it was counted its coordinates the integrated density value IDV for each spot the area number of pixels for each spot and the contribution to the total Note manually added spots show areas of 1 pixel 128 Multiplex Band Analysis Tools In the ToolBox Analysis Tools a tab labeled Multiplex Band Analysis opens a set of tools with which the density of bands spots or other objects can be measured A two dimensional area of interest or Object is created and the density is obtained through the corresponding pixel intensity values designated as IDV or Integrated Density Value Region Bkarnd Control Std Curve Protoc A Region Bkarnd Control Std Curve Protaci a single Region Tools Use the single region single Region Tools Use the single region 3 tools to draw regions tools to draw regions 0O around the bands of around the bands of interest interest Multi Region Copy To make multiple capies Multi Region Copy To make multiple capies of a region highlight a of a region highlight a region and select the
17. 9 eee eS X 9X9 Figure 5 63 ARRAY Template 201 The tool box work area displays a number of buttons and controls for specifying the number of objects along with their orientation and sizes Analyze arrays with Circles or Squares Wells Count Radius Horizontal 3 Y Inner 3 x Verical 3 i Quer 18 4 Scan Blank Skewing aL Result Values Actual Values C Percentage Switch between Use Circle objects and Use Square objects The controls labeled Hori Wells and Ver Wells let the number of horizontal and vertical objects be set to a maximum of 100 and 100 respectively The controls labeled Center and Outer let the size of the center and outer circles be set Click on an arrow to increase or decrease the number of objects per row or column or the size of the objects As the number and size are adjusted the template displayed over the image is updated to reflect the changes Aligning the Template Once the number of objects has been set adjust the placement of the template If the image of the sample is not parallel with the template the template can be realigned by adjusting the green line so it is parallel to the top edge of the sample Place the cursor on the box at either end of the line Hold the left mouse button down and drag the line until it is parallel to the image Next adjust the placement of the template so the objects coincide with the areas of interest on the image Move
18. NOS and Cede ect EM ELI MEDIA M MUI ML M ULM EL M EUM 72 TAE GY aE SHAY cis G rere amet eo S nu IM I M I uM Xu M IM E M 73 VOT AY TFI nS 73 EXIIICODCHanmnels ne e aa a aa e a Ae aa Ea eee ae a a a D Latest pd 73 CITE L VIEW Luc 74 NG A A ANAA E E AAAA EA NAA LR A PCM E IU AE AEA EA A AET pA EA A LE 74 ATUN OA a E EE NE E e EE eg ee x asta case aol ex stele a edh 74 CONCOS ara E a eta apse AE ATE E Ee le ee E A E a 75 Flai eld COLD TALE VIIA ee E a etos a a a a a OO a dea A a 76 Reor CATV r ate el sal alse ube Sale lee heros a a a e Sec eg sees a a era ER 76 DIOS ICSC Aarne LE M UM I M ME i 77 Dhocc dg cc mu UR EE E E I E 78 Bh ery 54 ON Sag Uc B flees tee ee or ene E ME d Ce ee d LC M ME te 79 Taur d csi cce I E uM E MI Iu I E T 79 FINI VIO Cee see hohe AMI EI M E LI M LE ME EL 60 PII D LERA A Te CIMA E MS Ri E c E T 60 PrE CL CNC ES er TNT TTE EEE EA A E A E a EE AA AA SO SV SICI 8000112271019 ERR PT 53 DHETOVEREAYANVIENU adttosdbtu Miet isd uote E suena seaeestis dea dee ute galt cq epa enters tanga DE suum dee 84 Loddo a OV CUI V Seg A EE E E Hr 84 SAV EA OV TAV siena A aN A IE A EA A EE A EENEN AE 55 Overlay LIDOT ES s te uscite dide ec teet abated ia oeste toutes uxpantuied i adore tac eau Me udbastedes ect fut ets etes ue dade 86 SOW OF DE aaea a aaa a a a iSc SUU D 86 TRUNET S MEN rae E T E E exe 87 IN OLD OG ea eE E EA T A T A N S A E 87 1035722 aE R EAE EET E A E lates 5S SSIES WWE Wo MEN Uaia
19. To displays or hides annotations in Analysis modules 86 The Utilities Menu A number of functions are now handled by Windows programs To access many of these programs while in AlphaView open the Utilities menu and select the program of choice Divide View Wil y Notepad 33 Explorer Figure 3 28 Utilities pull down menu Notepad The Notepad is a blank screen that allows the user to make notes about the experiment and save them as an ASCII file The Notepad is useful for saving any imaging comments or experimental conditions with the saved image for future reference P Innotech HD2 def Notepad File Edit Format View Help DRLimits ABL 200 IETIC 100 AvudImcgcaunmtzse 25 15 8 AECOompensatiansz 0625 General iDefaultFilter 1 sbefaultrFilter l i Lightcabinet l iDisableoverwritePrompt o iDisableunsavedPrompt o DisableNunROIPrompt PPawersaverz iIDr iverPathaec 5 pisablesecureralderPrompt z ToolBar AtTtrx 1168 Atte y 0 width 104 Height 321 M HE Rotate_angle 90 Too B0x Attrx 910 AttrY 27 45 Annotation FontTypes Arial i FantsT1zez 27 FDAPart11 Statusz i DantAskz Enhancel TonalAdjustmaent J Figure 3 29 Notepad Display Window 87 Explorer Windows Explorer allows access to files and other information saved on the local machine or the network if applicable St My Documents File Edit View Favorites Tools
20. move the cursor into the inner box Click and hold down the left mouse button The cursor changes to a hand use it to drag the green box until the desired region of the image appears on the screen Alternatively use the scroll bars in the image window to move the image up down and left right On screen Zoom tools is also available located on the main ToolBar Window in This function duplicates the Zoom tool in Tool Box and also allows for Image Drag to easily pan the image during any analysis functions located in Tool Box Analysis Tools Ss OS Image Drag icon Zoom Icons in ToolBar in ToolBar Histogram The histogram is a graphical display of the proportion of pixels assigned to each of the 4 095 gray levels This tool is found in Tool Box Enhancement Tools Scale oom Linear Scale G1 4x C3 Log Scale O 16s 64s Figure 4 2 Histogram display in the Tool Box The image is made up of picture elements pixels having brightness levels ranging from black to white A very bright image will have most of its pixels registering high gray levels and conversely a very dark image will have most pixels with low gray levels approaching zero The histogram is displayed in the lower left corner of the screen below the image window The horizontal axis represents the gray scale range black at the left end and white at the right end with levels of gray in between The number of pixels registering a particular gray level determ
21. pX X PX ane n PX X PX Lane 1 The Fold Change in phospho specific protein is calculated relative to the Baseline expression levels G 1 The image below shows a portion of the image that contains the primary bands Section of blot showing the primary bands of interest In above Figure lane 1 contains the untreated reference sample The ratio of pX red channel data to X pX blue channel data serves as the Baseline level of phosphorylation In this case phosphorylation does not greatly impact molecular weight so phospho and non phospho bands overlap on the blot Launch AlphaView v3 0 or later and open the image phosphor tif Y Alphalnnotech Potatoe File Edit Image Setup Overlay Utilities View Window Help eu GOV Goa eo Be phospho tif Gamma Linear Log L Show Grid Black I LNA White 4 WLna Gamma 9 D Auto Enhance Level Luz LLL sj Enhancement Tools Analysis Tools T Open the Analysis tab Select Multiplex Band Analysis Select Protocol tab and Load Protocol Browse to phospho1 sda protocol G2 Y Alphalnnotech Potatoe File Edit Image Setup Overlay Utilities View Window Help BOW CONES desi so OA Main mox phospho tif Gamma Linear rr C Show Grid Bek 99 IL White 9 9 24 Gamma 4 T J m 9 1 00 TTE Auto Enhance Level CELEC eps Load Protocol Look
22. 12 86 15 0 32 Fj 67 1 22 1 454 11 57 4 0 09 8 62 1 16 1 2342 9 80 a 0 17 E 2B 0 542 5 4 04 2 094 45 13 10 39 0 73 94 0g 75 1 335 2B 77 11 39 0 73 216 1 72 1 247 26 87 12 59 1 10 135 1 07 2 924 50 08 13 67 1 25 357 2 80 3 061 65 96 14 55 1 03 328 2 51 1 969 42 43 15 52 0 97 371 2 95 3 689 73 50 16 20 0 37 218 1 73 2 199 47 39 17 1 315 24 55 916 7 29 141 3 04 18 1 508 28 15 1 090 8 67 55 1 19 13 1 536 28 67 1 140 9 07 43 0 93 20 1 408 26 20 1 002 7 97 fa 1 62 Zl 2 005 37 43 1 449 11 53 102 2 20 FF 1 810 33 9 1 361 10 99 62 1 34 23 1 671 31 19 1 358 10 80 154 3 28 Fa 1 702 31 77 1 417 11 27 0 1 51 Figure 5 22 Band Correct Values The data table showing the respective Background Corrected BC Average values for each channel after regional background correction Note that the Signal Noise ratio is greater than 3 indicating a significant signal level for regions 17 to 24 in the Blue channel regions 2 8 and regions 17 24 in the Green channel and for regions 9 to 16 in the Red channel corresponding to the visible bands in the image Be aware that the actual biological variation best seen by observing the signals from replicate samples will be much greater than the signal noise ratios from the image data 139 Control Tab Control tab is used to apply loading controls and a positive control to the data Note that while the background regions are not displayed in the control tab the background corrected data values are maintain
23. 14 maps the gray scale levels to an orange palette Values of 0 are mapped to dark orange saturated to white and values in between to shades of orange This palette may be useful when printing an image of an EtBr stained gel onto a color printer 104 Image Filters Direction GISISISYSIIZISES dm Dia Figure 4 14 Filters Toolbox with 3 D contour selected AlphaView includes a variety of enhancement filters that can improve the appearance of an image Some filters sharpen detail others smooth and reduce random noise Still others help visualize edges and separate closely spaced bands or objects Depending upon the unique characteristics of an image the results of each filtering operation vary Assess the characteristics of the image and then select the filter designed to minimize its imperfections When an image is filtered the original image information is replaced with the results of the filtering operation As a result the original image information is altered To avoid losing the original image save it as an original TIFF file before applying a filter When the FILTERS button in ToolBox Enhancement Tools is selected a pop up box appears If the desired filter is not shown choose MORE to display more filters One or many filters can be applied to a single image High GEXGJSESISXESTS Figure 4 15 The Filters Toolbox with Sharpen highlighted 105 Smooth High Edge High Geni Ra Gem Figure 4 16 The F
24. 4 285 5 307 9 329 2 222 8 244 1 255 4 285 5 307 8 329 2 Position Position Example of a Point to Point Graph Example of a Least Squares Fit Graph Figure 5 6 Example of Point to Point and Least Squares fit graph The graph is useful to verify that the molecular weight data is entered correctly and can help determine the most linear region of the molecular weight standards Further if a query band is clicked its position will be shown on the graph by dashed lines as shown above 122 Colony Count The Colony Count tools in the Tool Box Analysis Tools make it easy to count the number of objects on an image such as colonies on a Petri dish or viral plaques Click on the COLONY COUNT The following sets of tools will be displayed in the lower left of the screen Threshold Count Protocol Area OF Interest Help on these tools Figure 5 7 Colony Count Tools Figure 5 8 Sample with Two Types of Objects 123 When two types of objects are counted the objects are highlighted in different colors They can also be marked on the image using a or an X Follow these three steps to perform an automatic colony count 1 Define and Position Area s of Interest There are three drawing tools with which areas of interest can be designated Click on the desired AOI Area of Interest button in the toolbox to define areas on the image containing the objects to be counted ROI Threshold Count Proto
25. 566 742 106 651 Green 4 Blue 20 3 803 2 545 620 2 233 2 056 017 1 804 Green 5 Blue 21 3 187 1 843 380 1 517 1 649 239 1 447 Green 6 Blue 22 3 024 1 657 560 1 454 1 606 350 1 409 Green 7 Blue 23 2 802 1 404 480 1 232 1 336 298 1 172 Green 8 Blue 24 2 651 2 387 180 2 094 2 939 701 2 579 Red 9 Blue 17 1 892 1 521 900 1 335 1 634 302 1 434 Red 10 Blue 18 1 804 1 421 580 1 247 1 498 744 1 315 Red 11 Glue 19 2 881 2 649 360 2 324 3 047 083 2 673 Red 12 Blue 20 3 618 3 489 540 3 061 2 818 391 2 472 Red 13 Blue 21 2 526 2 244 660 1 969 2 008 258 1 762 Red 14 Blue 22 4 246 4 205 460 3 589 4 075 534 3 575 Red 15 Blue 23 2 756 2 506 860 2 199 2 385 162 2 092 Red 16 Blue 24 2 152 1 499 100 1 315 2 245 1 719 120 1 508 2 2 3 1 751 040 1 536 2 245 1 605 120 1 408 2 842 2 285 700 2 005 2 647 2 063 400 1 810 2 9508 1 904 940 1 571 2 539 1 940 280 1 702 Figure 5 25 Loading Control Normalization Data Table The Loading Control Normalized values LCN are displayed in the Max channel format By multiplying the normalized values by the mean of all the loading controls the normalized values are scaled so that the LCN Average and LCN Sums can be compared to the BC Average and BC Sum to see the effect of normalizing to the loading controls Clear Loading Normalization Function clears all normalization currently active on the image Data Definitions LCN Sum LCN Average LC Regions Loading Control Normalized Sum
26. Band BlueBCAverage BlueLCNAvg RedBCAverage Red LCN Avg Clear Band Normalization 1 12 874 3 338 238 231 2 11 629 9 998 921 792 3 3223 9 998 1 507 1 633 4 3 333 9 998 2 043 2 189 5 8 918 9 998 1 966 2 204 6 8 500 3 398 1 638 1 928 C Help on these tools 7 8 786 9 998 1 878 2138 8 10 114 9 998 1 857 1 836 E 10 602 9 998 1 911 1 802 Zoom 79 X 2 1241 Y 188 cul Appendix X doc Mi Y AlphaInnotech Pot Microsoft Photo Edit Click the Identify Loading Controls button to de activate the selection tool Notice the data columns in the data table show the blue and red values for the background corrected average BC Average Local Background correction was pre defined for this protocol The data table also displays the Loading Control Normalized data LCN Avg Note that all values for Blue LCN Avg are identical This is expected because the Blue channel is designated as the Loading Control and the calculation for the Loading Control determines the mean intensity for all Loading Controls In this example the Blue Loading Control values are calculated from the Blue BC Avg which represents total protein X pX for each lane Ideally identical quantities of total protein are loaded in each lane but in reality slight variations in loading usually occur from lane to lane Note that the Red LCN Avg is calculated Red BC Avg Blue LCN Avg Blue BC Average using the respective values for the band
27. Control e g Red1 Green 12 Positive Control Normalized Sum NCN Average Area PCN Average Positive Control Normalized Average LCN Average of experimental band LCN Average of control band 100 PC Regions Specifies the channel and region number used for each Experimental Band and corresponding Band Control e g Red1 Red 6 Fold Change Fold Change of experimental band relative to control Note If LCN Average experimental band gt LCN Average control band then fold change LCN average experimental LCN average control If LCN Average of the experimental band lt LCN Average of control then fold change 1 1 LCN average experimental LCN average C3 APPENDIX D BIAS AND DARKMASTER UTILITY The FluorChem Q is shipped with a set of darkmaster and bias files that meet the requirements of most commonly encountered imaging applications 1 4 and 8 mins Some applications however such as extremely low level signal detection with lengthy exposure times may benefit from generating specific darkmaster files matched to the exposure time of the application for optimum performance AlphaView M software includes a utility for generating bias and darkmaster files Note that a darkmaster for given time need only be constructed once Subsequent imaging applications will then apply the optimum darkmaster from the set of all darkmasters in the system CCD images exhibit a non zero black level bias and temperature time dependant
28. Darkmaster generator Seles IMPORTANT Please put cap on camera lens turn off all cabinet lights and close cabinet door before starting this operation and untill ts end 7 ib Enter exposure time for the set of master images 1 to 60 1 min You may alza enter comma separated list of exposure times for generating of several sets of master images together Estimated time for building the set of master images 20h 10m Estimated time of process completion Thursday May UF 2009 at 11 49 4M Cancel 9 A final notice is given when the process is complete D3 APPENDIX E FLAT FIELD CALIBRATION The flat field calibration procedure is performed at installation or at the factory Flat field files are provided for the most commonly encountered imaging situations The purpose of the flat field calibration is to remove significant illumination and detection path non uniformities from acquired images Properly calibrated fluorescent images will be uniform for accurate quantification of signals levels within 5 at any position in the image A flat field calibration is only valid for images acquired under identical imaging conditions specific to that flat field file not including exposure time or binning factor A specific flat field file calibrates both the illumination intensity field and the image formation optics lens and filter and sample position within the cabinet Consequently any change to either the illumination field or the ima
29. Each pixel is assigned a brightness or a gray scale value level between black and white A very bright image has most of its pixels registering high gray level values and conversely a very dark image has most pixels registering low gray level values approaching zero The distribution of these gray values to the image is determined by the Contrast Adjustment Controls These controls regulate the Black level White level and Gamma setting brightness linearity allowing adjustment of the display to obtain the best image possible AlphaView software can als The AlphaView Software automatically detects this process and the Contrast Adjustment tools are configured for color image adjustments An image can be enhanced using these tools and then saved as a Modified file for publications However to preserve the original image information it is recommended that the file be saved as a different file name when using the save modified Using the Contrast Adjustment Tools for Grayscale Images There are three sliding scales found in the image control area to the right of the image Below each scale is a box displaying a number that corresponds to the position of the slider By adjusting these sliding scales the image display can be optimized Show Grid Bamma Linear Log Black 4 J H 0l white 4 JD Gamma 4 J Lr Equal Reverse Auto contrast Auto Enhance Level 2st islet Less Figure 2 14 Contrast Adjus
30. Help O Baci B J2 Search lig Folders Fea Address L My Documents Folders x B Desktop B My Documents i O AdobeStockPhotos i23 Alpha Images O Dotfuscated H Downloads D Folder I3 Logo IO My I50 Files a My Music A My Pictures D My Received Files a My Shapes a My Stationery 5 My Videos O My Virtual Machines E O Research Works Ez e SQL Server Management Studio O Test O TestDotfuscator i O Updater B O Visual Studio 2005 X My Computer Ez 3 My Network Places Recycle Bin O iPhone UUUWUUCUU amp AdobeStockPhotos File Folder Downloads File Folder My ISO Files File Folder My Received Files File Folder My Videos File Folder SQL Server Management Studio File Folder Updater File Folder Report AutoCount rtf Rich Text Format 31 KB Report SpotDenso rtf Rich Text Format 37 KB Test 2 xml XML Document 301 KB VUVLLUU m Alpha Images File Folder Folder File Folder My Music File Folder My Shapes File Folder My Virtual Machines File Folder Test File Folder Visual Studio 2005 File Folder Report BandAnalvsis rkF Rich Text Format 49 KB Rotate3d zip Winzip File 48 KB Dotfuscated File Folder Logo File Folder My Pictures File Folder My Stationery File Folder Research Works File Folder TestDotfuscator File Folder Report 1dMulti rtf Rich Text Format 500 KB Report MolecularWeight rtf Rich Text Format 5
31. Level 0 51 Device Corrected Negative Enable Auto Levels NegativeChange 1 Figure 3 23 Preferences Auto Enhancement Tab 82 To make changes to the preferences select your change by typing in a new value or select de select the appropriate box with a check mark and then select apply Some settings may require that the software be re started for the change to take effect 5 Analysis Tools used to show hide additional image analysis tools Any change in the settings will require software be re started for the change to take effect Preferences General Image Acquire Cabinet Settings Auto Enhancement Analysis Tools JN a Please uncheck those analysis tools which you don t need to view in Analysis Tools Tab Ruler Molecular Weight Scoring Manual Count Colony Count Array Multiples Band Analysis Lane Profile Figure 3 24 Preferences Analysis Tools Tab System Calibration In order to use system calibration it will be required the administrator log in of the AlphaView software program tup Overlay Utilities View Window Help d amp ample tif Print Info Prink Mode Print Date Preferences tem Calibration Darkmaster Utilitw Flat Field Utility Figure 3 25 Setup System Calibration menu Darkmaster utility see appendix D Flat Field Calibration Auto see appendix E 83 The Overlay Menu The Overlay menu prov
32. Selection Window Note To choose Greek symbols such as a p A x and 0 choose the Symbol font abcdefghijklmnopqarstuvwxyz apyxo soynioxAnuvonxOpotvuooSvZa Text Angles In the TEXT ORIENT window select whether text should be oriented vertically horizontally or at an angle in 15 increments sanr Color Line Ends Pen width Text Style C3 Pen Style Text Orient Clock Wise Ande D 4 C3 Anti Clock Wise Figure 4 10 Text Orient Selection Tools Note only rotate fonts that are True Type indicated by TT in front of the name other fonts such as Courier and Fixedsys do not re scale properly giving unpredictable results 99 The Drawing Tools Once the object attributes have been defined click the cursor on any of the drawing tool buttons to assign the function associated with that button to the mouse The cursor will change from an arrow to a cross indicating that AlphaView is in drawing mode After selecting a drawing tool move the cursor to the correct position on the image to begin drawing Press and hold the left mouse button and move the mouse to the end point of the object to be drawn Release the left button and the object should now appear Boxes will appear at the corners of the new object and the cursor will revert to an arrow indicating that AlphaView is now in edit mode At this point the object can be resized or repositioned The color pe
33. Sums or BC Sums for that region and are intended for analysis of dual labeled bands or spots as may encountered in dot blots These ratios are somewhat arbitrary as they depend on the relative exposure times of the channels Do not confuse these ratios with the LC regions or BC regions which indicate which regions are selected as control and experimental bands respectively See Appendix X for a detailed example of analysis of a phosphorylation study using multicolor western methods using loading controls and Band controls Mass Standard Calibration Curves for Quantitative PCR The button in the Multiplex Band Analysis toolbox labeled STD CURVE opens a set of tools that create a calibration curve for applications such as quantitative PCR and Western Blot band quantization The calibration curve functions allow quantization of the bands on a gel based on a set of standards A minimum of two standard bands must be input but the accuracy of the calibration curve increases as the number of standard bands and their range of values increase Defining the Bands as Objects Before generating a standard curve define the bands using the object drawing tools in the Multiplex Band Analysis toolbox As described in Drawing Objects above the size of an object can influence the integrated density value Therefore we recommend using the same sized box ellipse or freehand drawing when defining a standard curve See Copying Objects above for instructions 1
34. UV transilluminator and mount onto sliding tray be sure to align UV transilluminator rubber feet into sliding tray open positions Inside the cabinet locate the power cord taped with RED tape Please uncoil and insert into the power socket for the UV transilluminator Plug the light cabinet into the surge protector turn on the cabinet and test each of the switches on the front of the cabinet to ensure that all connections were made properly Note Do not plug a transilluminator or the Multilmage Ill light cabinet into the same power strip as the computer use a different circuit whenever possible FluorChem Q provides cabinet controls through software An RS 232 cable that is supplied with the cabinet is required for this purpose Please located the RS 232 cable insert it into the corresponding socket next to the power connect and power switch on the rear of the DE500 see picture following page A 3 foot long 8 pin miniDIN cable connects the camera to the rear of the cabinet 18 O lt e oor Figure 1 4 Cabinet Setup The DE 500 and DE 500FC Multilmage Light cabinets connect to the back of the computer via an RS232 cable WARNING IF EQUIPMENT IS USED IN A MANNER NOT SPECIFIED BY THE MANUFACTURER THE PROTECTION PROVIDED BY THE EQUIPMENT MAY BE IMPAIRED CAUTION POWER SUPPLY CORD IS USED AS THE MAIN DISCONNECT DEVICE ENSURE THAT THE SOCKET OUTLET IS LOCATED INSTALLED NEAR THE EQUIPMENT AND IS EASILY ACCESSIBLE
35. Your image has been acquired save wizard protocol OQ Yes No Ca Jens Selecting the Yes option and clicking Next gt will give a save protocol Ul The protocol will be added to the list of existing protocols Selecting the No option and clicking Next will generate the save image UI 29 AlphaMavigator cave acquired image te Yes C No Selecting the Yes option and the clicking the Finish button will generate the save image UI and exit the Navigator Selecting the No option will close the Navigator The next time the Navigator is run the user can run the saved protocol AlphaMavigator E New image capture D Yes need assistance with image acquisiti Mo load an acquired protocol Selecting the No option the following screen is generated with the last saved protocol selected AlphaMavigator Selec saved protocol tiy Ih B cd ride The user can browse for other saved protocols by clicking on the Ja button a list of existing protocols will be displayed 30 AlphaMavigator Selecting the protocol will go to the preview screen for user to verify setting or acquire 31 Camera Setup and Preview Window Clicking the Camera Acquire Icon launches the Camera Setup amp Preview window 8 S TE Setup and Preview AE cmoa 0 0 8 W a Exciaton Judsesisnced Channel assign n T ais istat Cpe Phen homala cw Haina medicion Now Figure 2 2 Came
36. a a i aa aaia lad 51 Figure 2 15 Black Level Adjustment Example ccccccssscceccssseecceeseeecseseecseueeeseaseeessauseeesegeeesssaseesssaaeees 52 Figure 2 16 White Level Adjustment example cccccsscccccssseecceesececeeuseeceseeeeeaseeessaeeeeseuseeessaseeessaaeees 52 Figure 2 17 Gamma Setting Adjustment example sseeessssessssssseee enne nne 53 Figure 2 18 Original and reversed Image cccccccesseccecceeseeeecceeeseeeeccsauseceeeseaueceessauseeeesssageeeeessananeeeeesaas 54 Figure 2 19 Orginal and Equal IMIE ieren oae de quier 2 050 odio Qa eese dote tais deco tos eon eaavens 54 Figure 2 20 Channel VIBWOL mstencsoat Mut teo a acad ioo Gori Cose dasstn faoc uses o inue doo iocos uunc edE eee 57 Figure 2 21 Contrast Adjustments window isses nnne nn nnns nn nnn n nnn nnns nna nnns 58 Figure 2 22 Multichannel iMag srsti cu tea s votar te basado ea stus dw div ba au cei bu PuvR VE DN a C cii avt OR vu Du E venus 59 Figure 2 23 The Enhance TOOS acciu a e a v Cu RO au og bam du ERE Da ai cb UN dal OR etat E ds 60 Figure 2 24 TOO Bal atien accede ance imde abite iato suut ue nce M estu ta teed done cM cin tdtsie deii tae ea R NEUES 61 Figure 2 25 POO BOXE paa neci ebat catis sted sic eut a be rima Edu ea tals thea eaten deti Edo M da eta 64 PIQUE 2 20 Status Dafosscenkutisesuteti ettet eo reete ett o a aet ec mtd et tu na eee ean Md E ced 64 Figure 3 1 AlphaView Drop Down Menu
37. a dialog box with all detailed image properties To remove this dialog box from the screen click on the OK button Image Information Property Acquisition Date Acquisition Time Madified Image width Image Height Image Type Bits Per Fisel Protocol Mame Cooler Temperature Acquisition Information Display Black Display White Display Gamma Display Inverted Value 2009 07 31 04 39 45 Ma bEz FAB Grayscale B n a Cooler n a M h 255 1 00 Ma Figure 3 15 Image Info dialog box 78 The Setup Menu This menu customizes the system settings by allowing users to save default parameter preferences and customize the software settings ZEE Overlay Utilities Print Info k PrinE Made Frink Date k Preferences System Calibration Figure 3 16 Setup Pull Down Menu Print Info Se Overlay Utilities View Window I a Frink Mode Eottom Frink Date k CFF Preferences System Calibration d Figure 3 17 Setup Print Image Info Dialog Box When printing an image basic image information is included on the print This includes the exposure time the Black level White level and Gamma setting the date and time the image file was generated an image ID number and the name of the file to which the image is stored To print this information at the top of the print choose Top from this menu To print at the bottom of the print choose Bottom Note Printing image information at the top or b
38. a gel blot or microtiter plate To score an image click on the SCORING button in Toolbox 4 Define the appropriate number of rows and columns of your image by using the appropriate controls You can also define the amount of spacing between your rows and columns to compensate for any variations between lanes or wells Columns B Spacing 10 Spacing 10 Figure 5 60 Scoring controls Once you have selected the number of rows and columns to be displayed you may adjust the template to fit your image Use the left button on the mouse to click hold and drag the outside borders of the template so they frame the lanes or wells to be scored Clicking holding and dragging on or within any border moves the entire template Clicking on the blue corners surrounding the template resizes it Scoring image with template positioned 197 Scoring the Sample Three different scoring options are available score with a positive negative or positive negative The software will display an X if you have a positive sample or three signs arranged in a vertical fashion if you score with a negative Nothing will appear if you score with a positive negative You may use the right and left mouse buttons to determine different scores The right mouse button will score the sample with an X The left mouse button will score the sample with three signs vertically arranged 9coring for positives negatives an
39. acquisition of images faster and more convenient than standard imaging software Note AIC requires motorized zoom optics Designing a User Protocol To utilize the single click feature of Automatic Image Capture a protocol specific to your sample must be defined and saved Click the Acquire button to reach the Camera Setup and Preview Window Acquire Below is an example of the design of a protocol for imaging DNA gels stained with Ethidium Bromide 44 Camera Setup and Preview Fajin ElirGel v Siren As Lateral Evolis Tiare Tt Next select Save As in the Protocol Tool to save your customized protocol Protocol Load protocol EtBr Gel w Figure 2 8 Save Load Acquisition Protocol The Save Load Acquisition Protocol Window will appear and allow to you Save and name your protocol Frotocol Load protocol EtBr Gel EtBr Gel 3channelsz Figure 2 9 Save Load Acquisition Protocol 45 This window is also useful for loading a previously saved protocol to make small modifications or delete existing protocols Auto Image Capture AIC User customized protocols can be recalled for single click image capture using the AlohaView software s Auto Image Capture feature First load the protocol of interest by clicking on the drop down menu from the Auto Image Capture Icon Acquire Auto Image Capture Drop down menu Figure 2 10 Auto Image Capture AIC
40. align the objects as described above Once the array has been set up click on the Scan Blank button The average pixel gray value for each object and torus is measured and set to zero 0 Now when samples are scanned the gray value of the blank is subtracted from that of the sample before it is scaled This ensures that the density reported only reflects the sample in each well When Scan Blank has been selected the background values obtained apply to all subsequent scans To indicate that a background is being applied the Scan Blank button changes to read Clear Blank To turn off this background subtraction function simply click on Clear Blank 204 Common Export Result Feature Once the analysis has completed then there is option to export the result Either the results can print directly to a printer or export the results as ASCII data for direct importation into Excel or other spreadsheet programs Export Data Export To Printer ApdeT HP Lasenet 1100 C3 File C3 Append Clipboard Excel Format Clipboard Text Format Figure 5 64 Common Export Dialog Box Sending Data to a Printer To send the data directly to the Video Printer or Default Printer click on the Export button and click in the circle next to appropriate printer Sending Data to a File If you would like to take the data from the system and import it into another computer the data can be saved to a diskette or network which will allow you to o
41. allow zoom control over the dendrogram window Zoom with Selection Tool The zoom with selection tool allows the user to zoom an area of interest within the dendrogram defined by mouse Grab Tool The grab tool allows the user to navigate the entire dendrogram in a zoomed display by clicking and dragging to window 189 Common Features Default analysis tool has three common features namely Protocol Report and Export Results Protocol ROL Threshold Count Protocol Report Save Protocol Load Protocol Help an these tools Figure 5 52 Protocol Tab The Protocol tab is used to save and load protocols for use on replicate blots Saving a protocol differs from saving an analysis in that protocols may be used on images other than the original image while a saved analysis is available for loading only on the original image Note that an analysis may be saved at anytime by using the file drop down menu mI Edit Image Setup Overlay Utilites View Wir EF Open ct0 A dm a 8M me Save Analysis Close Ctri F4 p Load Analysis Figure 5 53 Saving an analysis A protocol or analysis contains all band and background regions created with the loading controls band controls and standard curve settings used A protocol or analysis may be saved at any point in the analysis workflow 190 Report Using Report Tab user can generate reports based on analysis data Reports offers different option
42. amp amp dwanced Chane ac mgnment Tabs best Soeed Hesohuho n Homma ba cw Mogsreduchon Hore Once MOVIE MODE is selected a display box will appear for independent control of all lighting filters and exposure delay for each image frame The TOTAL FRAMES setup provides you with the ability to determine how many individual frames images you want for the movie There is a maximum of 50 frames images and a minimum of 1 frame that can be captured with each movie The FRAME selection is used for setting up the conditions for each frame image For example if three 3 images are to be captured you would choose FRAME 1 and setup all of the desired lighting and filter requirements You can then click on FRAME 2 and repeat the above Or you can click on COPY TO NEXT to help speed up the setup process COPY TO NEXT copies all settings from the previous frame to the current frame Usually for chemiluminescence imaging all lighting is off and the filter wheel is positioned for the chemiluminescence position for all frames Thus the only variable that is changing from one frame to the next is the exposure time In this situation COPY TO NEXT is a useful tool to save time in the setup process 111 If you are performing kinetic experiments where you want to have a predetermined delay between captured images then you can use EXP DELAY to configure this function The default EXP DELAY is set for the shortest possible del
43. and then point to the individual bands and click once on the left mouse button on the band of interest Continue to do this until all of the markers have been loaded Select apply to calculate the unknown mass values in the rest of the image 169 Band Scoring Lane s to score Choose the lanes to score by highlighting the lanes to be included Score based on Select the parameter to base the scoring on The choices are Area Mass and Height The Area and Height are calculated automatically in Lane Profile In order to use the Mass as the scoring parameter the user must calculate the Mass of every band by applying a Mass Standard This can be done separately or in conjunction with MW calculations See the section above on Molecular Weight and Mass integration Select Scoring Method Select the scoring method of interest The choices are Present Absent High Medium Low Absent of control and Quantity e Present Absent this method is used as a qualitative measurement for review The researcher is interested in whether or not a lane has a specific band of interest based on some criteria area mass or height e Additional Criteria When choosing Present Absent as the scoring method the user must enter a value for determining the presence or absence The value is a percent When the value of an unknown band falls below that percentage value it is classified as Absent when it exceeds the value it is classified as Present Tools
44. button and then click on the image to apply the curve Clicking within the lane boundaries will produce a three point curve with an anchor point each on the boundary of the first and last lanes and one at the position of the mouse click Once a curve has been applied following other Gel Smiling options are enabled Scoring Band Watchin Protocol IL Report Gel Smiling Mol wt Mass Std Std Tools Result aaa Goan Delete Anchor Delete Curve elete Curve DeeeAlCuves elete All Curves a 2 Help on these tools Add Curve Cure Add Anchor Anchor 180 Adjust the curve by dragging the yellow anchor points 181 Adding Removing Anchor Points Anchor points can be added by first Selecting the Add Anchor option from UI and then clicking on or near any Gel Smiling Curve When the cursor changes to a cross T it will add an anchor point to the nearest curve by clicking the left mouse button once Additionally anchor points can be deleted by first selecting the Delete Anchor option from Ul and then clicking once on the anchor will remove anchor point Remove Curve A Curve can be deleted by first selecting the Delete Curve option from UI and then clicking on the appropriate curve will delete the curve Adjusting Curve using Anchor Points Click Edit Curve to adjust anchor points by clicking and then dragging the anchor point from one location to the next Additional Gel Smiling Curves Apply en
45. cereus eese e eee en eene een nnns eee tna aaa a nno APPENDIX E FLAT FIELD CALIBRATION rite eron te ERE Rope neo e Pe ena RER e CH En opa Pea Ce aav eopeR Pa Rae APPENDIX F DATA INTERPRETATION 3 pesce vae npn bb e eee ae epa nep Pessoa opa E PERS e eaa ce bene pb Ue e rear en APPENDIX G FULLY WORKED EXAMPLES OF MULTICOLOR BAND ANALYSIS APPENDIX H REGULATORY COMPLIANCE ee eee eese ee etta sees etes sess teens sese etna sees eno TABLE OF FIGURES aera me oridic V AbE Medqec mE m 14 Figure 1 2 Computer monitor mouse and keyboard setup sssesssseeeeeeeeennn eene 16 Figure 1 3 Pictured with the cabinet top DE 500FCQ seseseeeseseeeeeeeeeenn nnne nnne nnn 17 Figure TA Gabin SCID m RT PME p I cece eeu adieeuioads 19 Figure 15 Ultta Violet RIG oder ett oe ee eee 19 Figure 1 6 CAUTION Risk of electric SNOCK cc cceececccccceeceeeesseeceeeeeesaeeeeeeeeeeseaesseeeeseesseaeseeeeeseeeseaaaaeses 20 Figure 1 7 HAZARD please take appropriate PreCaUtiOns ceecceceee cece cette eee eeeeee reese saa aaaeeeeeeeeeeaaaaaeees 20 Figure S arti QrOUnG lermlplaissqiseneoroc cea uisu A DU Dd M ctos OL o tdi a epa D edi US Ioa 20 Fig re 1 9 Camera Setup Step T i3 9 sand ee ee eh ici ieee 21 Figure 1 10 Camera Setup oleD2 us eemter Tee one Dod prem ctus Dona E Diam tcl n qute eeoe ee ee b DU Poe eda 21 Fig re 1 13 Camera Selup 9l6D eer Dieu n dum p
46. graph the regions that were peaks are now closer to the baseline and those that were near the baseline are now peaks Original graph inverted graph Since the baseline is determined when the image is initially scanned it may be necessary to use the Reset Baseline function to set an accurate baseline for newly inverted data When the Invert Data function is activated from the pull down menu the menu option changes to Uninvert Data Selecting this undoes the inverting operation Unlike the REVERSE button described in Chapter 2 this function does not alter the appearance of the image but does change the data Overlaying Graphs When comparing the bands in one lane with those in another it is often convenient to display the graphs so that one is superimposed over another After clicking on a graph so that it is enlarged in the image area select the Overlay Control function from the Data menu to display the Lane Overlay Control dialog box In this dialog box specify the lane s whose graph s should be superimposed and the color and line pattern in which they should be displayed e The first column in the dialog box contains the numbers of the graphs which can be Superimposed e The second column reports whether or not the graph of a particular lane is currently superimposed These ON OFF settings toggle back and forth by clicking on them e The colored bar in the third column indicates the color in which a graph will be displayed
47. is saved in an unaltered form This option should be selected if the image will be analyzed later If the Black level White level or Gamma settings have been adjusted the new values are saved but the pixel values are not altered When this file is opened at a later time AlohaView will display it with the values that were displayed when the image was saved however it is still possible to revert to the original raw image file by selecting Reset on the Tool Bar Annotation information cannot be saved with the Original image option It can however be saved as an Overlay See section 3 5 for more information If the image was saved as an original file usin fl Wit vel ard arma ceto Al with the new information 68 Note If the image is saved as a Modified file it is converted to an 8 bit image 69 Print This function sends the image to the default printer specified in Print Setup Print Setup This function displays a dialog box in which the settings for the parallel printer are specified When all the pertinent printing preferences have been specified click on the OK button If you purchased a printer with AlphaView this will be preset from the factory Page Setup Paper Size v Source Default tray hal Orientation Margins inches Portrait Left Fight Landscape Top Bottom Figure 3 6 Printer Setup Dialog Box Pape Setup Printer S Latus Ready Type MITSUBISHI P33D Where X USBOO1
48. object box to contain a few pixel values that are true representations of the background level If the object is too small and the band fills the object box then local background correction will result in significant errors 130 Magic Wand and AutoSpot Single Channel Only These two selections are automated detection features designed to recognize sample in an image and draw Object boxes around it They are found under the Auto Detect heading in the main Multiplex Band Analysis Tab Once an object is created the dimensions can be altered and the background subtracted similar to manually drawn objects Auto Detect Auto Spot Figure 5 14 Magic Wand and AutoSpot Tools Magic Wand Selecting Magic Wand will activate a wand like symbol and the following data table Magic Wand Parameters Magic Wand Sensitivity 0 100 Sensitive a J Less Sensitive More Sensitive Spot Type Bright Spots C3 DarkSpots Outline Type Border Outline Box Outline Figure 5 15 Magic Wand Parameter Window The tip of the wand should be centrally positioned over the sample spot on the image and then simply click the mouse The software will use either an edge detection algorithm to trace an area of interest around the spot or draw a box around the sample A right mouse click will reactivate the wand icon for another selection Magic Wand Sensitivity The Magic Wand Sensitivity slide bar will alter the parameter for how much of t
49. on screen buttons and menus in an intuitive interface Images can be printed using a 256 level gray scale thermal printer or any printer with a Windows driver The low cost high quality prints are ideal for lab notebook records or publication About This Manual This manual uses different fonts to indicate certain conditions e Arial font indicates the name of a button a menu or a function found in a menu Courier font indicates an entry that is typed indicates key points and useful hints indicates actions that may either harm the system or affect the data quality e cons or buttons to be pushed are placed next to their respective descriptions in the text Questions or Comments For questions or comments please use the following contact methods For general information e Email info alphainnotech com e Phone 510 483 9620 For technical support e Email support alphainnotech com e Phone 800 823 0404 Business hours are Monday through Friday 7 00 AM to 4 00 PM PST TABLE OF CONTENTS INIRODUCINGALPHA VIEN Re ei eS erat POR dU Mn EORTC Pee ARI Lee Tn PnSrICe een ere nS 2 ADON LUS MONU ttr 2 Oues tions Or C Om ENIS o a EEE E ET eae MM tag OAN EEE LS LA DU eae EE LEA 2 CHAPTER 1 INTRODUCTION AND SETUP ii ttes teer S detccsasaseavesscatetoctedbeueie desnsestucsarevedebotececseussecstenes 10 INTRODUCTION TO THE FLUORCHEM Q IMAGE ANALYSIS SYSTEM eese eene nenne ene nnne e sean enean 10 DU TRY FAUT To
50. on the REGION tab If the image has dark bands and light background then INVERT should be selected by placing an X in its box If the image has light bands and dark background the INVERT option should not be activated Note This function does not alter the appearance of the image unlike the REVERSE button described in Chapter 2 136 Background Tab Calculating background values Region Bkamd Control Std Curve Protoc Background Region Tools Multi Region Copy LOJLOJLA Regional Background Applies a background region to all bards Multi Regional Background Applies background region to a subset of Bands Link Background Unlink Background Local Background Uses 10 lowest peels inside each region None Help on these tools Background tab Background subtraction is an important part of image densitometry analysis The Background tab is used to subtract the background in the image from the regions of interest The background is unwanted signal in the region arising from the fluorescence detection chemistry the membrane and quality of the sample itself Because fluorescence detection is extremely sensitive high background levels in the image can be a common problem especially in the early stages of protocol development Fluorescence protocols require careful attention to cleanliness and sample handling to minimize background problems Regional Background Background region applied
51. on the curve The heading of this column reflects the units entered as in Specifying the Quantization Units above Standards have an s next to the object number and are highlighted in green to differentiate them from unknown bands Values that are outside the range defined by the standards are not quantified and simply have a dash entered in the corresponding column Quantization Values of Unknown Bands across Color Channels A specially constructed control sample Color Channel Normalization Control or CCN is required to make quantitative comparisons between different color channels in a multicolor image One such sample is a mixture of a single type of protein or appropriate size similar in size to the other proteins of interest that has three different fluorescent labels The GE DIGE minimal dye labeling kit can be used to label aliquots of the protein with CY2 CY3 and CY5 The aliquots are then mixed together in equal amounts and loaded into a lane When the gel is run and transferred to a membrane the resultant band generates signals in each color channel and serves as a reference The Color Control CC permits making a correspondence between a specific amount of signal from one color channel to the other color channels Given that each channel may be acquired with different exposure times excitation energy variations emission filter variations the actual signals can be quite different By normalizing the respective signals t
52. over the objects 203 Measuring Density Once the objects are sized and positioned correctly click on the SCAN button in the tool box The density values will be calculated and displayed under each object The values are the average pixel values within the object adjusted to a scale of 0 to 100 0 corresponding to black pixel values and 100 corresponding to white pixel values If both a center and an outer circle have been specified two values are displayed The upper value corresponds to the inner circle and the lower value corresponds to the torus The INVERT Box The INVERT function reverses the relative density assignments so that 0 corresponds to white and 100 corresponds to black If the image has dark areas of interest on a light background then INVERT should be selected by placing an X in its box If the image has light areas of interest on a dark background the INVERT option should not be activated Unlike the REVERSE button described in Chapter 3 this function does not alter the appearance of the image Removing Background using the Scan Blank Function Since a microtiter plate is not completely transparent it contributes to the density measured when samples are scanned A non uniform light source also affects the density measured To ensure that the density measured is attributable only to the sample in the wells scan a blank before scanning samples Load an image of a clean unused microtiter plate and
53. pull down The choices are Neighbor Joining UPGMA WPGMA Single Linkage Complete Linkage Ward Median and Centroid These are standard statistical algorithms references can be found in most statistical text books Clusters Select the number of clusters you would like displayed in the dendrogram The values are 1 to maximum number of lanes in the image This option is disabled when Neighbor Joining is chosen for the cluster method since the method determines the number of clusters OutGroup When Neighbor Joining is chosen for the cluster method the OutGroup option is enabled allowing the user the ability to set the number of Out Groups to display Exporting and Printing the Dendrogram Print and Export are selectable under the File menu Select Print to send the report to a local or network printer Select Export to export the results to the clipboard or to file When exporting to a file there are two file types that can be saved standard text file txt or a comma separated value file csv When exporting to Excel use the csv file type to skip the Excel import wizard or use export to clipboard followed by Paste in Excel Display Type Under the View menu the user may select to view the dendrogram in the Phylogram or Cladogram format Collapse Expand The Collapse Expand tool allows the user to collapse or expand the dendrogram by clicking on a red intersection marker th fon Zoom In Out Tools The zoom in and out tools
54. seconds minutes and hours are available The following list describes ideal exposure settings for different applications e For most white light applications using a 50ms exposure is sufficient Final aperture adjustments can be made to optimize the image quality an exposure range between 8ms and four when performing any is recommended for these applications adjustments Selectio e Fo such as chemiluminescence a longer exposure time may be appropriate In these cases push the red Acquire Image button to directly acquire the 34 image upon setting the desired exposure time If a good exposure time is unknown an alternative method is to utilize the Auto Expose feature configured to super sensitivity Auto Expose image generation with signal intensity showing generally completes in less than ode can be selected to generate a better resolution image For further images the software automatically calculates the new exposure time NOTE When the system switches to Expose Preview mode the image may flash or change brightness due to camera photon collection from the image over a longer period of time prior to sending the image to the computer s display readout 35 Light Source amp Filter Controls The mechanical controls on the cabinet and the linked FluorChem Q software virtual cabinet control interface labeled as Cabinet Controls provide near real time synchronization and updating of light source a
55. the count by clicking on the ZERO button This will erase all of the count markers from the screen and zero the count data to allow you to begin counting again with the same image or another image that you have acquired 200 Analyzing Arrays The ARRAY analysis tools found in the ToolBox Analysis Tools make it easy to measure the relative gray levels of objects in a uniformly spaced array such as microtiter plate wells or dot blots These tools allow spot number orientation and size to be specified For microtiter plates two options are available measuring the density within a circle well or measuring a central spot and a surrounding torus halo separately Wells Count Radius Horizontal 2 Inner 9 Verical 3 i Outer 10 4 Scan Blank Skewing Ces Result Values Actual Values 3 Percentage Figure 5 62 ARRAY Toolbox Once these parameters have been set a template can be saved in a default file and recalled at a later time See Section 3 5 for details on saving defaults Setting up an ARRAY Template To access the ARRAY tools open ToolBox Analysis Tools and click on the ARRAY button A template is displayed on the image depicting the layout of the objects to be measured Up to 10 000 objects in a 100x100 array can be measured The default is an array of 96 circles arranged in 8 rows of 12 eee 99 S 9X 9 9 9 9 9 9 9 9 9 o o 9 9 9
56. the image These can be changed to symbols or x by holding the shift key and clicking the right mouse button Repeat to return to numbers Spots can be hidden from view by checking the Hide Spots checkbox Specify the Size Range of the Objects If the image has objects of different sizes but only those in a certain size range are of interest use the Area Controls opecify the size range by adjusting the minimum and maximum diameter settings The numbers below the Min Area and Max Area headings indicate the minimum and maximum diameter settings in pixels Any object whose diameter falls between these two numbers will be counted As these number are changed objects will be added to or deleted from the display and the Count windows will be updated Using the ADD SPOT Tool Occasionally the automatic counting function may fail to count an object on the image This could occur for a variety of reasons including inappropriate Density and Size Threshold parameter settings Objects can be added to the total count numbers using the Add Spot button Click on the appropriate count box red or green Next click on the Add Spot button Point the cursor at the object to be added and click the left mouse button The object will be highlighted and the count will automatically increase Figure 5 11 Results of Colony Count After Manual Addition of Three Spots To add a second object click on the right mouse button to re ent
57. this is a user defined vs automatically detected peak To define a second peak click the right mouse button to reactivate the cursor Deleting Peaks A peak can be deleted by clicking on the peak and selecting Remove Peak from the Integration menu The selected peak s integration disappears and the integrated peaks to the right are renumbered accordingly A peak can also be deleted by clicking on its entry in the data table window and hitting the Delete key on the keyboard To prevent accidental deletions a dialog box will open asking for confirmation Visualizing Peak Placements To determine exactly where a peak is located relative to the original image it is often helpful to see the graph and the gel image in the same orientation The Show Strip function found in the Options menu displays the area of the gel that has been defined as a lane across the length of the x axis Graph With Show Strip Selected Note If Show Strip fails to show a portion of the image check that the appropriate driver for the VGA card is selected See Appendix X or Section 1 3 for more information Resetting Peak Edits The Auto Peak function in the Integration menu recalls the automatic peak finding algorithm used when the graph was first opened Note If the Auto Peak Detection Parameters height width and area have been changed these will be the variables used with the algorithm and the peaks may differ Clearing All Peaks To delete
58. using the calculated exposure time until a different mode is selected Switching from Expose Preview mode to Acquire Image mode does not reset the calculation If the system changes from Expose Preview mode to Acquire Image mode after calculating the correct exposure time in Expose Preview mode images are acquired using the last calculated exposure time Similarly if a different sensitivity binning mode is selected after finishing the exposure calculation the calculation is quickly converted and no recalculation is required nit is the same as used in film photography also called a stop 1 EV will double the exposure time while 1 EV with halve the exposure time The general exposure time using EV is found by the following formula E VCompensatedTime Time 2 Where Time is the typical exposure time for the sample under test to reach full exposure as determined by the auto exposure tool Setting Exposure Time Without Auto Expose Once the sample is positioned and the lens is properly focused close the Multilmage light cabinet door and check to ensure that the appropriate illumination source is turned on In addition check to ensure that the cabinet door indicator in the Cabinet Control software interface indicates Closed Uncheck the Auto Expose option Click on th and select the desired exposure time in to configure the system for the desired image intensity quality Individual adjustments for milliseconds
59. 06 jh 8 7 EE r 10 120 Fag 11 79 10 Ts T cc m3 RB 1 E 11 146 ee 110 561 12 04 12 165 11 33 503 10 80 IE 205 3 51 ral fal 13 te 15 133 3 61 v 17 23 no C1 v3 1 Ac 17 Figure 5 37 Example of a Quantitation Data Table PEAK is the number assigned to each peak on the graph beginning on the left and moving right DIST is the distance in pixels that each peak starts from the beginning of the line scan WIDTH and HEIGHT refer to the size of each peak AREA is the integrated area under each peak and represents band intensity is the percentage each peak contributes to the total density The sum of this column will be 10096 for each lane Adjusting Peak Detection Parameters Minimum Area Height and Width Once the graph to be analyzed has been selected and is displayed in the upper right of the screen the tools in the lower left of the screen change to allow adjustment of the automatic peak finder parameters Auto Peak Adjust Min Area 100 Min Height 1U Min Width 9 4 x Figure 5 38 Tools For Adjusting Automatic Peak Finding Parameters There are three attributes that determine whether or not the automatic peak finder recognizes a region of the graph as a peak minimum area minimum height and minimum width Click on the 157 arrow s associated with the MIN AREA MIN HEIGHT and MIN WIDTH headings to adjust these settings As these parameters are adjusted the automatic peak finder r
60. 1 Jo eo ee tm Figure 3 10 AlphaView interface after CROP has been selected Reset and Clear The Reset option configures the Black White and Gamma settings to default settings Clear removes any annotations that are present on the image 72 The Image Menu The Image menu option provides the ability to perform a variety of image processing functions Setup Overlay Utili Overlay Extract Channels Channel viewer Equalize Arithmetic kr Conversion Flat Field Calibrate Register Channels Resize Image Information Figure 3 11 Image Pull Down Menu Overlay To ing d Mc use the OVERLAY function under the des File menu This function appropriate color channel images added together A simple way to acquire multiple images for this function is to use the Movie Mode function in image acquisition and acquire a series of identical images The Overlay Images option allows you to overlay up to three different images with three different color channels You can select the BROWSE button for each color channel and select the appropriate images to be used for generating a color image For example if you have a saved grayscale images of an identical gel taken with a SYPRO red filter for the red stain and a SYBR green filter for the green stain you can choose these images in the appropriate Red and Green Channels to generate a composite image with the red and green colors mapped onto the compiled i
61. 1 KB Test 1 xml XML Document 301 KB 1 28 MB Y My Computer Figure 3 30 Windows Explorer Dialog Box 88 The View Menu The View function provides the ability to control the display of the on screen control tools as well as provide image enhancement abilities GESE Window Help My Default tools position JP Zoom In P Zoom Out Zoom 1x Fit in Screen Figure 3 31 View pull down menu Default Tools Position Four 4 main control windows exist within AlphaView Tool Bar Contrast Adjustment Tool Box and Status Bar dO BONES Gea ele ae m Tool Bar Gamma Linear Log Show Grid Black YL white 3 90 25 Gamma 2 9 J 189 Auto contrast Auto Enhance Level mphnug nDne uUuu Contrast Adjustment Window 89 Enhancement Tools Analysis Tools ToolBox Window Ready Intensity 67 Zoom 100 X 180 Y 340 Status Bar These control windows automatically open when AlphaView is launched for additional ease of use and to generate a common look and feel Since these items are floating tools you can click on Default Tools Position to move all tools to the default locations for more intuitive operation Lastly except for the status bar it is possible to select and move any of the other windows to a custom location Zoom Functions Additional options provide the ability to Zoom In an
62. 100 Area of Band Region in pixels Average pixel level of Band Sum Area XY position of Band Region centroid Standard Deviation of pixel gray levels in Band Average X Y Position SD Pixel Levels Background Tab BC Sum Background Corrected Sum BC Average Area BC Average Background Corrected Average Average Bkgd Average Note Subtracting the Bkgd Average from the Average accounts for differences in area Bkgd Sum oum of all pixel gray levels in Background Region Bkgd Area Area of Background Region Bkgd Average Average pixel level of Background region Bkgd Sum Bkgd area Signal Noise Signal to Noise ratio of the band region Average Bkgd Average Bkgd SD Control Tab p LCN Sum Loading Control Normalized Sum LCN Average Area Loading Control Normalized Average BC Average of Experimental Band BC Average of corresponding Loading control mean of all loading controls LC Regions Specifies the channel and region number used for each Experimental Band and corresponding Loading Control e g Red1 Green 12 Positive Control Normalized Sum NCN Average Area Positive Control Normalized Average LCN Average of experimental band LCN Average of control band 100 Note If loading controls have not been applied BC Average is used If a background correction has not been applied Average is used PC Regions Specifies the channel and region number used for each Experimental Band and corresponding Band Con
63. 3 K5xP5 P4 P5 P6 X K4 K5 K6 K6xP6 P7 P8 P9 K7 K8 K9 K7xXP7 K8xP8 3x3 pixel neighborhood convolution kernel K9xP9 P5 is being calculated New Value for P5 106 Each element of the convolution kernel is a weighting factor also called a convolution coefficient The size and arrangement of these weighting factors determine the type of transformation the image will undergo Changing a weighting factor influences the overall sum and therefore affects the value given to the pixel of interest Sharpening Filters These filters can increase image sharpness and provide edge enhancement However image noise may be enhanced as well These filters accentuate the high frequency details of an image while leaving the low frequency content intact High frequency portions of the image get brighter while low frequency portions become black Sharpen level 9 high has the largest effect on the image Sharpen level 5 has an intermediate effect Sharpen 1 low has the most subtle effect on the image 9 different sharpening levels are available for optimization of the image Noise Filters This filtering process uses the values of the pixels contained in the area surrounding a pixel to determine the new value given to the pixel of interest The noise filter sorts the pixels in the neighborhood into ascending order and picks the middle or median pixel value as the new value for the pixel of interest 3 levels of noise reduction are available
64. 44 Specifying the Quantization Units Once the objects are defined click on the STD CURVE button The Standard Curve Toolbox will appear Enter the units in which the results should be reported e g ng ul pg in the Enter units box Enter units no Load Save Add items Remove selected 10 200 100 i578 40 5 51 55 Curve fitting Model Linear Equation ly 0 01069 7 519 Y Axis ec Average lw Graph Color control normalization Identify CE band ear normalization Amounts A G B PlotUnknowne Help on these tools Back Next gt gt Figure 5 28 The Standard Curve Toolbox Designating the Standard Bands Select Add Items and select the color channel in the above figure the Red channel has been selected and select the appropriate regions created using the object drawing tool Continue to select regions until all regions used for standard curve calculation are listed For each band whose value in the above figure Red background corrected average is selected is known enter the concentration in the last column as shown in Figure 5 40 Band Red BC Average ng 4 10 200 100 4578 40 Ear B5 Figure 5 29 The Standard Curve input for known concentration expressed in nanograms in example above In the Band Sum concentration box displayed enter the value for the band When the value is inserted click on the OK button The band number
65. 45 angle until the circle encloses the area of interest To draw a freehand object select the freehand icon the mouse will change to a pencil Draw an object of the desired size and shape When the mouse is released the start and end points of the object will be connected Once the mouse is released the cursor changes to an arrow indicating that AlphaView M is in edit mode and the object is shown in gray with handles around it The object can now be resized using the handles or repositioned by clicking within the boundaries and dragging to a new location To draw another object return AlphaView to drawing mode by clicking the right mouse button If a box or ellipse is large and includes significant background area the corresponding density value may be large The higher the background the greater its contribution to the total of pixel values in that object Therefore objects should be drawn so that they fully enclose areas of interest but do not include an excessive number of background pixels or pixels from neighboring regions s M u Ge Object too Large Object too Close to Band Recommended Object Placement Hint Keeping the data window open can reduce the speed at which objects are drawn and manipulated on the screen Therefore it may be preferable to hide the data window until all objects and backgrounds are drawn and in place Hint Accurate Local Background Correction method requires the
66. 5 4 Molecular Weight Cursor Box The MW CURSOR box found in the Molecular Weight toolbox reports the location on the y axis the molecular weight and the R value of the current position of the cursor These data are updated as the cursor is moved This information can be helpful for positioning the cursor in a precise location or for a quick estimate of a band s size 121 The Graph Tool The Graph function is found in the lower left corner of the Molecular Weight tools box E PU D Markers Query Graph Protocol Report Calculation Method C3 Least Square Fit Show Graph Point To Point Fit Molecular Weight Cursor Position Mal weight Relative Freq 470 0 000 335 Shaw Band Number Mol Weight Help on these tools M Figure 5 5 Molecular Weight Tools Clicking in the Graph box displays the semi log graph of the molecular weight data that is used to calculate the molecular weights of unknown bands To remove the graph window from the display click on the Graph checkbox a second time The x axis corresponds to the band s vertical location on the screen and the y axis is a log representation of molecular weight By clicking on the appropriate button the data can be toggled between a least squares fit and a point to point fit MW Markers n x MW Markers nx Molecular Weight Point To Point Fit Least Squares Fit Molecular Weight j I j I 222 8 244 1 255
67. 603 36 717 885 4 482 2 531 1 952 18 947 077 2 313 5 85 923 183 10 489 8318 1571 36 074 316 4 404 2 456 1 949 18 321 893 2237 6 82 182 354 10 032 8 500 1 532 36 259 025 4 426 2 427 1 999 15 673 527 1 913 C Help on these tools 7 84 440 555 10 308 8 786 1 522 36 574 588 4 465 2 466 2 000 17 645 765 2 154 8 95 538 570 11 662 10 114 1 543 42 956 527 5244 3 019 2 225 17 591 983 2147 9 99 154 899 12 104 10 602 1 502 47 595 698 5 810 3 361 2 450 18 224 839 2 225 AGB 346 2459 1685 Zoom 79 X 849 Y 263 3 05 PM m Appendix X doc Mi Y AlphaInnotech Pot Select the Blue channel and then select all Regions to identify them as Loading Controls G5 Y Alphalnnotech Potatoe File Edit Image Setup Overlay Utilities View Window Help Boles oOo e eo Be LU Main A x phospho tif Gamma Linear Log C Show Grid Back O97 LNA White 9 4 Gamma 9 9 JL 1 00 mmi Display composite Auto Enhance Level BBB DEORE Enhancement Tools Analysis Tools Loading Control Normalization s loadin Identify Loading Controls iden ro Identify Experimental Bands Clear Loading N ormalizatio lt Band Control Normalization Band analysis results All Channels Identify Control Band Normalizes AY us experimental CN Display Style deny Experimental Bands bandslo 3 i
68. 9 Exam SOS TUT RESUS oaeiai n O EEANN AE NE AEE t EEA E A 110 MOVIE MODE eainnt rm 111 SAVING An INGIVIGUALTING GE T TOM MOVIES sis e ETE E E T oe dor ee vt ri dca niae dede idt ind 112 SaypupgsPartolty acquired MOVE coire a outeotoin fubtus dut loft dde Pe dlc bebo t tofu e dez cet au dd 112 Movie Mode Save Load Movie Mode setup routines eee eeeeeeeeeeeeeeeeee eene nnn hann nnn nnn n ns 113 Frane SACAT T Sau ok apeeuae 113 CHAPTER 5 WHE IMAGE ANALY SIS TOQUE i5 aeeeeeeccodvsse cbues ae eee eae eo eu vas etes oboe aae see ene aee asus eccca deseos 114 DEFAULT ANALYSIS TOOL S I TE 114 MOLECULAR WEIGHT DETERMINATION meriete Talai tuse niaaa dpe dent bon santa meme letancnenntemegedeuebonsuatieomalantees 114 TTT OAV OM ste sesh ctv te TEA E ily testes ss gaa a DLL rp 114 Entering Known Molecular Weights for Markers cesses eene eene enne nennen nnne nnne eene nns 116 Determining Molecular Weights of Unknown Bands cies eene eene nnne ess na annee essen nns 117 Using ihe Molecular Weight Standards Libr ry uai cn eae a 116 CCI COIS vote eL IMEEM d LAM UE LE UM Rc A IR ee 121 COCON T C OLDNIE S itte dte Dt lA tus AMA rM d tte 123 EE OO See ee eae LI E IM II I Iu M E 126 SPOR COUN UNG sed sd MM E MEME MEM ME EE 126 NIUETIPEEX BAND ANALYSIS TOOLS prdor iini detto Epp nett ne vane Eb uhr iR b RE bEopdu e be M sette dite EE S E HE e ERE ERU 129 Cr
69. BlueLCN Avg RedBC Average RedLCN Avg PCNAverage NC Regions Fold Change 2 11 629 9 998 921 792 34247 Red2 Red1 4342 3 9 229 3 398 1 507 1 633 70588 Red3 Red1 7 06 4 9 333 9 998 2 043 2 189 94604 Red4 Red1 9 46 5 8 918 9 998 1 966 2 204 95261 Red5 Red1 9 53 6 8 500 3 398 1 639 1 928 83351 Red6 Red1 4834 C Help on these tools 7 8 786 3 398 1 879 2 138 92435 Red7 Red1 9 24 8 10 114 9 998 1 857 1 836 79371 Red8 Red1 7 94 9 10 602 9 998 1 911 1 802 77832 Red3 Red 7 79 Ready RGB 414 2439 1732 Zoom 79 X 790 Y 7 fe C Documents and 5 Zu cul Appendix X doc Mi Y AlphaInnotech Pot Microsoft Photo Edit Select Identify Experimental Bands and select regions 2 9 De activate this tool by pressing Identify Experimental Bands or right clicking Note the data column Fold Change is populated The Fold Change column represents up down regulation in phosphorylated protein relative to baseline levels of phosphorylation in the reference sample Lane 1 The Fold Change value is calculated using the Red LCN Avg values for each band compared to the Red LCN Avg value for the control band band 1 e g for band 2 Fold Change Red LCN AVQBand 2 Red LCN AVQ control Band 1 792 231 3 42 Recall that Red LCN Avg Red BC Avg Blue LCN Avg Blue BC Average for each band and recall that Red BC Avg pX Blue BC Avg X pxX When the value for Fold Change is calculated Red LC
70. Comment Figure 3 7 Printer Dialog Box For more information on using the Print menu see the Windows manual 70 The Exit Function The Exit function closes AlphaView To restart AlohaView from Windows double click on the AlphaView icon The Edit Menu The Edit menu provides the ability to copy crop and remove any annotations or filters that have been added to the original image males Image Setup Edit Activation Copy Crop i i Reset Clear Figure 3 8 Edit Pull Down Menu To activate the Copy and Crop functionality place a check mark next to EDIT ACTIVATION This will turn the mouse cursor into a sign that will allow you to highlight the region of interest for the image After Edit Activation is highlighted the desired area of interest is drawn using the mouse Y Alphalnnotech AlnhaVicew re RET trace Setup Overlay tities wew window Heip p ejes GOBUCO a S E d UR Reset Figure 3 9 Ready to Crop or Copy 71 Once this is completed you can select either the COPY or CROP function in the EDIT menu options COPY will copy the desired area of interest into the Windows Clipboard and allow you to paste into any desktop publishing package i e Word Excel Adobe Photoshop etc CROP will display just the region of interest as the active window in the AlphaView interface N Alphalnnotech Alphat bow D ETE IM en ee eee og eem Zample br Sample Crop
71. F file will allow users to double click TIFF files from Windows Explorer and automatically launch the application on any machine that has AlphaView loaded on it Users may customize this in the preferences section covered in section 3 4 of the manual if they wish to change the default file type extension MAC so they can be easily distinguished from Windows TIFF files Most software can distinguish between Mac and Windows TIFF formats and can accept either AlphaView offers the option of both formats in the event that only one of the two is acceptable GLP is a proprietary file format that allows changes to only be made in AlphaView M programs It will accept 8 bit and 16 bit images and can not be opened in any other software program BMP PCX TGA PIC JPG GLP are additional graphic file formats which may be useful when saving an image for desktop publishing These file formats can be imported directly into many Macintosh and PC programs See Appendix A for more information Do not use these formats to save images that will be analyzed later since pixel data can be lost or altered when saving files in these formats Note Not all of the file types listed above can be saved as a 16 bit file The AlphaView software allows 8 bit Tiff images to be saved as BMP JPG or Tiff format Ceres naa some may require you to convert the image to an 8 bit file first Original versus Modified Files An Original image file is one in which the data
72. FLUORCHEM Q USER MANUAL Alpha Innotech Gel Imaging Systems for Life Sciences Copyright C 2009 Alpha Innotech Corporation All Rights Reserved Introducing AlphaView AlphaView provides the utmost ease of use while offering comprehensive and versatile tools for capturing analyzing and annotating images With a simple to use graphical user interface coupled with new and improved features Alpha Innotech has pioneered the most intuitive image capture and analysis software available AlphaView s new and improved features include multiple image viewing the ability to save analyses and an enhanced movie mode With our suite of analysis tools you can perform molecular weight calculations R determination lane profile densitometry multiplex band analysis microtiter plate reading object distance measuring gel scoring and automatic colony counting In addition AlohaView s image optimization tools can adjust contrast automatically or manually convert images from positive to negative using digital filters apply false color and utilize many other techniques to clarify difficult to see portions of the image Notes labels arrows lines and other drawing tools can be recorded directly onto the image using AlphaView s annotation features Annotations are superimposed on the image upon hard copy printing and can either be saved as a template file or as part of the image itself All AlphaView features are accessible via convenient
73. File Edit Image Setup Overlay Utilities View Window Help BOWee GOSS eS e Be Main A xj Quant tif 4 bx Gamma Linear Log al C Show Grid Black LNA Whte 7 9 9L s Gamma 9 9 9 1 00 Display composite Auto Enhance Level LeeLee Enhancement Tools Analysis Toos Colony Cout J Bkgmd Control Std Curve Protocol Repo gt M Loading Control Normalization Identify Loading Controls ro Identify Experimental Bands E Clea LoxingNomakao e controls Band Control Normalization Band analysis results All Channels RX Identify Control Band Normalizes i Un experimental Export View Display Style Identify Experimental Bands bands to a F positive control Band Blue LCN vg Green LCN Avg Red LCN Avg Clear Band Normalization 1 7 360 7313 11 053 2 7 960 8 738 11 153 3 7 960 7 434 11 102 4 7 960 7 213 11 252 5 7 960 6 868 11 788 J 6 7 960 6 792 12 985 C Help on these tools 7 695 3181 943 8 785 13 622 1 128 3 673 5515 1 027 10 l 11 j 12 RGB 6912 5689 5832 Zoom 113 X 2 659 Y 101 cul Appendix X doc Mi Y AlphaInnotech Alp Microsoft Photo Editor website fi C Program Files Alp SAn Notice the data table shows the Loading Control Normalized LCN values for all th
74. IN CDD D OE 98 Figure 4 S Texr Style Selection T OOS isis cereale aou e ca dt uto a tus ect el aoelo Uca n Ue p DEN UE DE 98 Figure 9 Font Selection WIBOOW accoist sueta e cltumuea t custode omen Ud Puscntbrd bout div ades t e Doct suem D Ss i e pMpE DUE 99 Figure 4 10 Text Orient Selection TOOS o erit toro o cue des bce qvae tus ccs utu ta etes NUM DE DUE 99 Fig re 4 11 Sample ADioLatlOfls modus secl ai sociata ques E cocti Landuan te tuc UN Uc MM OIL a 101 Figure 4 12 Ee i eisROle rd et 102 Figure d 1 3 False Color Selection BOX icm di ea seas ee o Pee te vor Ye oes euet roov ea ue t vese e eu eue tee Ns 103 Figure 4 14 Filters Toolbox with 3 D contour selected seeeesseeeeereeeenneeeennnn 105 Figure 4 15 The Filters Toolbox with Sharpen highlighted cccccccsseeeeeeeeeeeeseeeeaeeeeeeeeeeeeseeeeessageeeeees 105 Figure 4 16 The Filters Toolbox with More selected ccccceeccccsesececeeeseeccseeecseuseecseseeessaseeessaneesssaees 106 Figure 5 1 Default Analysis Tools in the TOOIBOX cccccccccssceeceeseeeceeeseecceeseeecsaueeeceeseeessaseesssagseesnsags 114 Figure 5 2 vlolecular VV GIQNE OO lS sass 5 9 5 adtrapiid c mic asbl perd dolia cado v dam paci amate dud scel nile da eau baut dd 115 Figure 5 3 Molecular Wagn Data BOX uice disodeiladexibstun a iuter liia siat ats te opaco Rutas 115 Figure 5 4 Molecular Weight Cursor BOX iue soot iet pesa ren isla gesunvt cma Cosa e stus ledbu s
75. ING FUNCTIONS oss casera tees bete ed feu abiit qutut feu bab ru a tat eua be ecd tp do teens 25 ACQUIRING AN IMAGE USING THE NAVIGATOR 2 55 nnu a ete pn rave debt date tee rae ud te p redde 26 CAMERA SETUP AND PREY IEW WINDOW amp doitusbtvi beh OM REA tous woah take sant CONDE bo CEU REQUE e IRAE OU ODE dob LSU E EDE OU Uis 32 AU OL DOSE ceste ordered ts mda SM en eee nae E nti ts t nune go Setting Exposure Time Without Auto EXPOSE a er itte etae ja RR ba te autas tede se te blatuuden esta T 34 Lohr SONrce o Jer C ONTO S messes ta ted ENE toten i tunt esca itta teure ee Salat cited meta Cet 36 Turbo Modes and Speed ResolufionSettugs s ue ea OE tdt ee edu ERN eoe ate cun vases aaa tede tet 27 Gray Scale Optimization for Saturation and Contrast Displays esee eene ener nennen 39 VADE 40 DOVME AICS s RE eo e NEU ee Mam etna a dst ROS 41 IVA OWS VIO s Lr M 41 AUTOMATIC IMAGE CAPTURE AIC SOFTWARE aise tect ies iene esu eras hue Bat eat pace curls E besote esL Id sue De eS LIE 44 D s enno User TOLO CON PE 44 Awo Image Cap UTE AIC NEE aw 46 COMPRARE VIEW unine I NEE eh acc E R A O O N 47 ALPHAV IEW SYSTEM OUICK OUDE rrenari AO ENNAN EEO NEN EE 49 CONTRAS TADIUSTMEN T stapes a a a a a ms auaaueeatneas el emaneiae 51 Black Lect Ad Une D T ETE ONE EE NEN E E DI lai dda OE E ETON 52 Winte Ty Cl AUASI ass E E E Mot adu MM EA EE E E O EN 52 GGG SOT E AO SIm
76. J26 1 fob Curve fitting Band analysis results All Channels l E Equation y 0 003445 x 2 217 Export View Display Style ie LCN Average ine Green LEM Avg Green ng Hed LEM Avg Hed ng PCM Average F Color control normalization B r3 12 385 14 38 943 1 03 3 181 Identity CC band Clear normalization 13822 1128 1 67 426 84 5 516 26 40 Band 7 Std curve units 14 38 32 172 85 Amounts Fi G B 2 263 9 56 11 70 90 1458 5 42 1 555 314 46 00 Help on these tools 1327 465 1 748 3 80 35 32 7 6 5 195 fra 4 002 ro 1 343 E53 J26 433 fob ofa tid In the figure above the values in the results data table display the nanogram concentrations of phosphorylated protein represented by the green channel data in the column labeled Green ng Hover the mouse pointer over the column Green ng for Band 7 and the tool tip displays the band number and the data value The value for Band 8 displays a indicating the concentration is outside the range defined by the known concentrations used from the red channel data to construct the mass standard curve Only values within the defined range are accurately quantified G13 APPENDIX H REGULATORY COMPLIANCE FCC Statement This device complies with part 15 of the FCC Rules Operation is subject to the following two conditions 1 this device may not cause harmful interference and 2 this device must accept ay interference recei
77. LCN Average Area BC Average of Experimental mean of all Loading Control Normalized Average Band BC Average of corresponding Loading control loading controls Specifies the channel and region number used for each Experimental Band and corresponding Loading Control e g Red1 Green 12 Band Control Normalization Normalize regions of experimental bands to a reference band control First select the channel and identify the region for the positive control and then identify the regions for the experimental bands The control band and the corresponding experimental bands must be in the same channel Multiple groups of positive controls and experimental bands may be identified and each set may be in a distinct channel Identify Control Band Identify the control band using the mouse pointer Select the Identify Experimental Bands tool to identify the experimental bands to be normalized to this control band Bands can be selected You can either left click on each of the regions or draw a rectangle to select a row of regions Deselect the Identify Experimental Bands or right click when done 142 Figure 5 26 Band control normalization In this example region 2 in the Green channel was selected as a control band and regions 3 to 8 were selected as the associated experimental bands Then region 9 was selected as a control band in the Red channel and regions 10 16 were selected as the associated experimental bands Note that the reg
78. LL MAU LI CU MILL E ME UESELE E E OU Dico E E iat sobud SES EES c Ma M E ad LE reel A Eb DS M fe rS E Re E Placute MAKETS TOC OUI LA assai ais inest udo odd inse dta oues ta elu eu tib NE Erasint and Hiding Count MUEKOES eem T des d mee uq ub eue Ext dass did dme conos Dee adus ud Erdsine the Count Markers and Dala s qeu ese desse oua eese totae t tue Conan uda tue dua ided ANC ZING ARRAY Saco edt Ea EEE E A EEEE E dr nura lee deli AE A p TE Lula Re EEE EM SCUING UO n ARIAY Template ssidodiadmustnes tta i i assa One tradotta du exa ues E QUOD Analyce arrays put CIICLES OF S JUNE S es aseotiie tisse tutius utet eise fer iudei dde curate uides UIP OWN E TP CIM DIGIC cette cc tiet obe oes edes tutibudu oe best tefle oe futi tudo orb audeo edad SPECI WING INE Aveds TO De MOAOSWIOG aceti octets o Pul a Tube idea ful toad ARA Meast nE POSU V deiade tesceive i SE a todo bu Tonanti da ld dose un Lpte Cute Roots apad IREINVERT DON ssid thie tease te T cet E went Removing Background using the Scan Blank Function eese COMMON EXPORT RESULT FEATURE Cu ospatictes tae tex ete vi omae E eoa ven Ma ed RO T CDL APPENDIX A OPENING ALPHAVIEW FILES IN OTHER SOFTWARE PROGRAMS APPENDIX B ALPHAVIEW MOLECULAR WEIGHT LIBRARY FILESNS APPENDIX C DATA TABLE DESCRIPTIONS eee eee e eee eee ette sees eee eet enss sese e ee eoss sese e ee eoo APPENDIX D BIAS AND DARKMASTER UTILITY
79. Mult region and select the Mult Region Copy tool Region Copy tool Auto Center Automatically center Auto Center Automatically center C Invert Data region around a band C Invert Data region around a band Inverts data for opaque Inverts data for opaque Auto Detect hands hands Auto Spot Help on these tools Help an these tools Single channel GUI Multi channel GUI Creating an Object Area of Interest Single Region Tools LOJLBULZ An area of interest can be created in several ways The user can manually draw an area of interest through the three OBJECT buttons Objects can be enclosed with a box rectangle ellipse or freehand drawing Select the one that most closely corresponds to the shape of the objects on the image Object Boxes To draw a rectangle or ellipse click on the button labeled with a box or a circle beneath the OBJECT header in the toolbox The cursor automatically changes to a when it is in the image area indicating that AlohaView is in drawing mode Move the to the corner of the object to be measured click the left mouse button move the cursor and release the mouse button when the box or circle surrounds the object Hint to draw a perfect circle around a portion of an image first visualize a Square surrounding the area of interest Position the mouse in the upper left hand corner of the square Click and drag 129 the mouse down across the area of interest at a
80. N Avgpana 2 Red LCN Avdgana 1 the following substitution can be made Fold Change Xganq 2 Blue LON Avg X OX panazo PXepand 1 Blue LCN Avg X PX pana 1 and notice the value Blue LCN Avg cancels leaving the desired result G7 PX pana X pX panas PX pana 1 X PX panas relative to the total amount of protein for band 2 compared to the baseline level of phosphorylation relative to the total amount of protein for band 1 the untreated sample Fold Chang gang 2 which is the level of phosphorylation Repeat the above for the other bands to complete the analysis The data table can be exported and Microsoft EXCEL utilized to prepare graphs Phosphorylation Response 10 ZL H T u s38 I Fi Bee ob ae E ts o a n 4c qi EFEHEHE ee BE BEBE it Baseline 15 minutes 30 minutes 45 minutes BD minutes 75 minutes 90 minutes 105 120 minutes mintes Time After Treatment Example 2 Quantitation across color channels In this example the multicolor blot includes a dilution series in the red channel and a treatment course indicating expression level changes for a phosphorylated isoform in the green channel There is also a specially constructed control sample that serves as a loading control as well as a control for color channel normalization Color Control In this example the goal is to determine the absolute concentration of the phosphorylated isoform green signal
81. Prone Ddbonnedites i os ba vod o bored fetu oce oscula asusta convi iacu ieu t man ares 162 Molecular Weight Mass and Band Scoring integrated into Lane Profile eeeessssssssseeeeeeeen 165 nod UN cM MIS M M LM C E m M EI 172 Data Table and Edine OT AiG Date o ste i bet m EEHEEEI ID rts vet ba od ntur ic edo coc edbadtt usa beet Pe oo bd opua 174 Gel Smiling Correction with Gel Smibline T00ladnscoein ida ree OE QU een Deva Seul obs uade enu qu A deers eiat 179 Band Matching Similarity Matrix and Dendtoprams eie ee Itt eevee ds vac eset den EdU aee eyed easel Vase tens acne eves 183 COMMON FEATURE Laude DRM NUR aiu ema sae a i aM Te DR M cUm aie 190 FOU OV pct m pm M 190 REVOT Rm 191 o LEID DIT Sc ccc P 191 Gencrdloesu onse aues MO UcU Oda A eer een Sent geen een Meee Tres erie er per E R er Ener Meer eee H UR eee 192 E XDOLDISOSUT esistente tolit ade t cate e attt tta holland iud reacts Coda E 195 Exportine Ouantitati ve Data Lane Profle uoa So Lr Er CEN ELT et Mus o Ter Eoo Lo LU CH EPI e ERR LER SL EDU SR DS ELI EAE 194 ADDILIONADAANAEXSIS TOOLS ees eas uua tS tto a cud MM mM M M E MM DI ep EIE 195 THEE 56 2 2 gl BAN Gale Gi epee ee ane Ecisi i rare enna area EL A VT OOUCH OM ee ate T A n Usine TNC Ruler F UN CHON eneas note tede ee bet edu Coe a HIE SCORING B UNCTIONSG d de era act ait ec hat umbrosis ioci ie ae t Lid SCOFINDUDHCHSQIIDI ORO oe me ner ere IE
82. Result Gel Smiling Mol wt Mass Std Scoring Band Matching Protocol Report Lane H s Score based on to Score Area v Select Scoring Method Present Absent M em otn elerence J CT LT ee Co ha Maximum X deviation of distance For band matching Select Heferene Object Band af sel Mw for all lanes Help on these tools Figure 5 43 Band Scoring 170 High Medium Low Absent This method is used to classify bands into groups based on Area Mass or Height The researcher will determine break points for each classification high medium low and absent The software will place each band into one of these classifications depending on the user entered criteria Additional Criteria When choosing the High Medium Low Absent method the user must enter in values to use as break points for each of these classifications The values entered are percentages of the reference band based on the parameter chosen in the score based on field Tools Result Gel Smiling Mol wt Mass Std Scoring B and Matching Protocol P leport I Lane H s Score based or to Score Area a I Select Scoring Method e High Med Law Absent v z Med Low lt Absent lt b Lo e ox Maximum X
83. Rf Matching is selected the software will match bands based on the data values in the Rf column of the Profile and Data window These values are the relative Rf calculated vertically where the origin of the lane is set to zero and the lane front is set to 1 Gel smiling or curvature is not adjusted for in this selection Matching Reference Select a lane to serve as the matching reference It is advised that you choose a lane that either contains most of the bands of interest or spans the range of bands of interest while also containing many of the bands of interest This will reduce the amount of effort required for post match edits Matching Tolerance Enter a value to be used as the matching tolerance The tolerance is converted into the amount of displacement two bands may have and still be matched by the software The larger the tolerance value the larger the range the software will use for matching The valid range is 0 10 A value of 1 5 typically works best A lower percentage works well for tightly spaced bands Auto Match After selecting a matching metric reference and tolerance click the Auto Match button for the software to perform auto matching When matching has been completed the Display Results and Generate Dendrogram buttons will become active Refer to the appropriate sections below for a description of Similarity Results and Dendrogram creation Editing Match Results Following Automatic band matching the user may need to ma
84. USB port is used The Mitsubishi CP700 and CP770 printers connect via the on board parallel LPT1 port Plug in the standard three prong power cable on the back of the printer and into the power strip then turn the power on Set the printer up for color or black and white printing following the directions in the owners manual supplied with the printer 22 Power up Sequence Once all connections are made between computer cabinet camera printer and monitor power can be applied in the following sequence 1 Monitor 2 Computer 3 DE500 cabinet 4 Camera power supply the camera will begin to cool to 10C 5 Printer After the cabinet filter wheel has finished homing launch the AlphaView software to begin deep cooling of the camera minimum cooling period is 30 minutes Starting AlphaView FC Q System with AlphaView software To launch the AlphaView Imaging System software from Windows double click the AlphaView System icon on the Windows desktop M Alpha View 23 System Information To display system information select the About option in the Help menu This button accesses a pop up box About AlphaView AlphaView Software Version 3 0 0 0 Product Rey XXXXXXXXXXXXXXXX Alpha Innotech Corporation Copyright 1993 2009 All rights reserved Wwiw lphalnnatech com Alpha IInnotech Figure 1 12 System Information This box shows the Software version number Use the information specifi
85. V Results may vary for different software versions and or hardware configurations Adobe Photoshop Adobe Photoshop Canvas Microsoft Word Microsoft Word Microsoft Excel Microsoft Excel NIH Image 2 51 LE Mac 3 0 Mac 3 5 Mac 6 0 Mac 6 0a Win 5 0 Mac 5 0c Win 1 59 Mac Open no use ResEdit Insert Picture Insert Picture no Insert Picture Open no Open no Insert Picture Insert Picture no Insert Picture no Open As TIFF use ResEdit Insert Picture Insert Picture no Insert Picture Open no no Insert Picture no Insert Picture no For instructions on using ResEdit see next page Additional packages such as Claris Works and PowerPoint have also been tested For these systems it is necessary to save the file with a TIFF extension in order for them to recognize the file as a TIF format If you have a specific software package that is not listed here contact Alpha Innotech for a diskette This PC formatted disk contains a gel image saved as five of the file types that AlphaView can generate Try opening each of these files in your software package to determine compatibility We recommend that you start with the TIF file as this is the default Alpha Innotech file format A 1 Using ResEdit 1 Save image in IS 1000 500 as a TIFF or MACTIFF 2 Obtain a copy of the freeware ResEdit by downloading from Apple Computers through the Intern
86. Values of 0 are mapped to cyan saturated to yellow and values in between to magenta Palette 6 maps the gray scale levels to a cyan magenta yellow palette Values of 0 are mapped to yellow saturated to magenta and values in between to cyan Palette 7 maps the gray scale levels to a cyan magenta yellow palette Values of 0 are mapped to magenta saturated to cyan and values in between to yellow Palette 8 maps the gray scale levels to shades of red palette This palette may be useful when viewing red color images Palette 9 maps the gray scale levels to a red palette Values of 0 are mapped to dark red saturated to white and values in between to shades of red Palette 10 maps the gray scale levels to shades of blue palette This palette may be useful when viewing blue color images Palette 11 maps the gray scale levels to a blue palette Values of 0 are mapped to dark blue saturated to white and values in between to shades of blue This palette may be useful when printing an image of a Coomassie stained protein gel onto a color printer Palette 12 maps the gray scale levels to shades of green palette This palette may be useful when viewing green color images Palette 13 maps the gray scale levels to a green palette Values of O are mapped to dark green saturated to white and values in between to shades of green This palette may be useful when printing an image of a SYBR Green I stained protein gel onto a color printer Palette
87. W standard Note the new Checkbox in the Molecular Weight Standard dialog window labeled Calibrate Mass using MW std lane Scoring Band Matching Protocol Report m Tools Result Gel Smiling Mal Wit Mass Std Select Mw Calc Linear Log iv pen Marker File Save Marker File Add Marker Delete Marker Clear Markers Calibrate Mass using Mw Standard lane The option must be set before applying marker file to a lane Enter total mass per lane Select Mass Cale east Square Fil Help on these tools Checking the checkbox activates the Enter total mass per lane field and reveals the choices for calculating the Mass values scoring Band Matching Protacal Report reru ER Tools Result Gel Smiling Mal wit Mass Std Select MY Calc Linear Log Ww Upen Marker File Save Marker File Add Marker Delete Marker Clear Markers Calibrate Mass using Mw Standard lane The option must be set before applying marker file to a lane Enter total mass per lane ooo Select Mass Calc Least Square Ft v F Help on these tools 166 Procedure If you would like to calculate Mass based on your MW Standard Lane follow these steps 1 Check the Calibrate Mass using MW std lane checkbox 2 Enter the total mass that was loaded into the MW sta
88. Windows conventions 255 characters or less enter a new file name in the text box below the File Name prompt AlphaView will automatically give the appropriate 3 character extension The current directory path is shown beneath the Directories heading Below this is a graphical depiction of the path and the sub directories of the current directory If the file is to be saved in a different directory than the one open double click on the appropriate folder icon Alternate disk drives can be accessed using the Drives heading Once a name has been entered and the appropriate directory has been accessed click OK All of the marker values that are currently in the Data window will be saved to this new file To access them in the future follow the steps in Applying a Set of Saved Markers above 120 Special Functions Snap to Peak This feature makes it easier to place the cursor on a band If the SNAP TO PEAK function has been activated the cursor jumps to the highest pixel value in the band as the cursor approaches it If bands are so tightly spaced that the line is not coinciding with the band turn off SNAP TO PEAK and place the marker on the band manually If the cursor is jumping to areas between bands instead of the bands themselves check to see that INVERT is properly set See below for more information The INVERT Button The INVERT function reverses the gray scale assignments so that 0 corresponds to white and 4 095 corresp
89. a box shape around the sample usually including a significant portion of background User preference and image particulars will determine which outline type is best Previous Location Previous Location will automatically select the spot on the image last selected for magic wand and apply any changes made for sensitivity This feature is designed to quickly evaluate how the different sensitivity numbers will affect the drawn area using the same selected point in a spot The X Y coordinates are given for the previous spot as well Exit Exit will remove the Magic Wand Parameter Window Auto Spot Auto Spot is a feature designed to find multiple spots within a region of interest It is accessed through AutoSpot under Auto Detect in the Multiplex Band Analysis tab Once it is selected the following tab will appear and the cursor will be ready to draw an area of interest 132 Auto Spot Spots Find Spots d5 Find Spots Q Dark e Bright Options pions Target Size Min Fisel Max Pixel Background Threshold 0 Saturation 2 Bound 4 p HO Suggested Upper Bound Figure 5 16 Auto Spot Area of Interest After selecting AutoSpot an area should be drawn on the image by clicking and dragging the mouse The smaller and more defined the Area of Interest is the better the resulting data The area can be manipulated using the manipulation tabs at the edge of the red box Options The Options selection allows ei
90. a r m 89 IET o MOTO FOSON T 59 ZooM TIT ERROR EUM en 90 THE E IUD SAI ns ENT 90 TIE HELEEM NU eec nc cT 9 CHAPTER 4 THE IMAGE ENHANCEMENT TOOLS iere esee eseevs vae eoevqxeestes est ue vere Pe se eeneioe Suave reves au asees 92 SKEIE EOD TOOL teet Uu dM uA DIE KU MEUM LIE UU uM 02 PPS POG RAW ues ND MI x UCM M IU cM A E M E 93 EEE ROTAPES PLIP LOO bc n ico cand cc thse toca i oM esten mni dd MM ME 94 ANNOTA TIONS esent ota On ne TY INE MH MS UE MM M terete 96 OEC AUD OS casi ota enna te EI MM M UM IE D a 96 The qoM TOO Eat E EE E ML E 100 The Edumo A OOIS kane ud E aate tale eu beate ro mute A ee tesa EEO AER 101 EDE COLOR cutem db iad teinte E UE S Ese D edULU t aon i SOL 103 NAGE EIET E oaea aa umma dietbensse codivbntuete inte dUbsdbeoused sedi ut E obolis tee dU LnE 105 Genera In Orma S ERR NETTEN 106 DI DOE ELLE Re OPE EE OE EID LAM DAMEN I M II ELM MUI 107 UT RCNH E N UR EET RE 107 Desnec e Mer netu LI RM MCI IM OL LOr oe Pret IMMEIMEE LEE T Pere 107 DELE COBIOI EUIS tsctou LO MICI LM aa M LM ME MM t EE 107 SMON OE HIG LITE UM MI EEUU ME RE CL M IM RU LU I EE ert ee 106 Ep T ES ceed IEAA S MOOR E ie Rd Ud ES Rott ce ue A AANE AAE ceased 109 HOn OAT ARET OE neis astu tote tana tu esae du auae fsa tse e E totis sa tio os abut d asada Pe QU E a e uiedd tud 109 WETICE 28 TII AEN NP 109 CONECT U OT RITE POTRETE 109 TREUN PO BUNOT MEET 10
91. ab and select Identify Loading Controls In this example Loading Controls are represented by the blue channel G4 Y Alphalnnotech Potatoe File Edit Image Setup Overlay Utilities view Window Help BOs COCHO eS o OA Main mox phospho tif 4bx Gamma Linear Log C Show Grid Black O97 WL White ____ 0 _N4 Gamma 4 T J dae 1 00 TTE Auto Enhance Level Ce BAEC Enhancement Tools Analysis Tools Select channel Please select appropriate channel Bkgmd Control Std Curve Protocol Repoi Loading Control Normalization Identify controls then Identify Loading Controls dis a single row o experimental Identify Experimental Bands baide NET correspondin Clear Loading N ormalizatio to the ds controls lt Jii 5 Band Control Normalization Band analysis results All Channels E E E _ 4 x Identify Control Band Normalizes c EP experimental F Se ey Ee ae Sus fln Band BlueSum Blue Average Blue BC Average Blue Bkgd Average Green Sum Green Average Green BC Average Green Bkgd Average Red Sum PENE AEAN 1 118 094 017 14 416 12 874 1 542 45 540 806 5 559 3 617 1 943 4 463 562 545 2 108 347 064 13 226 11 629 1 598 41 364 218 5 043 3 146 1 903 9 744 097 1 189 3 88 801 405 10 840 9 229 1 612 36 376 321 4 440 2 559 1 882 14 543 474 1 775 4 89 586 621 10 936 9 333 1
92. aches it If bands are so tightly spaced that the line is not coinciding with the band turn off SNAP TO PEAK and place the marker on the band manually When the horizontal line is positioned correctly click the left mouse button A dialog box appears requesting the band s molecular weight Using the numeric keypad in the dialog box or the numbers on the keyboard enter the known molecular weight Alternatively enter the value using the keyboard After entering the molecular weight press either the OK button in the dialog box or the Enter key on the keyboard The dialog box disappears the number of the band appears on the image and the band s data is added to the Markers section of the data box The horizontal red line remains indicating that the cursor is ready to select the next band as described above After entering the value for the last marker click the Add Markers Complete button instead of OK This will deactivate the value entering function and will return the cursor to its normal mode To reactivate the value entering function select Add Marker from the menu again or click the right mouse button The Molecular Weight Data Box The data box is located near the top of the image Initially no values are displayed in the data box When a molecular weight value is assigned to a band the following information is displayed in the data box e Band numbers are assigned beginning with the first band continuing in the order sel
93. ae voce e Pee cR Yan N 193 Figure 5 57 Lane Prolll Export Dialog BOX daa peUt ioa casa ruv o cou EUR o e adu ve ric ip dca nac eve vus ra de ME Eus 194 Figure 5 58 Additional Analysis Tools in the TOOIBOX ccccsseeeeeeceeeeeeeeeeeeeeeeeeaaeeeeeeeeeeeesaeaneeeeeeeeeeaaas 195 Figure 5 59 The Ruler Tools and Ruler Toolbox 2 Iooeesk etre e bu aa voca baut Ue Unies 196 Figure 5 OO SCOLING COMUOIS segipen OO m OU E EET 197 FigureS 61 Manual Count WOO T E 199 Figure 5 52 ARRAY TOO DOX 5 tiendas bad ucs Ls a aT A 201 FIQure 5 63 ATTESA Y TeTTIDIBIS oat osindusibdeents bs dur og oa uk O rale raO t oder id pom oU O SOR dU TE VE PaadE0qe 201 Figure 5 64 Common Export Dialog BOX cccccccccssesseeceeeeeeeaeseeeeeeeesaeesseeeeeeeseeaeeseeeeeeesessuaaseeeeeeeseeaaas 205 Chapter 1 INTRODUCTION AND SETUP Introduction to the FluorChem Q Image Analysis System Introduction Th system is designed specifically for the acquisition of high quality images from multicolor Westerns The FluorChem Q produces uniform and consistent fluorescence images with the sensitivity and dynamic range needed for accurate quantification of multicolor Westerns Together with the advantages of multicolor fluorescence labeling for Western blots the FluorChem Q provides the most cost effective solution for your imaging requirements The FluorChem Q includes all the features of the FluorChem HD2 plus the light source
94. ails easier to see 3 D level 9 high has the largest effect on the image 3 D level 5 has an intermediate effect 3 D level 1 low has the least effect on the image 9 different 3 D levels are available for optimization of the image 107 In addition 3 D allows the user to control the direction of the 3 D shadowing effect Once the level is refined just click on a direction arrow to visualize the shadowing adjustment The default shadow direction is in the lower right hand corner direction and 9 different directions are available MEE AUTRE Oriainal Imaae 3 D Image Level 5 amp lower right 3 D Image Level 5 amp upper g g shadow middle shadow zn High LIP Low High rials Low High Pan Weesie od HEHEHBBHDUE on HBHHEHBBHDBE on Original Filter Toolbox Lower right shadow Upper middle shadow Smoothing Filters These filters are very useful for reducing the visual noise present in an image When a smoothing filter is applied to an image rapid changes in intensity are averaged out with the remaining pixels in the neighborhood thereby decreasing the high frequency content The visual result is a slight smoothing of the image because sharp pixel transitions are averaged with their surroundings omooth High has the largest effect on the image Smooth Med has an intermediate effect omooth Low has the most subtle effect on the image 108 Edge Filters These filters use Laplacian enhancement to highlight edges regardl
95. alization data table In this example region 2 in the Green channel corresponds to a negative control band and Region 9 in the Red channel corresponds to a negative control band The PCN average values for regions 3 to 8 show the signal level relative to the control band and regions 10 to 16 show the signal level relative to the control band 143 If a loading control has been applied to all selected bands then the loading control normalized LCN average values are used in the PCN calculation If loading control normalization has not been done then the BC values are used and if no background correction has been applied then the Average values are used Data Definitions PCN Sum Positive Control Normalized Sum PCN Average Area PCN Average Positive Control Normalized Average LCN Average of experimental band LCN Average of control band 100 PC Regions opecifies the channel and region number used for each Experimental Band and corresponding Band Control e g Red1 Red 6 Fold Change Fold Change of experimental band relative to control If LCN Average experimental band gt LCN Average control band then fold change LCN average experimental LCN average control If LCN Average of the experimental band lt LCN Average of control then fold change 1 1 LCN average experimental LCN average Note In the Data table there are columns available for Blue Green Blue Red etc These columns display the ratio of the indicated channel
96. all the defined peaks on a graph at once click on the Clear All Peaks function in the Integration menu 159 Adjusting the Baseline As a default the baseline value is set to Auto Base As the baseline is adjusted the values for all peaks are updated to reflect the new baseline All of the following functions are found in the Background Subtraction menu Auto Base This method breaks the red line defining the left boundary of the lane into sixteen regions and outputs sixteen points which reflect the average backgrounds across those sixteen regions The user can manipulate the 16 points by clicking and dragging The user may also designate more points than the 16 and manipulate those in the same fashion Intra Lane This method uses the lower of the two values at the edges of the lane along its travel as the background If the section of the image is used is wider than the bands then this is a very effective way of removing co migrating material from your measurements This method should only be used if the bands are completely enclosed This is because when the edges of your lane area intersect any of your bands band material will be considered as background severely affecting your results Rubber Band This method can be thought of as stretching a rubber band underneath the lane profile This option does not work well if the values at the ends of the intensity profile are lower than the rest of the intensity pixels We do not recommen
97. allows the user to change the visual display of the lanes and bands on the analyzed image e Under Band Representation the Cross selection designates bands with a cross symbol The Box selection will display a 2 dimensional box around each of the bands e Lane Property gives the option of either showing the lane name by number Numerical or by letter Character e Show Center Line selection displays the vertical line which runs through the center of each of the bands e Show Lane Bounds displays the perimeter of each of the lanes found in the gel e Show Band Properties turns the band property display on off The Band Property value to be displayed is selected under Band Property e Band Property allows the user to select what value to display as the bands property The choices are Position from top to bottom in Numerals Position from top to bottom in Characters Rf Adjusted Hf or Molecular Weight 178 Display Options Lane Property Show Lane Center Line Show Lane Boundary Show Band Properties Band Property Mame 9 Numeric 1 2 3 Mame 9 Numeric 1 2 3 C3 Character a b c Character a b c O Relative Frequency RF Band Representation 7 Q Ad QUE J Adjusted Relative Frequency C2 Box CO Molecular Weight Options Gel Smiling Correction with Gel Smiling Tool The Lane Profile Analysis module of the software contains the Gel Smiling tool which allows the user to adjust fo
98. ands Band Matching dialog window is accessible through the Analysis menu on AutoLane s Profile and Data window Select Band Matching from the Analysis menu Tools Result Gel Smiling Mal wt Mass Std scoring Band Matching Protocol Report Automatic o T Matching Metric Adjusted RF Matching v Matching Reference Lane 1 v Matching Tolerance 0 00 pi Manual New Band Type Display Option Show Result Help on these tools Figure 5 48 Band Matching Dialog window 183 Automatic versus Manual Band Matching Functionality is provided for both Automatic and Manual band matching Each will be explained in detail Note that these are not exclusive After Automatic Band Matching has been performed the user is able to use the Manual matching features to edit or correct the automatic findings Automatic Band Matching Matching Metric Select either Adjusted Rf Matching or Generic Rf Matching as the matching metric Adjusted Rf Matching When Adjusted Rf Matching is selected the software will match bands based on the data values in the Adjusted Rf column of the Profile and Data window These values result from the gel smile curvature rather than the absolute vertical Rf value Adjusted Rf is calculated based on the addition of M W markers and or the Rf Curve lines which serve to correct the Generic Rf values for gel smiling Generic Rf Matching When Generic
99. ard measurement within the image such as a ruler Using the mouse click and drag one end of the line and then the other end into position Once the line is in position click in the SCALE LENGTH box and type the length of the standard Press enter to save this value Distance is measured from the origin designated by the circle with a cross in it to the mouse location Place the origin marker appropriately and then move the mouse The distance will be constantly updated in the DISTANCE box To hide the scale and origin click on the HIDE ORIGIN SCALE checkbox Cartesian Coordinates The Ruler function can also be used to create a Cartesian coordinate system The origin icon has default values 0 000 0 000 These can be changed by clicking on the ORIGIN box typing in new values and clicking lt enter gt Once the origin is placed on the screen in its desired location the relative coordinates of the mouse at any location are shown at the right hand side of the screen In the example above the cursor is 0 747 inches to the right of the origin and 1 071 inches below To hide the coordinates click on the HIDE COORDINATES checkbox 196 The Scoring Function The Scoring function is designed for gene expression applications to check for presence or absence of specific samples The scoring function can be used for a quick manual identification of several different sample types The software allows for up to three different scores on
100. ating synchronize function scroll and zoom can be applied on both images together To switch back from compare view to tab view simply clicks on the Compare View icon CGM CONS dan oo ac w mmi deems ii 47 Another way is by drag one image tab to see the docking options as shown in the following figure LEAR B iy ig P om Bi Dem p pem 20cm C8AN oO daa Qui Be EN Sc m ed Ps BD j ssim E ec inm E ru lam bemm deka CE eo IY Tm Abr T mE omma OH RES aaa me ere h amm MER Xa val Figure 2 12 How to enter Compare View After selecting appropriate place when you drop the image tab compare view will be opened T iiptaiemi ek lpkal um eic BAS daa oo Be i IE a ME Pami del b erum i amm MER Figure 2 13 Compare View 48 Note This is intended as a quick reference guide for acquisition For more detailed information on the individual features reference section 1 6 of this manual 1 Power on the system a b C Turn on the computer monitor and optional printer After the computer has finished loading the Windows desktop turn on the power to the cabinet The AlphaView software is activated by double clicking on the AlohaView icon 2 Positioning and focusing on the sample a b C In the Tool Bar select the Acquire icon to activate the image acquisition software
101. ay 19 milliseconds but can be configured up to 50 minutes between each image Also if your exposure delay and or exposure time and or lighting options filter position is consistent for the entire movie then once you setup the first frame you can select the COPY TO END selection to automatically choose the first frame settings for the entire movie of frames images Once the movie is setup to the desired configuration click on the GO button The movie will then begin the image acquisition for each frame of the image When it is complete the movie setup box will disappear and the TOOLBOX window will automatically configure to the MOVIE tools This will allow you to playback the movie save or load the movie or record a new movie Once all images have been captured the above Movie display box will become displayed The remaining buttons will perform the following tasks HEC Move to Camera Setup and Preview Movie Record setup functions to record a movie PLAY Display a continuous loop movie of all of the captured images STOP Stop the movie at the current frame display PAUSE Pause the playback of the movie at a user defined image REW rewind Rewind the movie to the first image REV reverse Play the movie in a continuous loop in reverse FWD forward Forward the movie to the last image DEL delete Delete the movie on the display THIS FUNCTION WILL NOT DELETE A MOVIE SAVED TO THE HARD DRIVE LOAD Load a previously saved mov
102. c to your instrument and software when calling Alpha Innotech for technical support and software upgrades To close the box click on the OK button 24 Chapter 2 GETTING STARTED BASIC IMAGING FUNCTIONS When the FluorChem Q system computer is powered up you can click on the FluorChem Q icon on the desktop to automatically open the AlohaView software The following screen appears Y Alphalnnotech AlphaView File Edit Image Setup Overlay Utilities View Window Help BOK COVES TOi ae A xj Sample tif Ready Intensity 33 Zoom 100 X 297 Y 453 Figure 2 1 AlphaView screen showing the image area and display controls Basic Imaging Functions AlphaView software has ws for all image acquisition contrast adjustment enhancement and analysis functions 25 Acquiring an image using the Navigator Clicking the Navigator toolbar icon will guide the user through commonly used applications in this example the Ethidium Bromide application is demonstrated AlphaNavigator L Mew image capture Yes need assistance with mage scquisition O Mo load an acquired protocol Selecting the Yes option AlphaMavigator Select sample type C Gel C Chemiluminescence Blot Colony Plate 3 Fluorescent Westem Blot Selecting the Gel option 26 AlphaMavigator Select acquisition channel s Cy5 or similar L3 er similar Cy ex si
103. ceeeeeeaeeesseseeeeeeeeseeeeeeeeeaaaaaaeeseseeeeeeeeeeeeees 146 Figure 5 31 Standard Curve cccccccccccccccccccccceeeeeeeeeeececeeeeeeeeeeeeeeeaaaeeeeeeeeeeeeeeeeeeeeeaaaaeesgeedeeeeseeeeeeeeeeeeees 146 Figure 5 32 Multiplex Band Analysis Data BOX cccccccssescecceeeeeeseeeeeeseueeeseeeeesseeeeeeseaeeeessansessaneeeeesaaes 147 Figure 5 33 Lane Profile tools eelllseeeeeeeee eee eeeeeeeee nennen nennen nennen nnn nennen nnns 150 Figure 5 34 Lane Profile Template cccccccseeeeceeceeeeeeeeeeeeeeeeeeeeaeeeeeeeeeaaeeeeeesaaeeeeeeseaeeeeesseaseeeeessanaeeees 150 Figure 5 35 Skewed Lane Profile Template Properly Aligned on a Gel eseeuusss 151 Flgure 5 96 Image Area or Sample SCAM qsesssdon ias icai idco E EAR E luta Denis ed 155 Figure 5 37 Example of a Quantitation Data Tableros n tota t totes br d eden pesas 157 Figure 5 38 Tools For Adjusting Automatic Peak Finding Parameters ccccccsceccseeeeeseeseeesaeeeeeseeees 157 Figure 5 39 Lane Profile Data Interpretation TOOIS cccccsssccccseseeeceesseecceseccsegeeecseseeessaseeessanseesseaes 162 Figure o 40 Using they EINE pP M 162 Figure 5 41 Molecular Weight Mass and Band Scoring integrated into Lane Profile 165 Figure 5 42 Saving and Loading Mass Standards in Lane Profile ccccccceeeeeeeeeeeeeeeaesssseeeee
104. ch results can be viewed in one of four configurations Select the configuration desired under the View menu In each view Band Type is identified in the left most column Band numbers appear in their Rf Position within each lane Lane numbers are identified in the first row The file location matching metric matching reference and matching tolerance are displayed below the tables Mixed Table This display will show you both matched and unmatched bands in one table Separate Tables This display will show you matched and unmatched bands in separate tables Matched Table This display will show you only the results of matched bands Unmatched Table This display will show you only the results of unmatched bands 186 Exporting and Printing Band Matching Results Print and Export are selectable under the File menu Select Print to send the report to a local or network printer Select Export to export the results to the clipboard or to file When exporting to a file there are two file types that can be saved standard text file txt or a comma separated value file csv When exporting to Excel use the csv file type to skip the Excel import wizard or use export to clipboard followed by Paste in Excel Dendrogram and Similarity Matrix Generation After bands have been matched dendrograms and similarity matrices can be generated from the data Click the Generate Dendrograms button to view these Dendrogram Sample tif File View Optio
105. changes from white to green indicating that it is now a Standard Figure 5 41 145 Band Hed BC Average Hedn 1s 10 200 EE 4 578 ds 6 57 4 020 67 55 F 1 345 bs 348 Sais Figure 5 30 The Standard Curve spreadsheet Once a second value has been entered a curve is displayed The points corresponding to the standard bands are labeled in green Points for the unknown objects on the image are displayed on the standard curve based on their selected values These are labeled in white Enter values for each band whose amount is known As more standard points are added the calculated values of the unknown points may change The relevant values for the best fit curve and the corresponding equation along with the coefficient of correlation is displayed Standard Curve Linear nx Linear y 0 01069 t R vilis Parm 0 Seer 112 0 sse T8 4 Ic 61 6 44 8 26 0 3 453 6 5027 5 6 601 9 o 176 1 9r Be 11 324 4 BC iveracge Figure 5 31 Standard Curve Green spots are the entered standard values Yellow spots are quantified unknown values NOTE Cubic spline and point to point curves have no equation nor r value These values are displayed only for linear regression and log linear regression fit models To remove a band from the set of standards click on it again The band number changes from green back to white indicating that it is no longer a standard To de select all the standard points at once click
106. click on the V Line checkbox A vertical line which can be moved across the graphs will appear Use this line to determine if two peaks are located in the same position Figure 5 40 Using the V LINE The line also contains hash marks which represent pixel intensities Use this information for a quick comparison of peak heights Note The hash marks are most useful when only four graphs are shown so that enough detail is displayed To accomplish this click on the selection button in the upper left corner of the window and deselect Show All Lanes Alternatively the intensity value at the point where the V Line crosses the graph can be displayed Hold the snift key and click the right mouse button to toggle between intensity values and hash marks 162 3 D View The 3 D View bed ure changes the appearance of the graphs to a nd adig display This can ine seful in understanding how data was obtained and in visualizing any anomalies that cS AN m The depth of the display is determined by the Scan Width i e the above scan has a width of 35 and is displayed in 3 D mode as 35 graphs This function acts like a toggle click in the box next to 3 D View to activate the function click in the box again to deactivate it and return the graphs to their original appearance E m E TL ome A D rr E D m i e Eua li Ux ume i S UM N AN i
107. col Report Area OF Interest uE aaa L4 Help on these tools Figure 5 9 Colony Count Tools Circles This is the most appropriate choice for counting colonies on a Petri dish After clicking on the button in the toolbox labeled with a circle move the cursor into the image area to the edge of the Petri dish Click the left mouse button and move the cursor to open the circle When the circle corresponds to the perimeter of the Petri dish release the mouse The circle has handles around it and can be moved or re sized at this point Hint to draw a perfect circle around a portion of an image first visualize a square surrounding the area of interest Position the mouse in the upper left hand corner of the square Click and drag the mouse down across the area of interest at a 45 angle until the circle encloses the area of interest Rectangles After clicking on the button in the toolbox labeled with a square move to one of the four corners of the region to be enclosed Click the left mouse button and move the cursor to the corner diagonally opposite Note the box that opens as the cursor moves Release the left mouse button when the box has reached the desired size The rectangle has handles around tt and can be moved or re sized at this point 124 Freehand Drawings To draw a freehand object select the freehand icon the mouse will change to a pencil Draw an object of the desired size and shape When the mouse is
108. commended 2 Puta cap on the lens mounting plate to block all light from the camera head A lens cap will leak light Creating darkmaster file with the camera and lens mounted to a cabinet is not recommended If the camera cannot be dismounted then close the aperture and position the most opaque filter under the lens and turn off the cabinet before proceeding 3 Access the utility from the application menus Owerlay Ubilities View Window Help Print Info d Prink Mode d Print Date Preferences tem Calibration Darkmaster Utility 4 Login to the utility call tech support for the password User Login User Mame master CE Password D2 5 Enter the times for darkmaster files creation separated by commas Bias and Darkmaster generator IMPORTANT Please put cap on camera lens turn off all cabinet lights and close cabinet door before starting this operation and untill its end Enter exposure time for the set of master images 1 to BI l minl You may also enter comma separated list of exposure times for generating of several sets of master images together Estimated time for building the set of master images 20h 10m 6 If bias files do not exist they will be created automatically If only bias files are desired then enter a time of 0 minutes 7 Notice that the times to create the calibration images are quite long so please plan accordingly 8 The estimated time of completion is given Bias and
109. ctivated to show up under the tool box as shown here To activate these tools 1 Goto Setup gt Preferences gt Analysis tools tab 2 Select tools to show Preferences General Image Acquire Cabinet Settings Auta Enhancement Analysis Tools d L Please uncheck those analysis tools which vou don t need to view in Analysis Tools Tab Ruler Molecular Weight Scoring Manual Count Colony Count Array Multiples Band Analysis Lane Profile This tool set allows the user to perform ruler measurements gel scoring manual counting of colonies and cells and perform high density array analysis 195 The Ruler Function Introduction The Ruler function allows the user to create a scale based on a standard and to measure distances from a set origin to a chosen location Orion ty 0 0000 ooog Seale Length 1 0000 Distance Hide Origin Scale ce Hide Coords Figure 5 59 The Ruler Tools and Ruler Toolbox Using the Ruler Function To access the Ruler function click on ToolBox Analysis Tools and then on RULER A line with a box at each end and a circle with a cross in it will appear on the screen Before any measurements can be obtained a standard scale must be established The scale that is created will serve as the standard for all measurements Therefore it is advisable to draw the scale line according to some stand
110. d Zoom Out on the image Zoom to 1X and to Fit to Screen Note Zoom In and Zoom Out are duplicate functions for the Zoom In and Zoom Out icons in the ToolBar and the Zoom options in the Enhancement Tools The Window Menu Qum Help E Next Window Ctrl Tab E Previous Window Ctrl Shift Tab El Close All Document s coomassie 16 tif Sample tif Figure 3 32 Window pull down menu The Window menu function provides the ability to navigate from one image window to next image window It also allows user to close all document 90 The Help Menu On Line Help About Figure 3 33 Help pull down menu How to use this software help is available in the On Line Help section of the Help menu Common tips are included for both Enhancement Tools and Analysis Tools detailed in Chapters 3 and 4 respectively The software must be registered before the end of the trial period Registration can be done when the application is run or from this menu Note The registration menu item will be grayed out after successful product registration To display system information select the About option in the Help menu This button accesses a pop up box This box shows the system serial number and software version number Use this information when contacting Alpha Innotech for technical support software upgrades etc About AlphaView AlphaView Software Version 3 0 0 0 Product Rey XXXXXXXXXXKXXXXXX Alpha Innotech Corporation Copyrigh
111. d by reducing the individual display windows Lane Profile Result Data BaseLine Options Lane Peak Distance width Height Ama S F Adi RE 2 4 131 g 32 117 2 29 0 3629 0 3629 E 5 140 g 53 217 4 21 0 3878 0 3878 Ea ee aes iz 7 162 12 99 541 10 42 0 4499 0 4499 H 17k 13 115 Fad 1 Aare Aare aT Figure 5 36 Image Area of Sample Scan Graph Display Graphs representing all the lanes on the template are shown in the lower left hand quadrant When AUTOGRID is first selected the active graph is the one corresponding to Lane 1 To select a different lane simply click on its graph and it will fill the Active Graph quadrant AlphaView uses the following color coding e the active graph is shown in white e graphs that have been viewed already are shown in yellow e graphs that have not yet been viewed are shown in gray When the template contains many lanes there may be too many graphs to see reasonable detail It is possible to view only four graphs at a time by deselecting Show All Lanes This function is found under the function button of the window 155 Lane Profile Result Options Background Color Data BaseLine ht Area x RF en Peak 1 1 Axes 1 Show Image Strip 1 4 11 2 28 0 3712 To 00v eH 228 444 3951 I 5 E Shwallaes
112. d data table windows to the clipboard for pasting into different programs e g Excel Lane Profile Result Data BaseLine _ Options Lane Peak Background Color Axes Est Show Image Strip Window P Shaw All Lanes Options drop down menu Editing Peak Boundaries The boundaries of existing peaks can be readjusted to make the peaks wider or narrower To adjust either peak boundary click on the peak to select it Next point the cursor at the vertical boundary line hold down the left mouse button and drag the line to the desired location When it is in place release the mouse button The data table is automatically updated to reflect any adjustments Both the peak and the data in the data table will be blue to indicate that this is a user defined vs automatically detected peak The Data Window Once a peak is defined its integration data and associated information are displayed in a table located in the lower right quadrant of the screen The data table is updated any time a peak is deleted or added peak boundaries are redefined or the background value is re set 176 Feak Distance 146 width Area Es 0 4044 0 4044 Lane Height Ad RF A E 4 134 3 32 117 2 29 0 3712 03712 1 5 143 10 54 228 4 44 0 3961 0 3961 1 153 12 79 411 7 83 0 4238 0 4239 E 7 165 12 38 535 10 31 0457 D4571 1 8 178 13 115 E41 1235 04958 04958 1 3 197 13 123 710 13 67 05457 05457
113. d positive negative nothing For faster scoring you may look at the entire image and visually determine whether the samples are primarily positive negative or positive negative By clicking on the All All or All boxes all of the samples on the image will have either an X three or nothing depending on the button you clicked Image after All button pressed and Image after All button pressed individual sample scoring Output and scoring options 198 Manual Count Click on MANUAL COUNT The following sets of tools will be displayed in the lower left of the screen Some samples are extremely difficult to count because of their shape size or lack of intensity variation from the background To accurately count these samples we have developed the manual counting feature within our software To manually count the objects in an image click on MANUAL COUNT in Tool Box Analysis Tools You may use either a green x or a red If you are only counting one type of sample you may simply use the x which is the default to begin counting in the software Count Sensitivity Figure 5 61 Manual Count Tools Placing Markers to Count Point the cursor at an object to be counted and click the left mouse button An x is placed over the object ensuring that the same object will not be mistakenly counted twice In the toolbox the number in the COUNT box automatically increases by 1 Click on all of the obj
114. d this method for images with poorly separated bands Minimum Profile This method takes the lowest value on the profile of each lane as the background for that lane We do not recommend this method for lanes where the beginning or end of the lane is the lowest point as this is dependent on where the lane boundary was placed when it was defined This can be hard to repeat between analysis of the same gel Valley to Valley This method requires that you have performed band detection first The background is taken as the line between the edges of the bands in the lane You enter a value see below in setting parameters of maximum slope in the accompanying entry box to avoid situations where the edge between overlapping bands which is not at the background intensity causes the background to climb too high Rolling Disc This method requires you to enter a parameter for the size of the disc see below in setting parameters in the entry box This option calculates the background as if a disc with the radius you have entered were rolling underneath the lane profile The larger the radius of the disc the less the background rises with the profile 160 Set Parameters Backeround Subtraction Parameters Enter the size of disc for the Rolling Disc algorithm in peels m Enter the masimum slope for valley to valley algorithm in pixels Set parameters dialog box Above dialog box is used to change values fo
115. de see the following pages for more details Annotation Colors Annotations can be displayed in a variety of colors The color options are displayed by clicking the COLOR radio button To select a color simply click the cursor on the button labeled with the desired color The color button appears depressed indicating that it is selected Any annotations subsequently entered will appear in that color It should be noted however that annotations are printed in gray scale on the video printer Further when an image is saved as a modified image the annotations are saved in gray scale not color 96 Line Thickness The PEN WIDTH menu specifies the thickness of lines when using the freehand lines box and circle drawing tools Click on the appropriate checkbox for the desired width All annotations subsequently entered will appear at that width Annotate Xj im Color Line Ends Pen Width CO Text Style CO Pen Style C3 Text Orient e e Figure 4 5 Pen Width Selection Tools Line Types The PEN STYLE menu specifies the style of lines when using the freehand lines box and circle drawing tools Click on the appropriate checkbox for the desired style All annotations subsequently entered will appear in that style Note these pen styles only work with a thin line see Line Thickness above Annotate Color Line Ends C3 Pen Width C Text Style Pen Style C3 Text Onent
116. deviation of distance For band matching Select Heferene Object Band of sel MWforallanes v Help on these tools 171 Auto Lane If Auto Lane is selected from the main Lane Profile tab the following interface will appear scoring Band Matching Protocol Report Tools Result Gel Smiling Mol Wh Mass Std Auto Lane Sensitivity J Less hore Sensitive Sensitive Band Detection Invert Help on these tools Figure 5 44 Auto Lane Invert Auto Lane will automatically detect whether the image about to be analyzed represents white bands on a dark background e g fluorescence or dark bands on a white background e g Coomassie Blue gel x ray film The user should always check to see that the software has correctly characterized the image as Black or White Band The user can override the automatic selection Vertical Lanes or Horizontal Lanes If the gel image has vertical lanes the Vertical Lanes option should be checked If the image s lanes run horizontally then the Horizontal Lanes option should be chosen The software will not automatically detect the lane orientation so the user should aware of this parameter Sensitivity Adjustment Bar This feature changes the number of bands the software will find using a sensitivity scale of 0 9 The lower the number slider bar left the less bands the software will find and the greater the number slider bar right the more bands th
117. doit tre situ e ou install e proximit du materi l et tre facile d acc s A English Warning This product relies on the building s installation for short circuit overcurrent protection Ensure that a fuse or circuit breaker no larger than 120 VAC 15A U S 240 VAC 10A international is used on the phase conductors all current carrying conductors e French Attention Pour ce qui est de la protection contre les courts circuits surtension ce produit d pend de l installation lectrique du local V rifier qu un fusible ou qu un disjoncteur de 120 V alt 15 A U S maximum 240 V alt 10 A international est utilis sur les conducteurs de phase conducteurs de charge German Warnung Dieses Produkt ist darauf angewiesen da im Geb ude ein KurzschluB bzw Uberstromschutz installiert ist Stellen Sie sicher da eine Sicherung oder ein Unterbrecher von nicht mehr als 240 V Wechselstrom 10 A bzw in den USA 120 V Wechselstrom 15 A an den Phasenleitern allen stromf hrenden Leitern verwendet wird H2
118. e This function closes the image currently displayed on the screen File Save Save As Save Modified and Save All Save allows original images to be saved in several different formats Save As allows images that have previously been saved to be saved in a different location or as a different file type without affecting the original image AlphaView has the ability to save files in several formats see the following figure Save Untitled 1 As Soe TII a My Recent Documents IIT E Desktop My Documents My Computer z Mo Network AlphaQuant 1 tif n lpha uant Examples tif n array template tif m Bhi tif coomassie 16 tif Dnalmage tif kepi forditott111 tif Sample blat with cherni tif n Sample Blot tif n Sample tif I Smile TIF I Testpatt tif Untitled 1 hull Tiff Files tif w eaf t Figure 3 5 File Save As Dialog Box File name Save as Ippe 67 save Cancel Enter a new file name in the text box adjacent to the File Name prompt Next choose a file type from the Save as type list AlphaView will automatically give the appropriate 3 character extension AlphaView will also create a file with the same base name an file saves information specific to this file File Types TIFF is the default file format for AlohaView files and was developed as a flexible and machine independent graphic file format Saving as a TIF
119. e bands and all the color channels Use the Band Control Normalization tool to determine the Fold Change in the phosphorylated samples relative to Band 7 Select Identify Control Band in the Band Control Normalization section and select the green channel and Band 7 Select Identify Experiment Bands and select bands 8 12 Fold Change values are populated in the data table G 10 Band analysis results All Channels Export View Display Style Band Blue LEM Avg 3 73b fA 4 r360 rela 5 DAS SE 6 26g b r360 bf d r b35 3 131 B fob 13 622 g Grd 5 916 10 303 2253 11 1 030 1 468 12 1 022 1 127 13 far falls 14 ro fra 15 72 r r 16 rig 553 17 496 133 Green LEM Avg Hed LEN Avg 11 102 11 252 11 738 12 385 343 1 128 1 027 1 547 1 556 1 f48 3 135 4002 1 343 g2 fob PLN Average 426 04 172 06 r0 46 00 35 32 Fold Change 4 27 1 73 1 41 217 2 83 Select the Std Curve tab to construct a quantitative curve Select Add ltems and select the red channel Select bands 13 18 Band 18 has a concentration of Ong De activate the Add Items tool by selecting Add Items again Multiplex B and Analysis _S a Bkamd Control Std Curve Protocol Repo Enter units nig EI Hed Sum bb 50 980 40 044 680 24 109 330 Curve fitting Model Linear Ww Equation y ax b Y Asis Sum Graph Color control normalization Identity CC band Clear nor
120. e Compensation Setting Normal exposure for image saturation Under exposure for chemiluminescence Over exposure for faint band detection Custom exposure compensations 0 1 8 EV Exposure Value EW Adjustment Close Figure 2 3 Auto Expose Clicking the Auto Expose Setup button launches the Auto Exposure Compensation Setting window This window offers four options e Normal Exposure for image saturation This setting is ideal for normal Colorimetric and Fluorescent imaging e Under exposure for Chemiluminescence Over exposure for faint band detection e Custom exposure compensation This setting allows the user to define th EV Th cet of up down arrow buttons change the EV valu while set of up down arrow buttons change the EV valu 33 Auto Exposure works in both Preview and Acquire Image modes The AlphaView software calculates the correct exposure time for the current situation In Expose Preview mode a status bar appears above the Auto Expose interface In Acquire Image mode a status window featuring a colored Auto Expose status bar for image acquisition appears The status bar indicates the current status of the calculation For exposure time calculations with saturated pixels in the image area the status In Acquire Image mode After achieving a green status the new image loads and the Camera Setup amp Preview window closes In Expose Preview mode the system continues repeated expose
121. e left mouse button and image will rotate to the desired angle To undo a rotation just click on the Undo button Also a Flip option allows for the image to be rotated 180 degrees in a vertical or horizontal fashion The Reset button on the main software interface will also remove any rotations or image flips and return to the display to the original image Image flipped vertically Rotated 11 degrees clockwise Rotated 11 degrees Counterclockwise Rotate Fip Rotate Rotate Rotate Angle 11 Angle 11 Ame 90 ng 11 90 50 nale 1 90 50 nate o 90 Flip Flip Fip Rotate Flip box with vertical Rotate Flip box at 11 degrees Rotate Flip box at 11 degrees Flip button pressed 95 Annotations The annotation tools found in ToolBox Enhancement Tools include a number of different options for adding text including Greek symbols drawing arrows and otherwise marking an image Note that these tools are for annotation only For information on drawing objects for quantitation purposes see Chapter 5 5 Color Line Ends Pen Width Text Style O Pen Style CO Text Orient lE mmm Figure 4 4 Annotations Toolbox Object Attributes Use the COLOR PEN WIDTH PEN STYLE LINE ENDS TEXT STYLE and or TEXT ORIENT menus to specify object attributes Attributes can be assigned to the cursor before drawing or typing Alternatively they can be assigned to an object while it is in edit mo
122. e menu or click on the Save or Save As icons EN Movie Mode If kinetic multiplex color or chemiluminescence experiments require the system to automatically capture several images at preset exposure times preset time delay between images preset lighting sources or preset filter choices AlohaView Movie Mode should be used Clicking the MOVIE box in the ToolBox s Enhancement Tools tab launches the Movie Mode controls Movie Mode setup is also accessible in the Camera Setup amp Preview window To launch the setup screen click the Movie Mode button in the Camera Setup amp Preview window Camera Setup and Preview M redd four o rome Deby a Cop to Mest 7 Stack Frames w il 7 FuulnEnd D Copy to End Salus Mere biun lr Tute Figre 7 Capes Auta expose ER abe oie 0 0 8 iinan T ab ish Speed leihen Homala Hongi Nore Juan Figure 2 6 Movie Mode Setup The Total Frames field sets the total number of individual frames in the movie Each movie can capture maximum of 50 frames images and a minimum of one frame The Frame field selects the active frame currently being used for condition customization For example if the movie uses three images selecting Frame 1 allows for lighting and filter setup of the movie s first frame selecting Frame 2 allows for setup of the movie s second frame All 41 frames can be adjusted manually using this technique or specific li
123. e software will find Upon changing the sensitivity Find Bands must be selected for the software to re find the bands Area of Interest Drawing 172 It is recommended that the user draw an area of interest on the image in order for faster and more accurate detection of lanes and bands An area of interest is drawn using the left mouse button to click and drag draw a box shaped region around the sample area Image artifacts well position marks etc should be left out of the area of interest as the software may record these as bands After an area of interest is drawn the Find Lanes button should be selected The software will automatically find lanes and bands within each lane An area of interest need not be drawn for the automatic detection Find Lanes to work The analyzed image and overlaid data table will now appear LY L3 a s a P P il n eg d m E 1 E Eri s cr o Was pee ae i Bai Figure 5 45 Auto Lane Analyzed Image Lane Frofile Result I x Data Baseline Options Peak Distance width Height Hf Adj Hf 4 131 g 32 0 3629 0 3629 140 g T3 l 0 3876 0 3875 162 33 4488 0 4458 176 11h 1 AR7F 1 AFF H MAT Ho Figure 5 46 Auto Lane Profile and Data Table 173 Data Table and Editing of Auto Lane When the AUTO LANE button is clicked the lanes are designated bands found and the information is displayed in three windows the upper window is the Active Graph the low
124. ead Alternatively if the exposure time is short enough Normal High may be selected for a full resolution 4 2 million pixels chemiluminescent image 5 Save the original image a b C d Select Save Image in the File menu or click on the SAVE or SAVE AS icon in the Tool Bar Enter a file name and select the directory to save image the directory path should be less than 100 characters opecify the file format TIF BMP PCX MAC color TGA Click OK to save the file 6 Enhance the display optional a b C Adjust the black white and gamma levels by moving the slider bars at the right of the image in the Contrast Adjust window or select Auto Contrast Apply the digital filters found in the Tool Box under the Enhancement and Filters to stop a filter hit any key on the keyboard to reverse the effects of a filter click Undo Add text boxes arrows etc to the image using the annotation tools in the Tool Box under Enhancement and Annotate 7 Print the image using the PRINT button in the Tool Bar or the pull down File menu option Analyze the sample using the analysis features in Tool Box ANALYSIS for quantitative analysis 50 Contrast Adjustment The Contrast Adjustment window allows for the best visualization possible of a sample utilizing the black white and gamma adjustments as well as image reverse and auto contrast The image on the screen is made up of picture elements pixels in an array
125. eatin dmoblect ATed OF INVENT CS Fz acest esos Ete evt a eese d QoS tad e eo Dieu deoa ado uU RR Vd Rouen ode 129 Magic Wand and AutoSpot Single Channel Only eee eee eeeeeeeeeeee nen nennt th nenas sense nens ns 131 MIGHT DUALS D DIOC cada ooo Miedo Ouid Raoul E A d tava AN dust eo esae sone ue umore mId RE pora Saut 135 Multiplex Band Analysis Measurements oues ana t eaaet e oae do Que Rn OE AA leta Re mese E RN a ttv E TUNE 136 Background Tab Calculating background values esses eene enhn nnne nenne enne nnns 137 Mass Standard Calibration Curves for Quantitative PCR e ceeesssssssseeeeeeeeeeeseee enne eene eee sss at 144 JEANECPROBIER CLANE DENSIPOMBE DRY 25 eresceediviazisiesescoede etis a Gad as ccu tuS sas ae esed ctu ta deuda circu esses N 150 D IDIORSI IM mer 150 setune up he Eanes Templie die e eoe cu cce eroe DRCEVE TE EU S E Tee sea uL Pd ao d e T ET o SR ELI URL PLUR IR E EL EN RER DUE SD 151 SPCC YIMe TRE SCAM M TOTIS S nubc sects caltentnea a bases e sch o biebas a tM nei ea Gunes abnseseauiuaial sound 152 Scannttiss he Ilde Sect ask chu ns Eee iet os tn iato Duva VeLUUS DO ot bae Teva RN E dard Ran og cae uou opt li vua ME EMT 155 Adjusune Peak Detection Paramelers ua eee et aot exei rd laeta abu ekle b dede tua etu e aeu oa aiu deae e a us etate uta pud 157 Eden Pell Seo dessus A el osa LA cel Mead co ee CLAU 158 Aduse the Baselie eire amt eri a etuer on Pee 160 Entetpreune Gane
126. ecific forms of a protein oX background corrected average from the red channel data Blue channel data represents the signal from an antibody that binds all isoforms phosphorylated and nonphosphorylated of the protein X pX background corrected blue channel data and is referred to as the total protein signal In this case the total protein signal can be used as a loading control The blue channel data represents total protein X pX while the red channel data represents only phospho specific protein So by designating the blue channel data as a loading control and the red channel data as the experimental sample an LCN for the phospho specific protein is calculated as LOCN pX X X anen Lane 1 contains the untreated reference sample and the ratio of pX X pX in this lane is used as a baseline reference Baseline pX X PX Lane 1 So the ratio of phospho protein to total protein in the untreated reference sample represents the Baseline All other lanes except the lane with markers are used to calculate the ratio pX X pX iane n relative to the Baseline The band control normalization tool is used to determine Fold Change The red channel of lane 1 is designated as the band control while the red channel of the other lanes is designated for experimental bands Since the red channel of these bands have already been subjected to LCN normalization this provides a Fold Change determination with the following relationship Fold Change
127. ecifications optional Windows operating system pre installed AlphaView image processing and analysis software pre installed and calibrated with computer and hardware system Multilmage III FCQ light cabinet with UV transilluminator and white light fold down transilluminator and interference filter Fast lens optional Epi illuminating UV lights optional 254nm or 365nm Printer optional e ChromaLight optional Upon receiving the system it is critical to check the enclosed packing list to verify that all components are properly included System Placement As with all electrical instruments the FluorChem Q System should be located on a table or bench top that is dry and stable and away from water solvents or corrosive materials In addition the system should be placed away from interfering electrical signals and magnetic fields If possible a dedicated electrical outlet should be used to eliminate electrical interference from other laboratory instrumentation Cable Connections The cable connectors and their respective mating ports are keyed or unique for each connection to eliminate potential wrong mating The connections are illustrated and described in section 1 3 WARNING Make sure that the power is OFF and all power cords are disconnected while connecting the cables and setting up the system 15 Hardware Installation All software peripheral drivers and operating systems come factory installed All compo
128. ected e The position corresponds to the band s location along the y axis ranging from O to 1030 e The value displayed under the Mol Wt heading is the molecular weight value entered for a known band or the value calculated for an unknown band e he H value for each band is also displayed Unless otherwise specified AlphaView assumes the origin R value 0 00 is located at the top of the screen and dye front R value 1 00 is at the bottom of the screen Using these points as a frame of reference AlphaView calculates R values for the intermediate bands 116 Molecular Weight Results Export MARKERS Band Position Mal Weight HI 1 E 14 00 0 496 2 2zhb Sz 00 533 3 z ul 43 00 583 4 314 4 D E54 QUERIES Band Position Mol weight 240 18 22 z0 50 50 203 41 32 al H A Molecular Weight Marker and Query Data If the data box obscures part of the image it can be resized or moved using Windows functions or it can be hidden using the Hide Data checkbox Repositioning and Deleting Markers Marker band indicators can be repositioned simply by positioning the cursor clicking and dragging the line to the desired location To delete a molecular weight marker point the cursor at the appropriate marker band and click the left mouse button This highlights the band in question by putting a red box around it Alternatively clicking in the data table highlights the marker s information and selects the ba
129. ects of this type or color to be counted To count objects of another type or color such as white vs blue colonies in a p gal assay use right mouse button to click on each object as above Count Sensitivity x aks EE Section of an Image After Manual Counting Corresponding Data Erasing and Hiding Count Markers To erase a marker that has been manually placed click on the marker on the image It will disappear and the count will be reduced by one The Sensitivity indicator determines how close the cursor must be to a marker in order to delete it Higher numbers allow the user to click further away from the marker to delete it however if Sensitivity is set too high it will be impossible to place markers on spots that are close together 199 Hiding Your Count Markers from the Screen The software will record all counts as you continue clicking on your objects If the counting becomes confusing you may click on the HIDE button to hide off of the x s and s that were used to count your objects on the screen If you wish to make a print of your image hiding the count make sure that the HIDE button has been pressed to high your count markers You can then go to Tool Box Enhancement Tools Annotations and label the image with the appropriate Click on the PRINT button to obtain a hardcopy Erasing the Count Markers and Data Once you have finished counting your objects and have obtained the data you need you may clear
130. ed in the Data Table and are used in the calculations Region Ekgmd Control Std Curve Protocs Loading Control Normalization Identify loading dently a singe row of Identity Experimental Bands experimental bands Clear Loading M armalizatio comespanding to the loading Band Contral Normalization control Normalizes esperimental Identify Experimental Bands bands to a positive contral Clear Band Normalization Help an these tools Figure 5 23 Control Normalization Tab Loading Control Normalization Tools Normalize experimental bands to the corresponding loading controls to adjust data for variations in the amount of sample loaded in each lane The loading controls may be in a different color channel than the experimental bands in a multicolor image In this case the loading control protein is labeled with Dye 1 blue and the experimental protein is labeled with another dye Dye 2 To perform loading control normalization first identify the row of loading controls and then identify a row of experimental bands A second and third row of experimental bands may then be identified by repeating this process Identify Loading Controls Select the channel for loading control normalization Identify each member of the row of loading control bands using the mouse pointer You may either left click on each of the regions individually or draw a rectangle to select a single row of regions Deselect the Identify loading co
131. eeeeeeeees 168 Figure 5243 Bane S6OFIteiaucu cesta re a ces Sgn DLL s De fence E E d dE 170 FOSS Ad AUO EAN m r PU 172 Figure 5 45 Auto Lane Analyzed IMage c cccccccssssececcceeseecececseseececseeeceesssuaececsssaaeeeeessaaeeseeessageeeessas 173 Figure 5 46 Auto Lane Profile and Data Table cccccecccccsssccceeseeeceesseecceeseeecsaueeeessseeessaeseesenseeseneass 173 Figure 5 47 Auto Lane Editing Features ui erp CI ee ge pedis bou ene laca qu eue tuno via dac geb veseE ge ten bu ai CUN d 177 Figure 5 48 Band Matching Dialog WINKOW ccceeeceeeeeeeeeeeeeeeeeeeeaeeeeeeeeaaeeeeeesaaaeeeeeesaaeeeeeessaneeeeeenas 183 Figure 5 49 Band Matching Results window displaying the similarity matrix sssssse 186 Fig re 5 50 Dendrogram WINGOW obs asit tag vag ap se a cad teu di uio Rosen tb phate Dali puinae RN S 187 Figire o 51 olmillariby WAX suec cies pue vu Copt a oen drea b quet a te on PEU ea ho RS VET AU va tuttaa Du RE 188 FIQUIG Es oral mire oleo ME omm 190 FIgure 529oaviligdriarialysiS iuiis seco aer eoe red erue opc en Wer en lube cs La i ve e pce eta va aes seuss a Po diia n Yd 190 Figure b5 54 Report Formatting Tabs aes toco a ue i un ta n et d de Co Po PR Or Eod 191 Figure o 55Heport Genetral Tab ei Eee e oO WE ecoa tust emet ava ec edi eus du eder iade eau d dese uS 192 Figure 5 56 Gommon Export Dialog BOXcxvscicvitiutcesi e todo Crit v curi Vebe cua Let eed een va
132. een and Blue intensities in the window are also displayed file This is detailed later in Chapter 3 4 of the manual Clear removes any overlays currently displayed on the image This function can be useful if annotations or other displays obscure parts of the image The Notepad icon opens up a dialog box to allow the user to quickly track experimental conditions comments and any other details to be saved as an electronic copy for future reference Detailed instructions are available in Chapter 3 as this Notepad function is duplicated in the Utilities function in the upper header bar The Open file explorer opens windows file browser The Compare Image tool allows comparing two or more images in compare view 63 Tool Box The Tool Box window contains an intuitive interface for performing all image enhancement and analysis functions Enhancement Tools Analysis Tools Figure 2 25 Tool Box The Enhancement Tools option contains the controls for enhancing and adjusting the image This includes software filtering false colors zoom factors and other unique features The Analysis Tools contain the controls fo including gel smiling corrections band matching Lane Profile densitometry multiplex band analysis molecular weight calculations colony counting and arrays Both the Enhancement Tools and the Analysis Tools are detailed in chapters 4 and 5 of the manual respectively Status Bar The Status Bar is located on t
133. er add mode then click on the desired object with the left mouse button Using the Erase Spot Tool This function removes any extra spots that were counted either automatically or manually For example if a portion of an AOI has high background or other noise a number of objects may erroneously be included in the count Draw a box around the object or set of objects Objects will be displayed with a white background to indicate that they are selected Clicking Erase Spot deletes all selected objects at once and automatically reduces the total count information 127 Spot Count Data When AlphaView opens the Colony Count data box is hidden To display spot count data click the checkbox to deselect Hide Data The following data window will appear ADI Na Ele EE Count lis UE Colony Count Result VIEW Export E 0 0 0 0 Eo i 148 255 db BBY Figure 5 12 Colony Count Data Window Showing AOI Summary Data This window shows the summary data for the area s of interest AOls This includes the threshold values that were set for each count type the number of objects found for each count type and the total pixels in all of the spots To see specific details on each spot select Spot Details from the View menu The following information will appear Colony Count Result A s p3 m vet 33 143 0 02 P 0 02 3 401 33 1 8 2 O4 4 454
134. er they are strongly not recommended for analysis as the pixel values have been adjusted Conversion Since AlphaView can generate 16 bit files the conversion option is useful when an image is to be imported into a program that only accepts 8 bit images Choosing this option will convert a 16 bit image into an 8 bit image A 48bit color image can be converted into a 16 bit greyscale image by simply averaging the RGB values for each pixel Setup Overlay Utilities View Window Hel al Overlay 59i A Alley ee ii eu e n x Sample tiFr Alph Equalize Arithmetic Conversion Flat Field Calibrate Resize Image Information i Figure 3 13 Image Conversion dialog box 19 Flat Field Calibrate Manual Flat Field Calibrate is a function that is used t across the entire image area so that the pixel data is even automatically as part of the acquisition process perform manual flat field correction only to images that have not already been flat field corrected Use the image info function see Figure 3 15 to identify the image processing operations performed look under Post Corrections Entry on the image in question before proceeding Creating flats can be art in itself there are many documents on the internet that can help users interested in this arena to create the ideal flat for the application However some useful flats that have been created in the past involve very
135. er Std File Save Std File Mate This will overwrite any mass standards calculated by the Molecular Weight process Help on these tools Figure 5 42 Saving and Loading Mass Standards in Lane Profile Select Add Mass Std to add the first marker Pull the cursor onto the image to select the band of interest and click once on the left mouse button to add the marker Type the marker value into the dialog box Continue this process until all of the standards have been loaded After the mass standards have all been entered select Save Std File Then move the cursor to the lane that contains the markers of interest A yellow line will appear in autolane To select the lane of interest click once on the left mouse button and a new dialog box will appear allowing for the name of the file to be saved in the directory of interest Select apply to calculate the unknown values of the markers in the rest of the lanes 168 Mass Standard files can be loaded by selecting Add Mass Sid in either autolane or autogrid Browse through the directories to find the marker file of interest then select the file name and click on open The user will have the option to add the markers manually or to auto load the markers Markers can be auto loaded by selecting yes in the dialog box and then pointing to the appropriate lane on the gel in autolane or the appropriate lane profile in autogrid To add the markers one at a time select no in the dialog box
136. er left is the Graph Display and the lower right is the Data Window The Lane Profile tab will also change to display the Auto Lane Editing functions The data and display of Auto Lane is very similar to that given in the AutoGrid feature described above Graph Display Graphs representing all the lanes on the template are shown in the lower left hand quadrant Lane 1 will be the default active lane when the scan is initially done To select a different lane simply click on its graph and it will fill the Active Graph quadrant AlphaView uses the following color coding e the active graph is shown in white e graphs that have been viewed already are shown in yellow e graphs that have not yet been viewed are shown in gray All of the lanes can be displayed in the graph display quadrant by selecting Show All Lanes under the Options drop down menu in the Profile and Data window By deselecting Show All Lanes only the first several lanes are shown providing more detail A scroll bar allows for the other lanes to be viewed 174 Lane Profile Result Data Baseline Options Lane Peak Background Color ht rea E Hf 1 Show Image Strip 4 qr ee 224 3712 i g UU 229 444 3961 ie s ET T HE 535 10 31 4571 1 17 13 115 E41 USERS n 48Ra i The Active Graph The active graph is shown in the upper quadrant The x axis represents the distance in pixels from the top of the temp
137. es are accessible via convenient on screen buttons and menus in an intuitive mouse controlled interface AlphaView software also includes a broad array of analysis tools including molecular weight calculation R determination lane profile multiplex band analysis quantitative PCR microtiter plate reading object distance measuring gel scoring and automatic colony counting FluorChem Q system is a complete package that includes all necessary hardware and software for image capture enhancement and analysis AlophaView software does not require a dedicated computer system and can operate simultaneously with other office related software such ED rcs hoe and desktop publishing software Images can be printed using a 256 level gray scale thermal printer or any printer with a Windows driver The low cost high quality prints are ideal for lab notebook records or for publication A list of journals featuring published prints generated from AlphaView software is available from Alpha Innotech 10 Chemiluminescent Detection The FluorChem Q has the same outstanding capabilities for chemiluminescence detection as the FluorChem HD2 The FluorChem Q imaging system incorporates the same 1 0 95 fast lens and dark enclosure as the FluorChem HD2 supporting quantitative chemiluminescent detection Chemiluminescence is a well established detection technique for quantification of protein abundance on Western Blots Chemiluminescence explo
138. escans the graph and defines the peaks based on the new settings A peak is recognized only if it meets these minimum criteria Note Using the MIN WIDTH control is the quickest way to regulate the number of peaks detected by the automatic peak finder Minimum Width is 10 Minimum Width is 17 Editing Peaks Editing Peak Boundaries The boundaries of existing peaks can be readjusted to make the peaks wider or narrower To adjust either peak boundary click on the peak to select it Next point the cursor at the vertical boundary line hold down the left mouse button and drag the line to the desired location When it is in place release the mouse button The data table is automatically updated to reflect any adjustments Both the peak and the data in the data table will be blue to indicate that this is a user defined vs automatically detected peak 158 Manually Adding Peaks Additional peaks can be defined manually using the Add Peak function in the Integration menu When this function is selected a vertical line appears Position the vertical line so it corresponds to the left edge of the peak click the left mouse button to define the left boundary of the peak then move the cursor to the right edge of the peak and click again to define the right boundary The peak area between the 2 limits now appears shaded and the data table is automatically updated Both the peak and the data in the data table will be blue to indicate that
139. esponds to a gray scale value When the slider is at the very bottom of the scale this number is 255 As the slider is moved upwards along the scale the number decreases and the image becomes progressively lighter This is because all pixels at the specified gray level value and above are shown on the screen as white pixels For example if the slider is set to 150 all the pixels between 150 and 255 are shown as white and the image appears lighter White Level set at 255 White Level set at 150 Figure 2 16 White Level Adjustment example o2 Gamma Setting Adjustment Changing the Gamma settin by adjusting the linearity of the image on the screen and printouts The camera sees objects linearly while the human eye does not By adjusting the Gamma setting the user can make the image on the screen correspond to what is seen when he she looks directly at the object We recommend a Gamma setting Gamma set at 1 0 Gamma set at 0 55 Figure 2 17 Gamma Setting Adjustment example The Auto Contrast Selection The Auto Contrast feature will automatically scale the black and white values of an image to more tightly fit the gray scale intensity profiles histogram This selection will use different black and white values for different images depending upon their unique histograms A more dramatic visual change will take place for low light level images such as chemiluminescence where smaller portions of the histogram are used This selecti
140. ess of direction All edge enhancement operations attenuate the low frequencies of the image Regions of constant intensity or linearly increasing intensity become black as a result of these transformations and regions of rapidly changing intensity values are highlighted Note The White level may need to be adjusted after using the Edge filters in order to see the result of this filtering process Horizontal Edge Filter This filter brightens horizontal edges This can be useful in pinpointing bands on a gel The horizontal edge filter Horz Edge enhances image edges by shifting an image vertically by one pixel and then subtracting the shifted image from the original In an area of constant pixel intensity the subtraction yields black pixel values At an edge which is an area with large changes in intensity the subtraction yields light colored pixel values The larger the difference in intensities the lighter the resultant pixels Note After applying the horizontal edge filter the entire image may appear black and might require reducing the White level in order to better visualize the results Vertical Edge Filter This filter Vert Edge brightens vertical edges using the approach described for the horizontal edge filter see above except that the image is shifted horizontally before the shifted image is subtracted from the original In this case the vertical edges produce light colored pixel values As in the horizontal edge filte
141. esults to Excel or other spreadsheet programs click on the Export button click on the clipboard option then click on OK If you have Excel or another spreadsheet program loaded on the system and running in the background you can simply press the ALT and TAB keys simultaneously to move into the spreadsheet program You can then import the data directly to the desired spreadsheet from clipboard Note The spreadsheet program must be installed on the computer in order to export the data to that program When the appropriate export source has been selected click on the OK button to send the data 193 Exporting Quantitative Data Lane Profile The exporting of Lane Profile is similar to what has been defined above with some extra feature The Lane Profile has following window to export data Export Data Export To C Printer Mpdel HF LaserJet 1100 Om C3 Clipboard Text Format C5 Clipboard Excel Format Output Lane All Lanes C3 Current Lane Only Figure 5 57 Lane Profile Export Dialog Box Output Lane This option allows user to export what type of data to be exported Selecting All Lanes will export all lanes data or selecting Current Lane will only export current lane data To send the data click on the OK button 194 Additional Analysis Tools Enhancement Tools Analysis Tools Figure 5 58 Additional Analysis Tools in the ToolBox Additional image analysis tools can be a
142. et under the Image pull down menu A prompt will appear allowing the user to select all of the images that for the set It is possible to browse the directories looking on the network drives and removable media if necessary Once all of the images have been selected click on the open button to finish the image set The resulting image is an average of all of the images together This is a useful function for extending the dynamic range on a set of similar images by allowing bright spots and faint spots to be seen on the same image The other functions are adding subtracting and dividing images together Subtracting images is often used to remove noise from a sample by running dark images first and subtracting them out of the final image The most common application for quotient is for those technical users who run their own flat field corrections This can be done using the Flat Field Calibrate selection under the Image pull down menu which will be described in detail later in this section All three of these arithmetic functions are performed by opening the main image that will be adjusted Next select the appropriate arithmetic function under the image pull down menu Then select the image that is to be added subtracted or divided from the original image and select open The dialog box will disappear and the resultant image will appear Note Images that have been arithmetically altered are ideal for publications and documentation howev
143. et 3 Open ResEdit An animated startup display will show up and continue until you click on the mouse or any key 4 Adialog box will appear Open the TIFF image Another dialog box will appear asking if you want to add a resource fork click on OK 5 Next go to the File pulldown menu and click on Get Info for This File 6 Inthe File Info window change the Type to TIFF instead of TEXT and the Creator to DAD2 instead of DOSA Must be typed in all CAPS as shown here Close the window and save changes Quit ResEdit 7 After this procedure the icon will change to a TIFF icon and the file may be opened in Canvas A2 APPENDIX B ALPHAVIEW M MOLECULAR WEIGHT LIBRARY FILES A library including the following DNA RNA and protein molecular weight standards has been incorporated into AlohaView For information about using these standard files see Section 5 2 Values of size standards are given in basepairs diii cu Rd ua pue oe idi edi ub an B 1 DNA Size Standards in bp HINDIII PHIX174 BRL10BP BRL50BP BRL100BP PRO100BP BRL123BP BRL1KB BRLHIMW B2 RNA Size Protein Markers in kD Standards BRLRNA BRLRNA BRLPRO BRLPRO BRLPRO BRL10 NOVEXM SIGMAHM SIGMAPM T3 B3 APPENDIX C DATA TABLE DESCRIPTIONS Single Channel Columns Region Tab Band Band Identifying Number Sum of all pixel gray levels in Band Percentage Percentage Sum Sum Total of all Band Sums
144. evels due to different labeling conditions reagent concentrations exposures times or photo bleaching effects When analyzing replicate multicolor blots it is good practice to acquire images at exactly the same acquisition settings In cases where this may lead to less than optimum images for subsequent replicates due to changes in sample loading labeling or other experimental factors follow the data analysis procedure described above to compare the data from replicates Note In the Data table there are columns available for Blue Green Blue Red etc These columns display the ratio of the indicated channel Sums or BC Sums for that region and are intended for analysis of dual labeled bands or spots as may encountered in dot blots or macroarrays These raw ratios are somewhat arbitrary as they depend on the relative exposure times of the channels but do provide the relative values of the channel signal levels for that region Do not confuse these ratios with the LC regions or PC regions which indicate which regions are selected as control and experimental bands respectively G2 APPENDIX G FULLY WORKED EXAMPLES OF MULTICOLOR BAND ANALYSIS Example 1 Phosphorylation The samples are a time course study that monitors the up down regulation of phosphorylation following treatment Red and blue channel signals are detected as overlapping bands in this example The red channel represents signals from an antibody that only binds the phospho sp
145. exposure information to sequentially add images to one another Normal Sequence will not perform this addition Please note that stacking frames will increase the noise level in acquired images Sample Case Sample case Capture 5 frames at 1 5 sec exposure for total time exposure time of 15 sec Display after summation of following frames Frame 1 Image 1 sec exp time Frame 2 Frame Image 2 sec exp time Frame 3 Frame2 Image 3 sec exp time Frame 4 Frames Image 4 sec exp time Frame 5 Frame4 Image 5 sec exp time uUum Um Umm E 113 Chapter 5 THE IMAGE ANALYSIS TOOLS Default Analysis Tools Figure 5 1 Default Analysis Tools in the ToolBox Default image analysis tools are contained within the Tool Box as indicated This tool set allows the user to perform molecular weight determinations automatic counting of colonies and cells multiplex band analysis and lane profile densitometry Molecular Weight Determination Introduction The button in ToolBox Analysis Tools labeled MOL WEIGHT opens a set of tools for entering the values of known molecular weight markers and determining the molecular weights of unknown bands on the image When the MOL WEIGHT button is selected a function box appears in the area to the lower left of the screen and a data box appears on the image If the data window obscures part of the image containing bands of interest it can be moved 114 Molecular W exih
146. f 4 bx Figure 2 22 Multichannel image Multichannel image with default contrast settings bottom image and with settings optimized for each channel top image Contrast adjustments do not affect the raw data but only change the visual appearance of the image Performing data analysis on a contrast adjusted image provides the same result as on a non contrast adjusted image 99 Automatic Enhancement Auto Enhance Level 123 4 Ls Ls ee Figure 2 23 The Enhance Tools This function is ideal for new or inexperienced users of the system since it offers 9 levels of automatic image enhancement of the black white and gamma levels simultaneously For an inexperienced user in can be difficult to adjust each black white and gamma buttons to their respective optimal positions By clicking on one of the nine Auto Enhance Level buttons the image is optimized according to a unique level Button 1 will make the image darker Each increasing button click will lighten up the image until button 9 is pressed which will make the image the lightest possible To undo any Auto Enhance Levels just press on the Reset button on the main interface Original Image Auto Enhance Level 2 Image Auto Enhance Level 9 Image Auto Enhance Level Auto Enhance Level Auto Enhance Level ULL JL Le ULL LLL HNHEHBHUOUHUSHEES Original Auto Enhance Toolbox Auto Enhance Toolbox with level 2 Auto Enhance Toolbox with level 9 60 Tool Bar The T
147. fect the original image capture data EG if a chemiluminescent blot is captured using the Reverse mode in order for it to appear as film the correct selection is still Bright Spots Find Spots The Find Spot button is selected once the correct area of interest is drawn and the Bright Spot Dark Spot selection is correctly made A green outline will be drawn around the detected spots Background Threshold The background threshold slide bar will adjust the criteria used for finding spots It is on a scale of 0 to 100 saturation The sliding bar maps the dynamic range of the camera By calculating the fringe pixels in the image or ROI the initial threshold value and suggested lower or upper bound value are calculated and shown on the sliding bar The user can always override the threshold value by moving the sliding bar or typing in the counter box Target Size The user can input the minimum and maximum area in pixels of the spots or bands of the desired targets The default values are 1 pixel minimum and no limit for maximum pixels Get Data Get Data is selected once a satisfactory spot outline is achieved This will convert all drawn objects into standard Spot Densitometry objects with associated density numbers 134 Manipulating Objects Selecting Objects To select an object click on it with the mouse Handles will appear at its corners Figure 5 18 Non Selected and Selected Objects To select a second object instead of the f
148. ffects of the treatments on the expression levels of experimental proteins and to determine the relative responses of one experimental protein to the other It is assumed that replicates will be analyzed to provide significance First create regions for the experimental bands and loading controls and subtract background as described above Then perform the loading control normalization to adjust the signal levels for the Cy3 bands and Cy5 bands for differences in the amount of sample loaded per lane Note that the loading control is in the Blue channel and the experimental proteins are in the Green and Red channels respectively The reason that the loading controls and experimental proteins may be in different color channels is that the relative difference between channels is factored out during the loading control normalization calculation For Green channel signals g1 g2 and g3 and Red channel signals r1 r2 and r3 and Blue channels signals b1 b2 and b3 where the values correspond to BC Sums the results after loading control normalization are g1 b1 bm g2 b2 bm g3 b3 bm r1 o1 bm r2 b2 bm r3 b3 bm Bm mean of the Blue loading controls Now consider that the Blue signal level doubles relative to the Red and Green signal levels now the ratios are g1 2b1 2bm g2 2b2 2bm g3 2b3 2 bm r1 2b1 2bm r2 2b2 2bm r3 2b3 2bm The twos 2 factor out so the relative change in Blue to Green and Red signal levels d
149. ge formation optics lens and filter and sample position within the cabinet Consequently any change to either the illumination field or the imaging optics will degrade the calibration Examples of such changes are positioning the sample at a different level within the cabinet changing lens focus and aperture settings replacing filters or repositioning the camera and lens bracket The flat field calibration is applied automatically as part of the image acquisition process when a matching flat field is present in the application directory C Program Files AlphaView Q If a matching flat field file is not present no flat field calibration is performed Also the flat field calibration may be disabled in the camera setup and preview window during acquisition Flat Field Calibration Procedure The appropriately named flat field file will be placed in the application directory and subsequently applied to image acquisition at those illuminations and filter settings The E 1 flat utility applies a smoothing function to the acquired image to remove irregularities due to mottling or fibers in the paper STEPS 1 Place a uniform target such a piece of white laser printer paper at the sample location that will be used for fluorescent imaging hk fy Vp T Flat field target inside FluorChem Q 2 Focus on the surface of the flat target paper target shown below Note It is important to set lens focus and aperture settings to those t
150. ghting conditions can be GND Copy To Next copies al settings from the previous frame to the current frame For chemiluminescence imaging and the filter wheel is positioned for the chemiluminescence position across all frames In these situations the only variable that changes from frame to frame Copy To Next is a useful tool that optimizes setup time The Delay field configures a predetermined delay between captured imaged for kinetic experiments GNESBEUE NE ia NC MBA ACME TERRE out can be configured up to 50 minutes between images Once the movie setup is properly configured click the Go button to begin the movie process The system begins processing image acquisitions for each frame When completed the movie setup box disappears and the Toolbox window automatically configures to the Movie tools This allows for movie playback saving loading or new movie recording The movie display buttons offer the following functionality REC Launches the Camera Setup and Preview window configured to Movie Mode PLAY Displays the movie in a continuous loop STOP Stops the movie at the current frame display PAUSE Pauses movie playback at a user defined image SAVE Images are automatically opened after completion of movie mode setup Save Load Setup Acquisition Individual movie parameters can be saved for future use The Save Setup and Load Setup buttons save and load all Movie Mode setup parameters This data is saved in fi
151. ging optics will degrade the calibration Examples of such changes are positioning the sample at a different level within the cabinet changing lens focus and aperture settings replacing filters or repositioning the camera and lens bracket The flat field calibration is applied automatically as part of the image acquisition process when a matching flat field is present in the application directory C Program Files FluorChem Q If a matching flat field file is not present then a flat field calibration is not applied Also the flat field calibration may be disabled in the camera setup and preview window during acquisition The flat field image naming convention follows the format FlatNxM tif where N is an index that lists the excitation light source and M is an index that lists the emission filter wheel position Flat Field Calibration The flat field calibration procedure is performed at installation or at the factory Flat field files are provided for the most commonly encountered imaging situations Flat field calibration corrects illumination and detection path non uniformities from acquired images Properly calibrated fluorescent images should have less than 5 non uniformity across the field of view 12cmx12cm Flat field calibration is appropriately utilized when no changes are made between flat field calibration and the imaging of biological samples A specific flat field file calibrates both the illumination intensity field and the ima
152. he Image pull down menu Browse the directories for the Flat image created Click on open Make sure to save the flat field calibrated image for future use A en N 3 Register Channels When bands of different channels are not aligned multichannel Images may be registered aligned using the Channel Registration tool 1 Select Register Channels from the Image menu 2 Place two ROIs in the image according the instructions 3 Click on the Register Channels button Image translations are calibrated and applied as part of the acquisition process for multichannel images not for gray images 76 Channel Hepistration By clicking on the image please create lwo small ROl sound two smal dittinet objects which ate airanged la hom each other n oppone comer ol Hye mage Then press Fiegister channel bulton Fiegister channels Image Resize The image resize function is to resize an image to a specific dimension for use in graphical presentations You have the option to Preserve aspect ratio to avoid image dimensional distortion or you can deactivate this function and configure the image resolution to the desired Width and Height dimensions Resize image width Height Maintain aspect ratio HesizB Cancel Figure 3 14 Image Resize dialog box Note It is recommended that you DO NOT perform quantitative analysis on resized images 11 Image Info The Image Info function provides
153. he bottom of the monitor and provides a real time display of the mouse cursor x y position the image zoom factor and the grayscale intensity at the mouse cursor x y position Ready Intensity 67 Zoom 100 X 2 180 Y 340 Figure 2 26 Status bar 64 Chapter 3 DROP DOWN MENUS Across the top of the screen is a Windows menu bar containing several system operation functions These include file saving and loading edit image setup overlay file utilities view window and help functions File Edit Image Setup Overlay Utilities View Window Help Figure 3 1 AlphaView Drop Down Menu The File Menu Use this menu to save an image as a file retrieve a previously saved image select different printers print an image to a parallel printer overlay multiple images in RGB color channels close an image log off of the system or exit the system mie Edi Image Setup Oy Open Ctrl o Analvsis d Close Ctrl F4 bel Save CErl4 5 I Save S d save Al cCtrie shift s Save Modified d ee Print Ctrl ShiFk P A Printer Setup Recent Files d Exit Figure 3 2 File Pull Down Menu 65 File Open This function opens an image which has been previously saved as a TIF GLP BMP PCX TGA PIC JPG or Macintosh TIFF MAC file Lock in 3mm z256colour FriF Spotter Noise tif 20050617 210904 s3 Cv3 LiF Spotter Sharpen tif hy Recent 20050701 175747 sl Cyv3 kif test band detection amp bit tif Documents conmassieB tif Tin
154. he line then release the mouse button The arrow can be adjusted by clicking on one of the boxes at the end the other will serve as an anchor point or by clicking in the middle to drag the entire arrow iz The button labeled with an angles arrows on its side is a line drawing tool very similar to the one described above The significant difference is that this tool limits the angle that the line can be drawn to increments of 45 x n The button labeled with a square draws a rectangle or square of any size on the image After clicking on the button move the cursor to the position that should correspond to one of the corners of the rectangle Press and hold the left mouse button Using the mouse move the cursor to enlarge the rectangle When it reaches the desired size release the left mouse button 100 The button labeled with a circle draws a circle of any size After clicking on the button move the cursor to the position on the image where the circle should be started Press and hold the left mouse button Using the mouse move the cursor to enlarge the circle When the circle reaches the desired size release the left mouse button Hint to draw a perfect circle around a portion of an image first visualize a square surrounding the area of interest Position the mouse in the upper left hand corner of the square Click and drag the mouse down across the area of interest at a 45 angle until the circle encloses the area of interest
155. he spot will be detected The slide bar ranges from 0 to 100 The smaller the number the less of the spot the software will define The greater the sensitivity number the greater the area of the spot included in data collection 131 HINT Selecting a brighter pixel within the band or spot will have magic wand draw a tighter area of interest Correspondingly a less intense pixel will draw a larger perimeter around the selected spot Spot Type Under the Spot Type heading the Bright Spots selection should be checked if the image contains bright spots on a dark background e g ethidium bromide stained fluorescent gels and chemiluminescent blots The Dark Spots selection should be made when the sample contains dark sample on a light background example film coomassie blue protein gels The software should automatically determine this selection but some manual intervention may be necessary Remember as in all portions of AlohaView software if the image has been reversed negative image using the Reverse selection on the Contrast Adjustment window the Dark Spot selection should not be made Reverse is a visual alteration only and does not affect the original image capture data e g a chemiluminescent blot is captured using the Reverse mode in order for it to appear as film the correct selection is still Bright Spots Outline Type Border Outline will use the edge detection methods to determine the spot area The Box Outline will draw
156. highlighted in the image area Adjust the slider positions until the objects to be counted are highlighted The numbers next to the Min and Max settings change to reflect the positions of the sliders These numbers represent the minimum and maximum gray scale values that are currently being detected Any object whose gray level falls between these two numbers will be counted If there are two sets of objects to count click on the second colony count button next to the COUNT button and adjust the density threshold so the second set of objects are highlighted 125 3 Count the Objects When the density threshold settings and size settings are satisfactory click on the COUNT button If two types of objects are to be counted click the second colony count button and click COUNT again The counts are shown for each area of interest and the totals are given in an colony count display window on the image Figure 5 10 Colony Count Sample Results for an AOI Editing Tools After the objects have been automatically counted AlphaView will automatically open the Edit Tools functions If editing is necessary these tools manually override decisions made by the automatic counter Threshold Count Protocol areen Apr Max Area 3 247 e Hed Mas Area Manual Tool Show Results Hide Spots Help on these tools Colony Count Editing Tools 126 Displaying Spots Spots are displayed as red or green numbers on
157. highlighted in yellow Now click on a band that belongs to this band type Repeat this process for all bands belonging to this band type Then repeat the process for each band type until all bands have been identified as belonging to a band type 185 Removing Bands from a Band Type Select the Minus Band tool by clicking on the Minus Band button Click on a band type line to select it the line will be highlighted in yellow Now click on the band that you would like to remove from the selected band type Undo Redo The Undo and Redo buttons will undo and redo the last action performed respectively Band Matching Results Display Results After bands have been matched a table is generated with the matching results Click the Display Results button to view the matching results Band Matching Results File View Tope Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 3 Lane 10 Adi HF 1 1 0 0271 z z D 0473 3 3 0 0813 4 4 O 1417 5 z D 1771 b b D 2228 T f 0 2604 d a8 Darr 9 g 0 3188 10 10 0 3583 11 11 0 3917 12 12 E 0 4208 13 13 f 0 4396 14 14 4 D 4813 15 15 g 0 5000 15 16 b 0 5396 T 1 3 4 0 5646 18 18 T 0 5938 13 13 0 6313 20 20 12 B 0 6604 21 a 13 J 0 6979 Ze Ze 0 7500 File OD Images sS ample tit Metric AdustedRelatyeFrequency Reference Lane 1 Tolerance 1 00 2 Figure 5 49 Band Matching Results window displaying the similarity matrix Viewing Result Tables The mat
158. ides a means of saving and retrieving annotation overlays This is especially useful when a standard gel format is run repeatedly Lane numbers molecular weight marker sizes and other pertinent information can be stored as an Overlay file and retrieved at a later date This eliminates the need to re enter the information each time a new image is captured Utilities View Load Overlay Save Overlay B Figure 3 26 Overlay pull down menu An overlay is any set of annotations text boxes arrows etc that have been drawn on the image They can be saved as a group and opened later If repetitive samples are being imaged an overlay eliminates the need to re enter the same information such as lane numbers standard sizes etc continually Loading an Overlay The Load Overlay function allows Overlay files to be retrieved and applied to the image currently displayed Opening an Overlay after an image has been captured places the annotations on top of the image They can be stored as part of the image by saving the file as a modified file See Save Image As in Section 3 1 for instructions Select the name of the file to be loaded If necessary change the directory or drive The file name is then highlighted in the list and appears in the text box below the Filename prompt Once the file has been selected click on the OK button to load the file Alternatively double click on the file name The dialog box disappears and the an
159. ie THIS FUNCTION WILL NOT LOAD INDIVIDUAL IMAGES PREVIOUSLY CAPTURED IN NORMAL CAPTURE MODE SAVE Save a movie of images Saving An Individual Image From a Movie After you load play and stop a movie at the desired image it is possible to save the individual image seen on the screen Use the SAVE AS button located on the tool bar to save the image in the desired location and file format on the local or network drives Saving Partially acquired movie It is possible to stop the movie in the middle of acquiring frames the user can exit the movie mode to save the acquired frames only Stack Frames Option 1 Go to continue movie Option 2 Close Window to view and or save acquired frames 112 Movie Mode Save Load Movie Mode setup routines Two buttons Save Setup and Load Setup allow you to save and load all Movie Mode setup parameters Files are saved as mvf files Camera Setup and Pnewerw Load pocion Lii piae x1 E Cherri D irplas Sheree Sarurahon Deyme Coprtetes O Stack Frames 3 Lead Sehu J Auto Content EI NCIACES EMO i a re lt Copsio End Stoker bovis sake Save Setup Tuta Zoom inal E ua erri Auto expose EN M a emo OVE 0 0 amp Hora Hore s Frame Stacking At the top of the Movie Set up window is an option for stacking frames If this selection was chosen during acquisition of the image Stack Frames will use all previous
160. ile tools ee es On oe st is a taito Tes LLLI e ee ui uM tw e TE ond Figure 5 34 Lane Profile Template 150 Setting up the Lane Template When the Lane Profile button is clicked a lane template appears on the image The red lines indicate the borders of adjacent lanes The green lines define the Scan Width described below Under the heading Grid Controls the number of lanes can be specified The number under the title Lanes indicates the number of lanes and can be changed by clicking the right increase or left decrease arrows As the lane number is adjusted the template displayed over the image is updated to reflect the changes Once the number of lanes has been set the template should be adjusted so it coincides with the lanes on the image If the lanes on the gel are horizontally oriented sideways the template can be rotated To change the orientation of the template to coincide with the image click the ROTATE checkbox The template will rotate 90 counterclockwise Clicking in the check box again X disappears rotates the template 90 clockwise restoring it to its original orientation After specifying the correct number of lanes and setting the orientation use the mouse to drag the outside borders of the template so they frame the lanes to be scanned Clicking on or within any border repositions the template Clicking on the blue handles surrounding the templa
161. ilters Toolbox with More selected General Information To enhance an image filters change the value assigned to each pixel The new value assigned to a pixel is determined based on the values of the other pixels in its local vicinity or neighborhood The neighborhood is a two dimensional matrix of pixel values where each dimension has an odd number of elements The pixel of interest is the one at the center of the neighborhood This is the pixel whose old value is being replaced with a new one as the result of the filtering algorithm The pixels in a neighborhood provide information about the brightness trend This information is important to the filtering process The brightness trend is also referred to as the spatial frequency Images with high spatial frequency content contain large closely spaced changes in pixel values For example on a black and white checkerboard the smaller the squares the higher the frequency content Images with low spatial frequency content for example images of clouds contain large areas of slowly changing pixel values Most of the filter options available with the exception of the Noise filters use a weighted summation process to determine the value assigned to the pixel of interest Each pixel in a 3x3 neighborhood is multiplied by a convolution kernel having the same dimensions The resulting sum is assigned to the pixel of interest K1xP1 K2xP2 K3xP3 K4xP4 P1 P2 P3 K1 K2 K
162. in 3 Appendix X Enhancement Tools Analysis Tools Bkgmd Control Std Curve Protocol Repor Save Protocol protocol contains all band and background regions created with the loading controls band controls and standard curve settings used amp protocol can be saved at any point in the analysis workflow Load Protocol Adjust regions after loading a protocol by File name phospho sda selecting moving and resizing regions My Network Files of type Multiplex Band Analysis Protocol Files sda C Help on these tools RGB 346 2459 1685 Zoom 79 cul Appendix x doc Mi Y alphaInnotech Pot X 849 Y 263 This saved protocol has Regions and a Local Background Correction defined In this example the Regions have been purposefully defined larger than the bands and contain many pixels of background signal G3 Y Alphalnnotech Potatoe File Edit Image Setup Overlay Utilities view Window Help BOW OCOY dowi 0O B mn ir phospho tif 4bx Gamma Linear Log C Show Grid Black 99 La Whte 7 7 9 9L s Gamma 4 T J dae 1 00 TTE Auto Enhance Level CELE sys Enhancement Tools Analysis Toos Bkgmd Control Std Curve Protocol Repor pro
163. ines the height of each bar along the axis 93 A Coomassie blue stained protein gel visualized with a white light box has a histogram reflecting mosily bright pixels Scale zoom 9 Linear Scale ix 4s C3 Log Scale C316 64s Histogram of a typical Coomassie gel Most of the pixels are found in the light portion of this histogram The dark bands represent a small number of pixels and include a variety of gray values and therefore do not show up as a single peak The histogram function is particularly useful to verify that an image spans the maximum range of gray levels When an image is to be used for analysis it is especially important that the gray level range be as large as possible If an image does not include most of the gray levels we recommend repeating the image capturing process The Rotate Flip Tool _Rotatefip Rotate Angle 9n je 0 90 Figure 4 3 The Rotate Flip Tool Flip 4 The Rotate Flip tool is found in Tool Box Enhancement Tools This function rotates the image in a clockwise or counterclockwise direction by 1 degree increments up to a maximum of 90 degrees in either direction This is a useful tool if the image is not aligned properly during the capturing process To rotate an image click and hold down on the center sliding bar with the left mouse button and move it left or right until the desired angle of rotation appears in the rotate box Release th
164. ions outlines are dashed to indicate the corresponding control and experimental bands After identifying the control and experimental bands the Data table is updated with four additional columns PCN Sum PCN Average PC Regions and Fold Change PCN Positive Control Normalized The control band is assigned a PCN Average value of 100 and the normalized experimental bands are adjusted to this value in PCN average column Multiplex Band Analysis Result Maximum Intensity Channel nx Export view Display Style Band Channel LCN Average NCN Sum NCH Average NC Regions Fold Change 2 Blue 128 2 Green 903 100 00 1 00 3 Green 1 643 207 342 181 38 Green 3 Green 2 182 4 Green 651 82 172 72 08 Green 4 Green 2 Ls 5 Green 1 804 227 659 199 70 Green 5 Green 2 2 00 6 Green 1 447 182 617 160 19 Green 6 Green 2 1 50 T Green 1 409 177 868 156 02 Green 7 Green 2 1 56 8 Green 1 172 147 966 125 79 Green 8 Green 2 1 30 E Red 2 579 100 00 1 00 10 Red 1 434 63 377 55 59 Red 10 Red 9 1 80 11 Red 1 315 58 120 50 98 Red 11 Red9 1 96 12 Red 2 673 118 165 103 65 Red 12 Red 9 1 04 13 Red 2 472 108 296 985 87 Red 13 Red 9 1 04 14 Red 1 762 77 878 68 32 Red 14 Red 9 1 46 15 Red 3 575 158 047 138 64 Red 15 Red 9 1 35 16 Red 2 092 92 495 81 14 Red 16 Red 9 1 23 17 Blue 18 Blue 18 Blue 20 Blue 21 Blue 22 Blue 23 Blue 24 Blue Figure 5 27 Band control norm
165. irst click on the desired object To select more than one object at a time drag the mouse around all of the objects of interest Note an object must be completely surrounded by the mouse operation in order to be selected To select more than one object where it is not feasible to drag the mouse around objects hold the shift key and click on each object to be selected To de select an object click the left mouse button outside of the selected object The handles disappear indicating that the object is no longer selected Copying Objects To draw boxes or ellipses enclosing the same number of data points use the COPY function First draw or select an object of the desired shape and size as described above Next click on the COPY button in the toolbox A duplicate of the selected object will appear on the screen This object can then be moved to the desired position To make multiple copies of an object continue to click on the COPY button until the desired number of copies is displayed Moving Objects An object can be moved to fine tune its position simply by clicking on it holding down the left mouse button and dragging it to the desired location Deleting Objects To remove an object or set of objects select the object s Select DELETE button on keyboard All the selected objects are removed from the image window and their associated data is cleared from the data window 135 Multiplex Band Analysis Measurements As
166. istinct fluorophores and imaged with the appropriate excitation and emission wavelengths With multicolor fluorescence you can resolve and quantify proteins that migrate in overlapping bands and also accurately quantify proteins with much different levels of relative abundance In particular dim bands adjacent to bright bands can be resolved and quantified For example y labeling the loading control and experimental proteins with different fluorophores the labeling conditions and image acquisition settings can be adjusted appropriately to bring each of the proteins into the quantitative dynamic range of the image The relative expression levels of each protein can then be analyzed in comparison to relevant positive controls and then compared to each other There are many labeling protocols available for multicolor fluorescence Western blots Choosing the optimal labeling protocol for a specific application depends upon the nature of the primary antibodies used and other factors One general multicolor protocol involves using primary antibodies raised in different species for each protein followed by the corresponding species specific secondary antibodies For example a generic combination of primary and secondary antibody combinations is shown in Figure 1 The antibodies chosen should be tested for cross reactivity to other antigens non target proteins and other antibodies utilized in the assay 11 goat a rabbit IgG Cy5 goat a mouse IgG C
167. ithm for automatically finding bands in query lanes To use this function select Auto Query from the Query menu A yellow vertical line will appear attached to the cursor Position the line over the lane of interest and click the left mouse button The bands in the lanes will be selected automatically and their data will appear in the Data box Repositioning and Deleting Bands Bands can be repositioned simply by positioning the cursor clicking and dragging the band to the desired location To delete an unknown band indicator point the cursor at the band and click the left mouse button The band in question is highlighted Alternatively clicking in the data table highlights the band s information and selects it on the image Click on the Delete Band function in the Query menu The band will be deleted as will the band s data in the query data table Bands entered after the one deleted are renumbered on the image and the data table To delete all the queried bands select the Clear Queries function in the Query menu Using the Molecular Weight Standards Library AlphaView contains a library of DNA and protein molecular weight standards Additional sets of markers can also be added to this library for later access For a complete list of pre loaded files and the band sizes in each see Appendix B Applying a Set of Saved Markers From the Marker menu in the data box select Get Markers from File A dialog box appears 118 Open Molecu
168. its the catalytic reaction of an enzyme and a peroxide based substrate to produce a light signal with very low background as no illumination is required The enzyme e g horseradish peroxidase is conjugated to a secondary antibody that binds to the primary antibody specific to the protein of interest Chemiluminescence is widely used by researchers because it is much less hazardous than radioactivity while achieving equal performance Fluorescence Detection The quantitative power of Western Blot analysis can be further improved by using fluorescence detection methods for many experiments Single color fluorescence detection methods compare favorably to chemiluminescence in terms of cost and ease of use While fluorescence may often have a higher level of background signal fluorescence has an intrinsically linear response to protein level whereas the enzymatic reaction producing the chemiluminescence signal may locally saturate causing a brownout Multicolor fluorescence detection methods offer significant additional advantages for quantifying multiple proteins on a single blot Multiple proteins can be accurately quantified using chemiluminescence or single color fluorescence detection methods only when the proteins are very similar in abundance and are also well resolved on the blot There are many experimental situations where these conditions are not met With multicolor fluorescence detection method using antibodies labeled with d
169. ject regions respectively Second note that the background correction is automatically adjusted for any differences in area between the background and object regions as fOO6Fllows Data Definitions Background Corrected BC Average Background Corrected Average Region Average Bkgd Region Average Background Corrected BC Sum Background Corrected Sum BC region Average Region Area For regions with different area the relevant quantities are adjusted for the relative region area to obtain accurate corrected values For example if local background correction is used the area is 10 pixels for the background region When this value is used to correct a data region the actual value used is scaled such that the 10 pixel area appears to be the same area as the data region In this way regions with different areas are accurately accounted for in all calculations 138 Figure 5 21 Multichannel image with regional background A multichannel image with regional background applied The background values from a single region are extracted and applied to each of the three channels respectively Multiplex Band Analysis Result All Channels Export View Display Style Band Blue BC Average Blue Signal Noise Green BC Average Green Signal Noise Red BC Average Red Signal Noise 1 45 0 84 104 0 83 23 0 50 2 74 1 38 841 6 69 27 0 58 3 77 1 44 1 558 12 39 29 0 62 4 74 1 38 566 4 50 22 0 47 5 153 2 86 2 233 17 76 13 0 41 6 102 1 90 1 51
170. l General Mote Generate Report Help on these tools Figure 5 55 Report General Tab 192 Export Results Once the analysis has completed then there is option to export the result Either the results can print directly to a printer or export the results as ASCII data for direct importation into Excel or other spreadsheet programs Export Data Export To Printer pdclAHP Laseulet 1100 C3 File C3 Append Clipboard Excel Format Clipboard Text Format Figure 5 56 Common Export Dialog Box Sending Data to a Printer To send the data directly to the Video Printer or Default Printer click on the Export button and click in the circle next to appropriate printer Sending Data to a File If you would like to take the data from the system and import it into another computer the data can be saved to a diskette or network which will allow you to open the data file on a separate workstation connected to the network Click on the Export button and then on the File option Specify the path and file name to send the data file The data is saved as an ASCII file and can be imported into most spreadsheet programs ASCII is a very common file format output option for numerical data Appending Data to a Same File Export dialog also allows you to append data to an existing exported file Note This feature is applicable to AutoCount and Array analysis modules Sending Data to a Spreadsheet Program To send the data r
171. lar Weight File Ij TestDotfuscator i O DatFuscated Updater My Recent 9 Downloads visual Studio 2005 Documents Lego 2 My I50 Files Amy Music Desktop Emy Pictures My Received Files Amy Shapes 7 Ely Stationery LM Videos My Documents math l My Virtual Machines Research Works OSOL Server Management Studio test Files My Network Files of type Mal Wr Files rwm Get Markers from File Dialog Box Using the left mouse button click on the name of the file to be loaded That name is then highlighted in the list and appears in the text box below the File Name prompt The current directory path is shown beneath the Folders heading Below this is a graphical depiction of the path and the sub directories of the current directory If the file of interest is in a different directory than the one open double click on the appropriate folder icon Once the file has been selected click on the OK button to load the file Alternatively double click on the file name The dialog box disappears and a new dialog box appears Load Markers 1 J 7 Markers found in file Auto Load File Auto Load Dialog Box This box gives the file name selected as well as the number of markers contained within the file It also give the user the option of auto loading the file If Yes is selected a vertical line will appear attached to the cursor Place the vertical line in the marker lane on the gel and click Al
172. late The y axis represents the average pixel intensity across the width of the scan Visualization of the Active Graph The drop down selections in the profile and data table Data BaseLine and Options allow for the user to perform editing visualization and outputting tasks pertaining to this window A 112 13415 16 1718 NH LII Data The data drop down menu allows for the user to superimpose different lanes onto the Active Window using the Overlay Control selection See Auto Grid Overlay Control above for detailed instructions 175 Baseline Background subtraction can be preformed using this menu See Auto Grid Baseline Control described previously in this section for detailed instructions Please note that background subtraction in AutoLane can have a dramatic effect on how many peaks are recognized as bands Try changing the background subtraction to optimize band recognition Options Background Color allows the user to select a variety of different background colors for the Active Graph Axes allows the user to display the axes on the graph x axis pixel distance down the lane y axis pixel intensity in gray scale Show Strip will display a strip of the lane being currently analyzed Select Window will allow the user to choose which of the data table windows to display Print will allow the user to print any or all of the profile and data table windows Clipboard will allow the user to send any or all of the profile an
173. les using the MVF format 42 Camera Selup and Preview spi Chers Dimple 7 Shows Sautan Auto Cerita M crawled Toli a C Tubo Zoom tool Fiia 1 Stack Frames E i Check box M Aulo expose J e AE come 018 10 5 2 Load Setup Su Save Setup vr E button Hoynablilha Hore Shiela Figure 2 7 Movie Mode Load Save Setup Frame Stacking The Movie Mode configuration of the Camera Setup amp Preview window provides an option for stacking frames If this selection is used during image acquisition GEEEEEEEEEEESEEEO exposure information to sequentially add images to one another Normal Sequence does not perform this addition Sample Case Sample case Capture 5 frames at 1 5 sec exposure for total time exposure time of 15 sec Display after summation of following frames Frame 1 Image 1 sec exp time Frame 2 Frame Image 2 sec exp time Frame 3 Frame2 Image 3 sec exp time Frame 4 Frames Image 4 sec exp time Frame 5 Frame4 Image 5 sec exp time uUum Uum Um Um 43 Automatic Image Capture AIC Software For molecular biology researchers engaged in high throughput and particularly high volume work involving repetitive assays Automatic Image Capture software is available This system automatically adjusts lens focus filter settings for fluorescent imaging light settings and exposure for perfect images All of this with just one click making the
174. levels primarily thermally generated electrons called dark current which are typically quite stable and can be corrected Once there levels are characterized they can be removed from the image under consideration This process is uniformly referred to as image calibration along with flat field correction to produce an image that contains only signals from the object under test and some residual noise from the original error sources The minimum requirements for accurate determination of bias and dark current levels is thermal stability and light tightness Any fluctuations in temperature will alter the bias and dark current levels Any light leaks will result in an additional source for errors that will not be correctly removed from images under consideration Bias and darkmaster files are created by collecting a set of 16 images that are combined in such a way to reduce the noise levels and reject spurious signals such as cosmic rays etc A set of such files for darkmasters are created at different exposure times to create a library of darkmaster files The files are logged into the program folder and used for all subsequent imaging sessions When an object is imaged the exposure time of the image is used to select the darkmaster with a similar exposure time and the darkmaster is scaled such that the dark current levels match that of the image under consideration The bias and scaled darkmaster files are then subtracted from the image under con
175. luorescence images acquired by the FluorChem Q ma 12 used primarily for samples that have been labeled with two or three fluorophores such as CY2 CY3 and CY5 Multichannel images are acquired in a 48 bit RGB format with 16 bit depth in each channel The FluorChem Q includes a number of advanced image acquisition features designed for optimal image acquisition of multicolor fluorescence Western blots Three spectral detection channels optimized for the most commonly available fluorescence reagents CY2 CY3 and CY5 Channel Excitation Emission Dye also Red Cy5 5 Alexa 680 DyLight 680 The acquisition of multicolor fluorescence images follows the same overall process as the acquisition of single channel images The objective is to acquire an in focus image of the blot with the features of interest exposed to levels suitable for accurate quantitative analysis In practice the presence of background signal arising from the blot and sample will reduce the assay dynamic range Generally using auto expose will produce an optimal image if the brightest feature in the image is a feature of interest Mouse Functions The FluorChem Q System comes packaged with a two button mouse The left button activates functions and makes selections when using the software In some cases the right mouse button can recall or reactivate the function that was most recently assigned to the left mouse button About This Manual This manual uses diffe
176. lution This mode decreases exposure times from full resolution images by approximately a factor of nine Fast Low Recommended for Chemiluminescence this mode captures images with a 4x4 pixel bin 512x 512 pixels Similar to Super Speed this mode decrease exposure time but with a tradeoff in resolution This mode decreases exposure times from full resolution images by approximately a factor of sixteen Super Speed Recommended for Chemiluminescence this mode captures the image performing an 8x8 pixel bin 256 x 256 pixels This mode significantly decreases the exposure time required for gr a long chemiluminescent exposure This mode s resolution is set to 256x256 pixels an recommended for precise quantitative analysis or publication Super Speed is ideal for Expose Preview functionality when using auto expose to determine the chemiluminescent blot signal level It is also useful for rapid visualization of signals for a qualitative assessment of the sample 38 Gray Scale Optimization for Saturation and Contrast Displays The following figure illustrates the check boxes available for modifying the displayed image Display Chemi Display Show Saturation Auto Contrast ht or under exposed too dark for Any images set for analysis must not be over exposed too li maximum clarity After inspecting the image s saturation the imaging controls can be adjusted accordingly to optimize the image The Show Sat
177. mage Note The images must be the same bit depth and resolution for the software to overlay the images Extract Channels Extract channels produces a separate image for each channel from a Multichannel image and display them on image screen window The Extract Channels icon is available when a Multichannel image is active 73 Channel Viewer The Channel Viewer icon is available when a Multichannel image is active Averages Red 5 385 Greer 5 951 Blue 25 478 The Channel Viewer opens a window that displays the multicolor image and each corresponding channel separately The window may be moved around the image by simply dragging the window The average Red Green and Blue intensities in the window are also displayed The Channel Viewer can seamlessly be used in Comparer View by moving Channel Viewer over any image Equalize The equalize option performs a duplicate function to the EQUAL option in the Contrast Adjustment Window Arithmetic The Arithmetic function is used to add subtract average and divide several images together to generate a compiled image Setup Overlay Ubilities View Window OPH Overlay Equalize Arithmetic Conversion d Subtract Flat Field Calibrate Average Ft arri Resize Image Information Figure 3 12 Image Arithmetic dialog box 74 To average a set of images together open one of the images in the set and then select Average a S
178. malization Amounts H 3 B Plat Unknowns Help on these tools In the Std Curve data table enter the known concentrations of protein for each standard Select an appropriate Curve Fitting model and select an appropriate Y axis parameter LCN Avg is preferable in this example Once the known concentrations are entered the curve fit is calculated and a graph plotted Y Alphalnnotech AlphaViewQ File Edit Image Setup Overlay Utilities View Window Help SOBE COo eS o Oe Main n Quant tif 4bx Gamma Linear Log Show Gn Back 9 8 84 White 9 y JL SE Gamma J 9 1 00 M i MN Display composite Reverse Auto contrast Auto Enhance Level CREE CIEE Enhancement Tools Analysis Tools Colony Count Bkgrnd Control Std Curve Protocol Repor Enter units ng 1 Band Red LCN Average ng 15 1 343 33 16 926 116 17 756 D4 v Curve fitting Model Linear v EH Band analysis results All Channels A x Standard Curve Linear n x Equation y 0 003445 x 2 217 EE ue Busse KETE Y Axis LCN Average Y Graph wA a Enne e a Wks Oa D DEA G EEE T Band Blue LCN Avg Green LCN Avg Red LCN Avg Red ng PCN Averac Color control normalization 2 zan Res nea Identify CC band Clear normalization 5 95 3181 943 103 s uu 8 786 13 622 1 128 426 84 Am
179. milar Select any of the filter combination Cy5 Cy3 and C2 are selected here for channels 1 2 and 3 respectively Camera Setup and Preview Change interface type Exposure Auto expose You are now previewing your image 1 To have the software automatically calculate your exposure time wait for the status bar to turn green or manually r exposure time by unchecking Auto Expose and entering a time in the box above 2 Focus on your image If zoom is available zoom in on your image 3 If required repeat step 1 until the image is ready to be acquired 4 To acquire image press Next The first channel Cy5 is displayed user can access the advanced mode or click next for the next filter combination The next screen is advanced mode for the Cy5 filter combination in this mode the user can modify any of the controls clicks Next to go to the next filter combination or back to the current filter combination preview mode 2 Camera Setup and Preview Zoom AOI Baoen Change interface type J l Load protocol Display T RN W Oe Show Saturation C Auto Contrast Tubo Exposure Auto expose in s AE comp 0 1 8 D 20 2 Cabinet Excitation Emission cys Cv5 Advanced haNavigator Channel assignment Tab label You are in Advanced mode now Speed Resolution Notmal Utra v Select Help to quide you in this screen or ss Back button to return to simple acquire screen Noise red
180. n thickness line type etc can also be changed simply by clicking on the desired choice as described in Object Attributes above To draw another object click the right mouse button to return to draw mode or click on one of the drawing tool buttons a w The selection tool allows the user to select all drawing for further operations such as vertical alignment asc The button labeled with an ABC adds text to the image Place the cursor at the location on the image where the left edge of the text should appear Click the left mouse button and begin typing To place another piece of text click where it should be placed Once all text is entered click on the right mouse button To edit text double click on it An edit window will appear in which changes can be made To change fonts see Text Background and Font above A The button labeled with a pencil icon allows the user to draw lines freehand After clicking on the pencil move the cursor to the correct position on the image to begin drawing Press and hold the left mouse button Using the mouse move the cursor as if it were a pencil When finished drawing release the mouse button w NJ The button labeled with a diagonal line and arrow draws arrows and straight lines After clicking on the button move the cursor to the position on the image where the line should begin Press and hold the left mouse button Using the mouse move the cursor to the other end point of t
181. nd on the image Click on the Delete Marker function in the Marker menu The marker is deleted as is the band s data in the marker data table Markers entered after the deleted bands are renumbered on the image and the data table To delete all the markers and start over select the Clear Markers function in the Marker menu Determining Molecular Weights of Unknown Bands After marker values are entered the molecular weight of any unknown band can be determined Manually Selecting Bands To indicate unknown bands manually select the Add Band function from the Query menu Just as in Add Marker above a line will appear attached to the cursor Point the cursor at the band of interest and click the mouse button The molecular weight of the band is automatically calculated and displayed in the Queries section of the data box To select a second band click the right mouse button and the cursor line will reappear Repeat this procedure for all bands for which molecular weights is to be determined Note that the molecular weight markers appear in blue while the queries appear in green 117 The molecular weight of a band is calculated based on the graph of the known marker bands Note If a query band lies outside of the markers it will be extrapolated in Least Squares Fit mode or given a value of N A in Point to Point mode See The Graph Tool below for more information Automatic Band Finding AlphaView includes an algor
182. nd 4 now shows ng quantities for each color channel These quantities are also shown on the standard curve graph Standard Curve Linear n x Linear deere 0 01069 eee ere Ra 0 9597 ILS 93 2 o 4 ng 61 6 44 8 26 0 3 453 6 5027 8 6 601 5 5 17641 9 750 2 11 324 4 BC Average 148 See Appendix X for a detailed analysis shown use of CCN to determine the amount of phosphorylated versus non phosphorylated protein isoforms 149 Lane Profile Lane Densitometry The Lane Profile button in ToolBox Analysis Tools accesses a set of densitometry tools with which bands on a gel can be scanned and analyzed in a lane format There are two different ways in which this can be done Auto Lane and Auto Grid Auto Grid allows the user to manually define the lane number lane shape and scan width of the Grid Auto Lane is a completely automated feature which will automatically define lane number and band finding parameters for the user Both detection methods provide similar data Image particulars and user preference will determine which method works best Auto Grid When the Lane Profile tab is selected the Auto Grid template appears on the image and the following functions are displayed S corr Band Matching Protocol Report Result Gel Smiling Mal wt Mass Std Auto Grid lanes 8 width Skewing Band Detection Invert Detect Peaks _ Help on these tools Figure 5 33 Lane Prof
183. nd filter selection Selecting a desired option via the software interface produces the same result as using the physical controls on the cabinet NOTE A slight delay occurs upon pressing the button and activation of a light source Filters selection is linked to appropriate sample visualization Filter options include Ethidium Bromide colorimetric stains film SYPRO Orange Filter Position 2 595nm Filter Position 3 CY2 Fluorescein SYBR Gold 520nm SYBR Safe 530nm Filter Position 4 CY3 SYPRO Red Texas Red 630nm Filter Position 5 CY5Filter Position 6 optional Hoechst Blue 460nm OR Empty Position NOTE 1 Each Filter has an approximate bandwidth of 40nm to allow for use with other fluorescence stains as they are developed For custom applications please contact Alpha Innotech directly to discuss custom filter design for specific applications NOTE 2 If exposure time is less than 100 ms yet signal is strong close the aperture by several f stops Turbo Modes and Speed Resolution Settings FluorChem Q system offers different speed and resolution settings to allow image acquisition across a variety of needs Tweaking resolution and speed settings allow image acquisition to occur as quickly as possible with the highest resolution possible or somewhere in between Available acquisition modes include 5 binning modes and 2 Turbo modes Change irtertace type Protocol i Load protocol Displa
184. ndard lane into the total mass per lane field 3 Select the Calculation method to be used for Mass calculation 4 Click the Open Marker or Add Marker buttons and apply MW standard markers in the normal way 5 Click OK or Apply The Mass for all bands is now calculated based on the total mass entered for the MW standard lane and reported in one report Lane Profile Result nx Data Baseline Options Lane Peak Distance width Height Area Hf Adi Rf il 2 4 131 3 32 11 2 29 0 3629 0 3629 I 5 140 J n3 21 4 21 3878 3878 ee ae XD RESO E X 2 f 162 12 33 54 10 42 0 4485 0 4488 2 H 17h 13 17145 Fad 17 AMN 427 1 4275 E E W based on Standard Sample Value Linear Log Mass based on Standard Sample Value Least Square Fit lt Note Legend Describes which lanes bands make up the MW and Mass standard curve values Also notes which regression method has been used to calculate the MW and Mass curves mws Molecular Weight Marker ms Mass Standard Marker 167 Saving and Loading Mass Standards Mass standard files can be loaded onto any image and saved for future reference To load a marker select Mass Standard from Lane Profile A Mass Standard tab will show on the screen Scarng Band Matching Protocol Fieport Tools Result Gel smiling Mal wt Mass Std Select Mass Cale Least Square Fit iw Op
185. ndation No Background or Manual Background is suggested for AutoSpot Magic Wand only the border outlines though or any object where little if any background is included in the object area of interest Background Link Unlink Tool Link Background Applied in conjunction with Multi Regional Background to link a background region to a subset of object regions First place a background region in an area of the image representative of the background specific to the subset of object regions Draw a rectangle by left clicking on the image and dragging to include the desired subset of bands and a single background region to be linked Alternatively use Ctrl left click to select multiple regions and the specific background region Then select the Link Background button The data table is updated with Background corrected values Unlink Background tool Used to unlink the background regions from the band subsets Figure 5 20 Unlink Background Tool B1 background and data box 2 region are selected and Linked as shown When using the region and multiregional background correction methods there are two points to consider First note that a background region applied to a multichannel image extracts and applies the background values on each channel using the respective background and region values for that channel For a three channel image for example Blue Green and Red background values are extracted and applied to the Blue Green Red channel ob
186. nds within the lane calculated As with most AlphaView features a right mouse click will reactivate the function and another lane will be ready to be added Edit Lanes This feature allows for the horizontal manipulation of the center line in each of the found bands The integrated area of each band will be altered as the curve is re figured to match the pixels surrounding the centroid mark Delete Lane This selection will activate the cursor to delete a lane on the gel image After selecting the button left mouse click on the lane in the image to delete As with all AlphaView functions a right mouse click will reactivate the function for further lane deletions Add Band This selection will activate the cursor to add a band onto the gel image After selecting the button left mouse click on the area in the gel in which a band should be added As with all AlohaView functions a right mouse click will reactivate the function for further band additions Find Bands This selection will re detect the bands in the lanes based upon a new sensitivity setting 0 9 The higher the sensitivity the more bands the software will find Delete Bands This selection will activate the cursor to delete a band in the gel image After selecting the button left mouse click on the area in the gel in which a band should be added As with all AlphaView functions a right mouse click will reactivate the function for further band deletions Display Options This feature
187. nel respectively Use the Contrast Adjustments to optimize the display to enhance the features of interest in the image Contrast adjustment tools modify the image display on the monitor only and do not change the original quantitative data Adjust the scales by moving the slider clicking on an increment arrow or by typing a number in the value box followed by Enter on the keyboard To adjust the contrast of a three color image begin by selecting Display Composite this is the default selection after acquiring a three color image Next select the Red channel selector Set the Black level to 0 and the White level to 65535 Gamma levels should be between 0 6 and 0 7 To reduce the normal background observed in the Red channel slowly increase the black level Once the Red background level appears visually Reduced use the Gamma selector to increase the intensity of the Red bands Repeat this protocol for the Green channel and Blue channel Single Channel Selection When Display Composite is checked the image display shows all three channels as a composite image An single channel can be shown independent of the other color channels by selecting clicking the appropriate color channel then de selection clicking of the display composite button Moving the sliders will now change only the display settings of the actively selected color channel 58 20080325 01_ ples Crop 1 tif qbx lt ji 20080325 01 mul rop 1unadjusedt
188. nents must be mated only to their correct ports during system installation Power Strip Surge Protector Setup Turn the power strip surge protector power switch off Plug the power strip surge protector into a wall outlet preferably a dedicated circuit and turn the power on CAUTION Using a separate circuit is highly recommended The cabinet must be turned on after the operating system has been completely loaded for the software to function optimally Computer Monitor Mouse and Keyboard Setup The computer must have the monitor mouse lg BS COMI pot for and keyboard connected in the correct ports CA m Multilmage Il Light see Figure 2 A standard three prong power 11 cable should be plugged into the back of the computer and the power strip Connect the monitors video cable to the monitor port on the back of the computer If after market video cards were preinstalled into your computer system connect the monitor s video cable to the video card connector Cabinet 9 LPT1 port for parallel port printers ee Connect the monitors power cable to the Keyboard Mouse power strip connections color coded purple and The mouse and keyboard connectors are color gre en coded and icon identified Attach the mouse and keyboard cables to the connectors on the EF LISB Printer back of the computer by matching the colors B connection color some systems have USB mouse and 8 coded with a red keyboa
189. notations in the selected file appear on the image To dismiss the dialog box without loading annotations click on the CANCEL button 84 Saving an Overlay Once annotations have been made select Save Overlay from the Overlay menu My Recent Documents 3 My Computer My Network Save as type Overlay Files ory v Canca Figure 3 27 Save Overlay Dialog Box Enter a new file name in the text box below the File Name prompt AlphaView will automatically give the appropriate 3 character extension The current directory is the one in which the new overlay file will be saved If necessary change the directory or drive as described in Section 3 1 Once a name has been entered and the appropriate directory has been accessed click the SAVE button to save the overlay file 85 Overlay Libraries AlphaView contains a library of overlays that can be accessed through the Load Overlay function described above This library of overlays is stored in the Image folder located in the AlphaView directory O8WHITE OVR 08BLACK OVR 8 lane labels in white black 1OWHITE OVR 10BLACK OVR 10 lane labels in white black 12WHITE OVHR 12BLACK OVR 12 lane labels in white black 15WHITE OVHR 15BLACK OVR 15 lane labels in white black 24WHITE OVR 24BLACK OVR 24 lane labels in white black HINDIII DVR X Hindlll label The objects in these overlays can be repositioned resized re colored copied or deleted as needed Show Annotation
190. ns Cluster Method Neighbor Joining Outgroup Default Root Clusters BET Distance Matris Method Dice Coefficient Cluster Method Neighbor Joining File Name D Images sS ample tif Metric AdjustedRelatweFrequency Reference Lane 1 Tolerance 1 00 Figure 5 50 Dendrogram window Dendrogram window where Dendrograms are generated and Similarity Matrices are accessed Similarity Matrix Select a method from the Distance Matrix pull down for calculating similarity The choices are Dice Coefficient Jaccard Coefficient Pearson Coefficient and Frequency Similarity These are standard statistical algorithms references can be found in most statistical text books Select the Display Matrix option from the Options to display the Similarity Matrix 187 Similarity Matrix Sample tif Mx File 2 3 4 5 B a J 10 1 100 37 0370 6 69565 5 69565 0 0 0 0 ar 0370 0 2 of 0370 100 40 3 B B3BER 100 100 4 2 69565 100 100 0 0 0 5 t LH 100 100 100 100 U 100 b 0 100 100 100 100 U 100 f 0 100 100 100 100 100 H O 100 100 100 100 100 3 of 0370 40 0 100 10 O 0 100 100 100 100 100 Similarity Matis Calculated by Dice Coefficient File D Images sS ample tit Metric AdjustedHelatieeFrequencu Reference Lane 1 Tolerance 1 00 x Figure 5 51 Similarity Matrix The similari
191. ntrols button when done or right click to deselect tool All loading controls must from the same color channel as would be expected for a protein represented in each lane identified with the same dye color Identify Experimental Bands Identify each member of a single row of experimental bands using the mouse pointer You can either left click on each of the region or draw a rectangle to select a row of regions The Identify Experimental Bands bution will automatically deselect when the number of bands identified in the row reaches the number of loading control bands 140 There may be multiple rows of experimental bands in the same or different channels corresponding to other proteins labeled with the same or different fluorophores Each row of experimental bands must have the same number of regions as the loading control row so that the correspondence between the loading controls and experimental bands can be established If the signal for an experimental band is absent in one lane of a row place a region in the corresponding lane where the signal is expected The software identifies the correspondence between the loading controls and experimental bands on the basis of their positions in the image The leftmost loading control is matched with leftmost experimental band The order in which bands are selected does not matter The lane correspondence for the loading controls and experimental bands is indicated by the matching colors of the
192. nually edit or correct some matches The tools under Manual Matching can be used for this purpose Refer to the section below on Manual Matching for a description of each tool 184 Manual Band Matching Tools Result Gel Smiling Mal wt Mass Std scoring Band Matching Protocol Report Automatic Matching Metric Adjusted RI Matching Matching Reference Lane 1 v zi Matching Tolerance 0 00 Manual New Band Type Display Option Show Result Help on these tools Band Matching Dialog window Manual band matching tools are useful for quickly matching lanes with only a few bands or for manual edits and corrections to Automatic Band Matching results New Band Type Select the New Band Type tool by clicking the New Band Type button A Band Type is a band with a unique Rf or Adj Rf Value Each band within a lane will be part of a band type and no two bands within a lane can be of the same band type After selecting the New Band Type tool click on a band to draw a band type line Repeat this process of selecting the tool and clicking a band until a band for each unique band type has been selected Deleting a Band Type To delete a band type select the Delete Band Type button and click on a band type line Adding Bands to a Band Type Select the Plus Band tool by clicking the Plus Band button Click on a band type line to select it the line will be
193. o be used for image acquisition Image showing flat field paper target Example flat field target image 3 Acquire a grayscale image of the target with the desired illumination and filter settings Use the top speed resolution setting possible Normal Ultra and Zoom ROI setting 1 The image should be generally bright with pixel gray levels between 50 and 90 of saturation intensity values 30 000 58 000 A few saturated red pixels are allowed E2 Tm Bene mA Example AlphaView screensho d 4 Select Flat Field Utility from Setup Menu Sed Overlay Utilities View Window Help Is coomassie16 tiF Frink Info k Print Mode d Print Date Preferences System Calibration Darkmaster Utility Flat Field Utility Flat field menu path 5 Complete login screen security entry Default is provided below User name master Password master User Login Security Login Screen E3 6 Image selection screen review images created for flat field calibration Images shows should match images acquired for flat field calibration Select Image s All selected images will be used as sources for creating a set of flat Field master images Title Resolution Bits PP File Path 204842048 16 EPEE Flat field file imager selection 7 Select OK or follow instructions AlphaView The system already has a Flat Field master image Far given Filtersligh
194. o the Color Control all the variations are removed This permits direct comparison of image data from multiple imaging systems possible A simple example illustrates the need for a CCN Image a blot and determine the blue green ratio for a band Reimage the blot with different exposure times and notice that the blue green ratio is now different The signal levels from the Color Control change in direct proportion to the exposure times Using the Color Control signals to normalize the experimental band signal levels corrects for the different exposure times and resolves the discrepancy 147 Identify CC Band Select the region containing the Color Control Band and enter the relative amounts of material in each color channel Color control narmalizatian Identify CL band Clear normalization Amounts H li G 1 B li In the case of using equal molar concentrations of DIGE minimal dye labeled materials the relative amounts are 1 1 1 for CY2 CY3 and CY5 A standard curve has been defined in the red channel using a dilution sequence Using the signals from the CC band and the known relative amounts of material in the CC band the color channel data is normalized across color channels and the unknown quantities for the green and blue channels is determined Band analysis results All Channels Export View Display Style Red BC Average ES sii 4 04 54 31 81 7 020 67 55 5 1 345 Bs 000 00 7 348 In the figure above ba
195. objects are drawn their density data is automatically calculated and displayed in a data window Any time an object is drawn or detected the data in the window are updated If the Multiplex Band Analysis Results window obscures a region of interest resize and or move it following Windows conventions Multiplex Band Analysis Result All Channels Export View Display Style Band Blue Sum Blue Average Green Sum Green Average Red Sum Red Average Area 1 1 987418 9266 8031506 7 6600 X 3 5448235 5218 L 2 11 391 701 10 911 8 756 504 8 387 5 673 756 5 434 1 044 3 9 808 616 9 395 6 318 603 7 968 5 471 463 5 240 1 044 4 11 025 467 10 560 8 671 133 8 305 5 577 774 5 342 1 044 5 12 246 198 11 730 8 137 708 8 752 5 753 006 5 510 1 044 6 10 413 079 9 974 8 277 805 7 928 5 244 043 023 1 044 7 6 907 973 6 532 7 945 679 7 610 5 Jz8 824 5 104 1 044 8 12 237 077 11 721 9 240 227 6 650 5 928 935 5 679 1 044 Figure 5 19 Example of a Multiplex Band Analysis Data Window Data Definitions See appendix D for a complete listing and description of the various quantities The basic set of measured quantities are sum average percentage area position and standard deviation These values are calculated for all regions in all color channels including the background regions Inverting Data The INVERT function reverses the gray scale assignments so that 0 corresponds to white and 65 535 corresponds to black This control is found
196. oes not effect the loading control normalization Note that even without multiplying by the mean of the loading controls the relative values of the Red Green bands would still factor out Next perform band control normalization for both the Cy3 Green bands and Cy5 Red bands G 1 Use the Cy3 Green negative control to normalize the Cy3 experimental bands and use the Cy5 Red negative control to normalize the Cy5 bands Note that band normalization uses the loading control normalized values The band control normalized data provides the relative change in expression level of each Cy3 labeled experimental protein relative to the its untreated control and the relative change in expression level of each Cy5 labeled experimental protein relative to its untreated control The relative changes of the Cy3 protein to the Cy5 labeled protein is then determined by comparing the changes of each to its respective control For example consider that the Cy3 band in lane 3 is decreased by 2 fold relative to its untreated control and the Cy5 band in lane 3 is increased by 3 fold relative to its untreated control This yields a relative change of the Cy3 band in lane 3 the to the Cy 5 band in lane 3 as a decrease of 6 fold Note that comparing the Red signal levels to the Green signal levels directly is only valid for the conditions of that single blot Replicate blots or even a second image of the same blot may have different relative Red and Green signals l
197. of interest e g for band 1 Red LCN Avg 298 9998 12874 231 The Red LCN Avg values now represent the pX X pX ratio for each corresponding band In this example it is not necessary to designate separate Experimental Bands The defined Control bands function as experimental bands without explicitly being defined as such G6 Select Identify Control Band from the Band Control Normalization section and select the red channel of region 1 primary band of lane 1 which represents the untreated sample Y Alphalnnotech Potatoe File Edit Image Setup Overlay Utilities View Window Help BOWHWe GBONKS GonWwes e W X phospho tif Gamma Linear tos C Show Grid Blck 99 9 LN White 27 OLN Gamma J L 1 00 ime nmi Display composite Auto Enhance Level L 12151 145 5 515 Enhancement Tools Analysis Tools _ColonyCout 0 Bkgmd Control Std Curve Protocol Repo gt Loading Control Normalization Lee jede controis then Identify Loading Controls i r Identify Experimental Bands i Clear Loading N ormalizatio zorrespond de controls lt gt Band Control Normalization Identify Control Band Normalizes e t Band analysis results All Channels Export View Display Style n x a Identify Experimental Bands bands to a pasive conned Band BlueBCAverage
198. on Hybond LFP and Hybond ECL GE Healthcare are low auto fluorescence PVDF and nitrocellulose membranes suitable for visible fluorescence applications with auto fluorescence levels as low as NIR fluorescence Advantages of the FluorChem Q The FluorChem Q includes preinstalled Blue Green and Red LEDs Blue Green and Red excitation and emission filters 50mm F 0 95 lens and HD2 camera for acquisition of high quality single color and multicolor fluorescence images The key feature of the FluorChem Q is the high power LED illumination system that produces a consistent illumination field and uniform fluorescent images A second key feature of the FluorChem Q is the direct acquisition of multichannel images By defining an image acquisition protocol all the system settings for sequential acquisition of multiple images of the sample are saved and executed automatically There is no need to manually Switch between LEDs or fiter positions during acquisition Bias dark and flat field calibration and image registration are applied automatically during image acquisition for a rapid and streamlined image acquisition procedure be eithe ne specific combination of excitation and emission settings and is used for blots labeled with a single fluorophore Single channel images have a 16 bit depth A multichannel image contains multiple exposures each exposure at a different combination of excitation and emission settings Multichannel images are F
199. on can be turned on or off and will adjust differently for each image The Reverse Button The Reverse button inverts the gray levels of the displayed image converting a positive image to negative or vice versa For instance an image with black bands on a white background is converted into an image with white bands on a black background by simply clicking the Reverse button Clicking the button a second time returns the image to its original form 53 Wu HIC gt Original Image Reversed Image Figure 2 18 Original and reversed Image Note ioe For example the bands in the above gel have the same density regardless of whether the gel is displayed as white bands on a black background or black bands on a light background For information on reversing pixel values see Invert in Chapters 4 and 5 The Equal Button The Equal button flattens the image to show all pixel values in the original image Equal automatically adjusts the image display for maximum contrast which is beneficial fof REGARD detection Original Image Equal Image Figure 2 19 Original and Equal Image 54 Making Linear Log or Equal Adjustments Original image of film with default BWG seitings Image of film with linear Contrast Adjustments selected Linear provides minimum and maximum adjustment tools from 0 to 100 range of 0 65 535 grayscales Gamme Liner Lon I ol eee Black GJS HL jJ white
200. on the Remove items button in the toolbox To change the value of a standard band click on it once in the spreadsheet Insert the new value and select Enter on the keyboard to input the value The value will be updated in the graph with a new value calculated for any unknown band if appropriate 146 Displaying the Curve If the box displaying the standard curve obscures a part of the image it can be resized using Windows operating system functions It can be hidden from view by clicking the Graph checkbox in the Standard Curve toolbox Quantization Values of Unknown Bands As the values for the standard bands have been entered the quantization values of the unknown bands are automatically calculated The calculated values of the unknowns are automatically updated in the spreadsheet It is possible that the graph display may need to be refreshed after changes in standards values adding deleting regions etc To refresh the graph display simply uncheck and recheck the graph control checkbox on the Std Curve tab Note The graph is based on a several different graph equations shown in the Toolbar Band Hed BL Average Hedn ls 10 200 10000 23 4578 4000 25 5 871 CE 4 020 67 55 H 1 345 bs 7448 Sass Figure 5 32 Multiplex Band Analysis Data Box The third column in the data box contains values for the standards entered by the user highlighted in green and the values for the unknown bands calculated based
201. onds to black If the image has dark bands and light background then INVERT should be selected by placing an X in its box lf the image has light bands and dark background the INVERT option should not be activated Unlike the REVERSE button described in Chapter 3 this function does not alter the appearance of the image This function is especially important when using Snap to Peak If INVERT is incorrectly set the cursor will snap to areas between peaks rather than finding peaks Calculating Rf Values To obtain accurate Rf values specify the location of the wells i e origin and the dye front using the functions Set Well Pos Start and Set Dye Front End both found in the Marker menu To designate the location of the wells point the cursor at Set Well Pos Start Position the cursor so it is pointing anywhere along the wells and click the left mouse button A horizontal bar appears defining the location of the origin By clicking and dragging the bar can be moved for better positioning Repeat this procedure using Set Dye Front End to indicate the location of the dye front Once the origin and dye front have been defined they are used to calculate the Rf values of any bands that are added lf dye fronts are not assigned AlphaView uses the top and bottom of the image as the boundaries for calculating Rf values The Molecular Weight Cursor Box Molecular eight Cursor Position Mol weight Relative Freg 29 0 000 0 052 Figure
202. ool Bar window provides intuitive icons for the most common functions in AlphaView aHa e deosgeo a The Navigator icon starts the AlphaNavigator Wizard The Open icon functions identically to the File Open function in the upper menu bar This function is used to open previously saved images Detailed instructions are available in Chapter 3 Hie The Save and Save All icons function identically to the File Save and File Save All functions in the upper menu bar This function is used to save captured images to the desired storage medium f Once an image is displayed it can be printed on the default printer by clicking the Print icon in the Tool Bar window display Most printers can be configured through the Windows operating system to be the default printer Refer to your Windows operating manual for more information on installing a default printer Sample Printouts 61 The Zoom Out and Zoom In icons provide easy zooming ability while you are active in image enhancement or analysis functions providing increased versatility Detailed instructions are available in Chapter 4 as this function is also available in the Tool Box Enhancement Tools Note The Status Bar always displays the image zoom setting in real time Assists the user to zoom in on a selected area the average pixel is displayed The Fit in Screen adjusts
203. or other applications such as fluorescent gels Since fixed focal length lenses do not have zoom rings adjusting an acquired image s region of interest is possible using the AlphaView M software interface When the sample s region of interest is smaller than the acquisition window s and the system is not equipped with a zoom lens the zoom ROI tool provides an ideal workaround The Zoom ROI tool allows for software based zooming without having to switch lenses To use the Zoom ROI tool focus on the image then position the Zoom ROI slider until the region of interest occupies the preview window s entire image area Use the Expose Preview and Acquire Image buttons to configure and acquire the final image The Zoom ROI tool eliminates the need for cropping an image down to a desired size post acquisition Button for Digital Zoom Camera Setup and Pnewierw f p i Derpy Cancel Moses Chai Digiap Mire coe Show Saturashon TM o Frwm i Dems a Copy to ed Stack Frames mmc Lead Satu Auio Coniraci i a a e eae UTR f i pus COE Copp to End Stoker Liria sakan Save Setup Tuta Zoom ied Fapa 1 E E apogr Accomp OWE 0 0 0 5 en Chesa amgen T ab ist Speed Hesculon Nomis Mogeceduchon Hore Figure 2 5 Zoom ROI Tool 40 Saving Images Once a satisfactory image has been captured it should be saved as an original file for safe archiving Use the Save As function in the Fil
204. ot include enough information and can result in a noisy graph By contrast a wide scan width can incorporate background pixels that will reduce the pixel average and dilute the actual data Therefore to generate the most meaningful quantitative data set the width to include as much of the bands as possible without including regions of background The scan width should not exceed the band edges and cannot exceed the red lane limit Ans hai rd ei ni Bac M a LIEN IM ee n 7 LIA 101 T1 snm ii Scan Width of 3 Too Narrow data noisy and graphs jagged 152 1 1 Casu 95 n MICUNNS Dacis erc quce nc aa Lane Profile R Scan Width of Entire Lane Too Wide peak heights reduced due to inclusion of background pixels 153 1 1 111 Uer cm uus mg weh mer n a L r w 2 R l LJ Lr L su Lane 1 e i ie A Recommended Scan Width middle of bands scanned smiling edges excluded 154 Scanning the Image When the AUTOGRID button is clicked the densities of each lane are measured and information is displayed in several quadrants of the screen e The graphs for each lane scanned are displayed in the lower left e The active graph is displayed in the upper quadrant of the screen e Peak information for the active graph is displayed in a data table in the lower right e The Image is displayed behind the above windows and can be viewe
205. ottom of a print may obscure a small portion of the image To print the image with no information on it choose Off 19 Print Mode AlphaView software provides custom printing options Printing can be achieved in three different methods Overlay Ubilities View Window Help Print Info RA mM Print Mode Full Image Print Dake k Screen Dump Image Window Preferences System Calibration d Figure 3 18 Setup Print Image Info Dialog Box FullImage Prints the original image Does not print zoomed images or images overlaid with data screens Screen Dump Prints the ae area Well suited for images overlaid with data Image Window Prints the highlighted window Print Date Under the Setup menu there is a selection labeled Print Info This allows the user to change the format in which the date is printed The choices are MM DD YYYY and DD MM YYYY Preferences In order to change the preferences of the system you will need to find the administrator of the AlphaView software program to log in the login is enforced only on data altering tool tabs image acquire cabinet settings and auto enhancement If you do not have an administrator of the AlphaView software program see Appendix C in this manual User Lopin User Name Password Figure 3 19 Login Dialog box for Preferences 80 There are five tabs in the Preferences menu AlphaView Stand Alone software doe
206. ough Curves to properly mimic the curvature of your sample The more curves applied the more accurate calculations will be for molecular weight and band matching After the initial curve is applied the software will attempt to mimic the curvature of the surrounding curves when new curves are applied This results in less editing of the additional curves 182 Band Matching Similarity Matrix and Dendrograms AlphaView provides features for matching bands between lanes The resultant data can then be used to generate similarity matrices and or dendrograms Band matching features are accessible under the Analysis menu within Lane Profile s AutoLane feature Prerequisites for Band Matching Prior to performing band matching a number of steps must be followed 1 Lanes and Bands must be detected and edited through the AutoLane feature 2 f Molecular Weights are to be calculated molecular weight markers must be loaded prior to band matching Refer to the section within Lane Profile on loading molecular weight markers 3 Optional A new Gel Smiling tool has been added to AlphaView This tool allows the user to adjust the software s calculations for M W Adj Rf and Band Matching by drawing the shape of the gel smile or curvature Applying the Gel Smiling tool will aid in the accuracy of automatically matching bands and will result in less user edits afterwards Refer to the section on Gel Smiling tool for further instructions Matching B
207. ounts R G B 9 673 5515 1 027 172 86 10 903 2 263 1 547 70 80 2 C Plot Unknowns Help on these tools 11 1 030 1 468 1 556 46 00 12 1 022 1427 1 748 35 32 13s 737 706 9 195 4s 73 773 4 002 15s 702 707 1 343 1B s 709 653 926 T2 les 4 433 Pb n 931 8 14314 3 794 0 6 157 0 8 519 9 10 8828 _LanePiofe 00000000 0 q i LCN Average Ready RGB 3812 3840 3599 Zoom 113 X 2382 Y 2 24 fi C Program Files Alp 9 c cul Appendix X doc Mi Y AlphaInnotech Alp Microsoft Photo Edit In the figure above the known concentrations appear in the table of the Std Curve section as well as in the results data table A new column appears in the results data table displaying the calculated concentrations for the Experimental bands for the red channel The standard concentrations are highlighted in green and the band number is marked with an s for Standard To determine the concentrations of phosphorylated protein the green signal in bands 7 12 use the signals for the Color Control bands to make cross channel calibrations of the mass standards Select Identify CC Band in the Color Control Normalization section and select band 1 In the Amounts section of Color Control Normalization enter values for Red Green and Blue In this example an equi molar ratio of 1 1 1 is represented G 12 Bkgmd Control Std Curve Protocol Band Hed LCN Average 15 1 343 15
208. own the left mouse button on the Channel Viewer window to select and drag the window to any position on the image An average of each channels intensity Red Green and Blue is displayed for the region in the window Red 2 708 Green 1 892 Blue 910 Figure 2 20 Channel Viewer The Channel Viewer is useful for scanning a composite image A region of the composite image is displayed along with a separate display of each underlying channel 57 Contrast Adjustments Gamma Linear Log MECT Baka 2 a White 3 J 17514 Samma 4 J HL 0 714 _ Ka Figure 2 21 Contrast Adjustments window The Red Green and Blue buttons select the channel and the Display Composite toggles the display between the color composite image and one of the single color channels The three sliding scales adjust the black level while level and gamma Multi channel composite selection When a multichannel image is active the contrast adjustments window allows the displayed image as a color composite of the component channels Multichannel images acquired with the auto contrast option selected will have the Black White and Gamma levels optimized independently for each channel and each channel will have a different combination of settings Multichannel images acquired with the auto contrast option unselected will have the Black White and Gamma levels at the default values of 0 65535 and 0 75 for each chan
209. pen the data file on a separate workstation connected to the network Click on the Export button and then on the File option Specify the path and file name to send the data file The data is saved as an ASCII file and can be imported into most spreadsheet programs ASCII is a very common file format output option for numerical data Appending Data to a Same File Export dialog also allows you to append data to an existing exported file Note This feature is applicable to AutoCount and Array analysis modules Sending Data to a Spreadsheet Program To send the data results to Excel or other spreadsheet programs click on the Export button click on the clipboard option then click on OK If you have Excel or another spreadsheet program loaded on the system and running in the background you can simply press the ALT and TAB keys simultaneously to move into the spreadsheet program You can then import the data directly to the desired spreadsheet from clipboard Note The spreadsheet program must be installed on the computer in order to export the data to that program When the appropriate export source has been selected click on the OK button to send the data 205 APPENDIX A OPENING ALPHAVIEW TM FILES INOTHER SOFTWARE PROGRAMS Alpha Innotech generated files have been tested in the software packages below For successful imports the command line is given Programs for the Macintosh operating system were tested on a PowerMac 8100 100A
210. phaView will automatically search for the markers in the lane 119 In the above example the brightest 7 bands in the lane would be selected and the values for Boe IPs 100 bp DNA marker ladder would be assigned to the bands If No is selected AlphaView will display a cursor with a red line attached Text in the upper right hand corner of the image window will tell the user how many markers have been entered and the value of the next marker to be entered Markers to be loaded 7 T amar m ER Manual Load Dialog To begin entering markers move the cursor to the top band in the lane and align the horizontal line with the band Click the left mouse button Notice that the position for that band is added to the markers data table Move the cursor to the next band click the left mouse button again to assign the next value in the series Repeat this process until the values of all the marker bands are assigned To quit this function before all markers have been placed click the right mouse button Adding a Set of Molecular Weight Markers to the Library If the same set of molecular weight markers will be run repeatedly there is no need to enter values on each new image Instead save the molecular weight values in a file and apply them to the marker lane of any subsequent images To save a set of molecular weight standards click on Write Markers to File in the Marker menu after the marker values have been entered Following
211. r it may be necessary to adjust the White level in order to better visualize the results of this filtering process Custom Filter This function also allows the user to customize filters to his her own specifications Using the weighting factors of the other filters as a frame of reference experiment with new weighting factor values The UNDO Button The upper right button is labeled UNDO This reverses the last filtering process applied to an image To revert the image to its original state after multiple filters have been applied press the RESET button on the main interface 109 Examples of Filter Results Original Image 3 D Contour Level 5 Horizontal Edge Vertical Edge 110 Movie Mode If kinetic multiplex color or chemiluminescence experiments are desired where you wish to have the system automatically capture several images at preset exposure times preset time delay between images preset lighting sources and preset filter choices the MOVIE box can be found in camera setup acquisition screen To access this screen select the acquire button on the tool bar and then select Movie Mode on the acquisition screen AlphaView stand alone software does not include Movie acquisition tools Camera Setup and Pnewenw Lapis Cancel Movieblote Cherri Dhipa Shere Eshan Lead Sau Auto Conti Zoom ina fx aa als a SS a S M T Auto expose az comp oie o0 0
212. r Rolling Disc and Valley to Valley background methods Base Lock The Base Lock function will designate the active graph baseline for all of the lanes in the data table To de select this option click on the Base Lock a second time The check mark will disappear Horizontal Base Activating the Horizontal Base function will move the baseline to the O position no background The baseline can be moved vertically by clicking the baseline and dragging it upwards Reset Base Activating the Reset Base function will move the baseline to the O position no background Unlike Horizontal Base the baseline cannot be moved Base Subtract This selection will subtract the current baseline from the intensity value line Un check Base Subtract to deactivate this feature 161 Interpreting Lane Profile Data AlphaView provides a variety of tools to help interpret Lane Profile scan data These tools are found in the toolbox at the bottom of the screen and in the Data pull down menu in each graph window lv Line Auto Peak pest 3 D View Min Area 1 Si Min Height 10 z Min width 5 si Figure 5 39 Lane Profile Data Interpretation Tools V Line Typically the peaks on a graph correspond to the bands in a lane The V Line tool can help determine if the peaks on two different graphs represent bands at the same molecular weight position i e the same distance from the top of the gel To compare the positions of peaks on different graphs
213. r gel smiling or curvature Using this tool will increase the accuracy of Molecular Weight calculations and Automatic Band Matching After the Gel Smiling have been applied a new column of data will be generated Adjusted Rf The Rf values reported are relative values from O to 1 where O is the origin and 1 is the lane front without correction for gel smiling as commonly occurs in gel electrophoresis The Adjusted Hf Adj Hf is the corrected value Suggestions The software calculates Adjusted Rf based on the bands position relative to the nearest Gel Smiling Curve When the band lies between two Gel Smiling curves with different curvatures a weighting is applied where greater weight is given to the nearest Gel Smiling Curve For the most accurate calculations enough curves should be added to properly mimic the curvature of the sample We also suggest placing at least one Gel Smiling Curve above the top most band and below the bottom most band If these curves are left out the software will use the lane origin and front as the first and last curves These are assumed to be straight Applying Gel Smiling Curves After identifying and editing lanes the Gel Smiling tool can be accessed 179 Scoring Band Watchin Protacal Report e E SSS Tools Result Gel omnis Mol Ww Umm Std Add Curve Add Anchor Edit Curve Delete Anchor Delete Curve Delete All Curves Help on these tools Add Curve Click Add Curve
214. ra setup and preview window This window provides camera exposure control sensitivity resolution control lighting filter wheel position control contrast display options and cabinet door status The active imaging channels are laid out in a tabbed architecture One tab for each channel that contains the complete acquisition configuration for that channel Live This window opens in Live control mode blue button Live control mode provides near real time readout for easy sample positioning and optics adjustments While in focus select the lighting filter and binning options appropriate for your sample You can then in on your sample select an aperture setting and focus on your image Preview Clicking the green Preview box allows user to preview what your final image would look like while still allowing you to make small adjustments to the options you select in focus Acquire Select Acquire Image to capture you final image You will then be able to save print or analyze your image Movie Mode 32 Auto Expose FluorChem Q features an auto expose function for ease of use The Camera Setup amp Preview window includes a check box to activate the auto expose function as well as a button labeled Auto Expose Setup to configure the function Checkbox for selecting Auto Expose Exposure Auto expose E uU Lo s gt f Exposure Time minutes seconds milliseconds Auto Exposure Setup Button Auto Expos
215. rds a Pe don The computer is now ready to be turned on Ro 9 COM mu Turn the computer on by pushing the power m m n button on the front of the unit Lens Figure 1 2 Computer monitor mouse and keyboard setup 16 Cabinet Installation Instructions Alpha Innotech 17 Cabinet Setup When you remove the light cabinet from its shipping carton it is already partially assembled The camera mounting assembly is packed separately in the same container The UV transilluminator and cabinet top are both packed in separate boxes Make sure you have received all the hardware before discarding the shipping carton 1 The largest box will be the DE500 cabinet This box will include camera bracket camera bracket gasket RS 232 cable magnetic pad and pen Set the entire cabinet assembly on a level flat surface There are indentations to the bottom of the cabinet please use them for lifting and placement of the unit The cabinet weighs 80lbs with the UV transilluminator please take appropriate precautions in lifting and moving the light cabinet The footprint dimensions for the cabinet is 20 wide X 14 deep Open door and remove shipping foam from above and below fold down white light table Note camera bracket gasket RS 232 and magnetic pad pen will be located in and around the shipping foam Fold up white light table Note be sure to remove the inserts from the white sidelight bulbs Slide out UV tray Unpack
216. regions Figure 5 24 Loading Control Normalization In this example the Blue channel regions 17 to 24 were identified as the loading controls The regions 1 to 8 were identified as one set of experimental proteins and the regions 9 to 16 were identified as another set of experimental proteins Note that region 1 was created even though no Green signal level is present so that there are equal numbers of Green and Red bands for loading control normalization The lane membership of each region is color coded for ease of identification Region 18 is the loading control for regions 2 and 10 all color coded yellow Notice in the above example that all the regions in the same vertical lane are color coded with the same color 141 Multiplex Band Analysis Result Maximum Intensity Channel Export X View I a Display Style Channel Sum Blue 1 006 046 Green 2 749 126 Green 3 566 145 Green 2 435 645 Green 4 335 657 Green 3 633 284 Green 3 448 302 Green 3 195 339 Red 3 022 677 Red 2 156 907 Red 2 056 935 Red 3 284 515 Red 4 125 500 Red 2 880 273 Red 4 840 962 Red 3 142 296 Blue 2 454 023 Blue 2 6 3 098 Blue 2 706 068 Blue 2 560 063 Blue 3 240 541 Blue 3 018 247 Blue 2 859 167 Blue 2 895 561 Average BC Sum BC Average LCN 5um LON Average LC Regions 882 51 300 45 146 002 128 Green 1 Blue 17 2 411 958 740 841 1 029 549 903 Green 2 Blue 18 3 128 1 776 120 1 558 1 872 529 1 643 Green 3 Blue 19 2 136 645 240
217. released the start and end points of the object will be connected Manipulating Areas of Interest Areas of interest can be moved copied and deleted To perform any of these functions an area must first be selected by clicking on it or dragging the cursor around it lts color changes to gray indicating that it is active More than one area of interest can be selected at any given time To move an area of interest click on the selected area While holding down the left mouse button drag the area to the desired location To delete an area of interest click on the cut button in the toolbox designated by a pair of scissors The selected area of interest is removed and any other areas of interest are renumbered accordingly To copy a selected area of interest click on the copy button found next to the cut button A second copy of the area of interest appears overlapping the first one Click and drag the copy to the desired location 2 Set the Density Threshold s The objects counted by the automatic counting routine are based on the difference in the gray levels of the objects as compared to the background e g the colonies on the petri dish are either darker or lighter than the surrounding area The density thresholds define the gray levels that are recognized by the counting routine To set the density threshold click and drag the sliders As the sliders are moved the regions of the image that fall within the gray scale range are
218. rent fonts to indicate certain conditions e Arial font indicates the name of a button a menu or a function found in a menu e Courier font indicates an entry that is typed e Letters or words found between lt gt refer to keys on the keyboard e Bolded NOTE indicates key points and useful hints e Bolded CAUTION indicates actions that may either harm the system or affect the data quality e Bolded WARNING indicates actions that can potentially be harmful to the operator e cons or buttons to be pushed are placed next to their respective descriptions in the text Questions or Comments The Alpha Innotech Corporation staff is available to respond to any questions or comments about the software For questions new software feature ideas or general feedback use the following contact methods e Email info alphainnotech com e Fax 1 510 483 3227 e Telephone 800 823 0404 or 1 510 483 9620 Telephone support is available between 7 00 AM and 4 00 PM Pacific Standard Time 13 Starting AlphaView software To start AlphaView software from Windows double click the AlohaView Software icon on the windows desktop Alpha view Figure 1 1 AlphaView shortcut 14 FluorChem Q Imaging System Setup System Components FluorChem Q System includes the following High performance high resolution CCD camera Manually operated 50mm f 0 95 lens or other optional lens Computer with keyboard mouse and monitor premium sp
219. romide gels e Positions 3 6 for other fluorescently labeled gels optional filters Select the illumination source LED UV or white light using the touch panel or software controls Select the green Preview button Check Show Saturation Check Auto Expose Select the Normal Ultra resolution setting Once the image in expose preview does not contain any saturation red false color palette for white bands green for dark bands select Acquire The exposure bar will turn green when this is complete If the exposure bar is pink in color saturation is still present in the image For extremely bright images particularly in white light applications it may 49 j k sQr7oQa205 be necessary to reduce the aperture setting until the saturation is removed from the image Close the cabinet door ther Select the Super Speed resolution setting Wait for the exposure bar to indicate that the proper exposure time has been found and that there is no saturation in the image the exposure bar will turn green when this is complete If the exposure bar is pink in color saturation is still present in the image Select Medium High or Normal Ultra resolution and uncheck Auto Expose Click Acquire Image NOTE The exposure time will vary depending on which resolution setting is selected in step 4 h If the exposure time calculated by the auto expose setting is too long it is possible to use the Fast Low or High Medium setting inst
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221. s and emission filters preinstalled for multicolor fluorescence imaging The FluorChem Q now provides outstanding performance in each of the most commonly used imaging modes for gels and blots chemiluminescence colorimetric UV trans fluorescence and multicolor epi fluorescence The FluorChem Q imaging systems are designed to meet the requirements of the most demanding chemiluminescence assays and also provide the most versatile fluorescence imaging system available today FluorChem Q systems integrate the latest developments in camera and lens technology for imaging chemiluminescence and fluorescence samples with wide dynamic range and fast image acquisition speeds The system is controlled by easy to use and intuitive software designed by Alpha Innotech AlphaView software performs image analysis and archiving and can also prepare images for desktop publishing AlphaView software includes image optimization tools that adjust contrast automatically or manually Converting the image from positive to negative using digital filters applying a false color map or other such techniques can be used to clarify hard to see portions of the image Notes labels arrows lines and other drawing tools can be recorded directly onto the image using the AlphaView M software s annotation functions Annotations are superimposed on the image upon hard copy printing and can either be saved as a template file or as part of the image All AlphaView featur
222. s not contain the Image Acquire or Cabinet Settings tabs 1 General Configure prompts and file saving opening formats Preferences General Image Acquire Cabinet Settings Auto Enhancement Analysis Tools i File Types D efault Save Tiff Files tif tif v Disable No ROI prompt Enable Part11 selection at startup Figure 3 20 Preferences General Tab 2 Image Acquire Inverts the image seen by the camera and adjusts the ROI values Preferences General Image Acquire Cabinet Settings Auto Enhancement Analysis Tools Lens Selection Post Processing Bias Dark correction Flat field correction Enable flash clearing nine 3 21 Preferences chang xote Tab 81 3 Cabinet Settings Used for adjusting the port settings and customizing filter positions on the cabinet Preferences General Image Acquire Cabinet Settings Auto Enhancement Analysis Tools Devices Cabinet Lights Filters Selection Maik Pal Selection LED 2 Pos 2 LED 3 Pos 3 Light 1 Pos 4 Light 2 Pos 5 Light 3 M ame Figure 3 22 Preferences Cabinet Settings Tab 4 Auto Enhancements Used to customize the Auto Enhance Levels located in the Image Enhancement tool box Preferences General Image Acquire Cabinet Settings Suto Enhancement Analysis Tools Black Level 0 00 White Level 7 00 Selection button Gamma
223. s to customize the report which can be saved on file as an Html Format html or Rich Text Format rtf User can also take print of report Reports also allows user to save report settings configuration as template or can modify existing templates Formatting Formatting tab allows user to format reports before saving or sending it to printer User can create new report or select existing report template to create a report using the drop down list If no report template is defined saved it will be disabled Choose image that will be part of report with the image size 1 4 is the default image size Also choose different information Data sheet Graph Acquisition information that will be part of report Report settings configuration can be saved using Save button in case of new or Save as in case of modifying an existing report template ROI Threshold Count Protocol Report Formatting General Select from existing templates Save Saves Image C5 Original Image Data Sheet Enhanced Image Graph with annotations F Acquisition O Analysis Overlay Image Infamatian Full 2 1 4 Help on these tools Figure 5 54 Report Formatting Tab 191 General General tab allows user to give report a title enter analysis notes comments or give user name Clicking Generate Report will create a report based on report settings configuration Threshold Count Protacol Formatting Genera
224. sideration which results in an image with no bias offset or black level errors and elimination of dark current signals As with any calibration method there are still noise artifacts that remain in the image after correction At the time of acquisition an image contains the actual bias level plus bias level noise also called read noise in addition there is the dark current signal and its noise signals The bias and darkmaster calibrations remove the bias and dark current signals but the noise from those signal remains other techniques must be employed to address the residual noise like frame averaging The best darkmaster images will have a similar exposure time to that which is typically used The utility allows building darkmaster files of any exposure time in 1 minute increments so a darkmaster with a time that matches the typical exposure time can be constructed It is best to have the darkmaster exposure time be greater than the time of interest For example if 4 5 minutes is a typical time then a 5 minute darkmaster would be best Shorter or much longer times will still work but optimal dark current correction occurs when using darkmaster exposure times greater than the test image exposure time but no too much longer round up to the next highest minute D 1 A procedure to use the utility to construct bias and darkmaster files is listed below 1 Allow camera to reach temperature and stabilize at least 30 minutes after power on is re
225. simple tools like a piece of 8 5 x 11 regular low quality copy paper higher quality paper contains watermarks that will show up in the final image It is essential that both the flat and the gel images be identical including the aperture zoom if applicable and focus settings on the lens Step by Step Manual Flat Field Calibration Applied to existing image a Place the gel or other application in the cabinet or dark room b Adjust the aperture zoom if applicable and focus on the lens c Use auto expose set to the normal selection and acquire an image of the gel Alternatively it is possible to select show saturation and then use expose preview and adjust the exposure time manually to just under saturation d Save the image of the gel e Next remove the gel from the UV transilluminator or white light tray and clean and or dry off the surface if necessary using glass cleaner f Place the white piece of paper onto the appropriate surface For example if the UV transilluminator was used place the paper onto the UV transilluminator if the white light tray was used place the piece of paper onto the white light tray g Turn on the appropriate light source used white light UV transilluminator epi lights etc Without changing anything on the lens acquire another image of the Flat image following step 3 again Save the Flat image Open the original gel or other application image Select Flat Field Calibrate from t
226. ss 85 Figure 3 28 Utilities pull down menu seeeeesssesesseeeeeeennnnnennn nnne nnn nnn nnn nnns nnn nnn nnn 87 Figure 3 29Notepad Display WIPIGOWas s oca mia nene pottiin od Un tco E E pite citt A e exte e na ous 87 Figure 3 30 Windows Explorer Dialog BOX a2 niianticku sr SebU uA I Sed asDu Musa Ded Qi aafeS uou oom p aqu ata aio LU HERE aA 88 Igure 3 91 VIew pull doWEIETTGNIl sss semana Mud ti ran baa E chat a De dua fud con Lco mae viaa duds aateaceaaues 89 FIGUIC 3 32 WIBGOW DLILOOWEPTITOUU as casis tasca n ii iutoT decade fus Dabei n etat a Ded a X can doin is csi Sotomuer Ae Dea A 90 Foure 3 33 Help DUIPGOW IM MONU S ssecbninat aia as aid rna ou buRa Bebo Za atu dud Ex eq wane dua ad aa o coss dtc due baie nae Rua 91 Figure 3 34 AlphaView About Help Dialog BOx lessen nnnm nnns 91 Figure 1586 709r TOOlS xtoiscosadear otii a E IN ET shoes tive baddag ctn g IURE nM UP Up 92 Figure 4 2 Histogram display in the Tool BOX ccccsccccccseeeeceeeeecceeeeeeceeeeeeneuseeesaueeessaueeeesaaeeeessaseeessaeeees 93 Figured o THE Rotate FID TOO edit iii eee ni Etat tsetd Ure EU NU in E 94 Eigure 4 4 Annotations T690lDOX oua pu omo Un E atomes hee Ia sedie cin seeds mee e Dunt 96 Figure 4 5 Pen Width Selection Tools aicea a E T 97 Figure 4 6 Pen Style elecllon TOO S ido eee sod eia ad sr Coe entoce su E Soa open Uus eude 97 Figure 4 7 Line Ends Selection T60lS o edo tn autism der biu vedette eue taU Pel E M
227. sse edv Usa tas i des adus 133 Figure 5 18 Non Selected and Selected Objects cccccccccssssseeeceeeeeaeseseeeeeeeeeseeeeeeeeeeeeeesueageeeeeeeeeesaas 135 Figure 5 19 Example of a Multiplex Band Analysis Data Window ccccssscecceeeeeeeeeeeeeeseeesaaeeesaaaeees 136 Figure 5 20 Unlink Background TOO c ccccccsseecceceeeeeeseeeeeeseeseeeeseueeeesaaeeeessaeeessaeeesseaeeessneeeseseaeeesseess 138 Figure 5 21 Multichannel image with regional DACKQrOUNGC ccccccsseeeeeeeeseeeeaeeeeeeeeeeeessaueeesaaeeessaaeees 139 Figure 5 22 Band Corect Valles cie tede o ad e head uiu doc ooo eene irai Ue dare t eed ieade 139 Figure 5 23 Control Normalization Tab irrito a 005 Dresd brit ue aUi dabo seen da beta ege 140 Figure 5 24 Loading Control Normalization ssseeeeeeeeessseeseee nnne nnne nnns 141 Figure 5 25 Loading Control Normalization Data Table cccccccccccccssseeeeeeeeeeeeceeseeeeeeeeeseseeeessaaneeeeees 142 Figure 5 26 Band control normalization ssns a a a 143 Figure 5 27 Band control normalization data table seessssesseeseeeeeeeeneeeennenne 143 Figure 5 28 The Standard Curve Toolbox ssesesesssesseeesssseeeseseennnen nennen nnne nennen nnn nnne 145 Figure 5 29 The Standard Curve input for known concentration sseseseseeeeeeeeeneennne 145 Figure 5 30 The Standard Curve Spreadsheet ccccccccccccecc
228. t Markers Query Graph Protocol Report Well Position Set Well Pos Set Dye End Detection S5napToPeak J Invert Markers Add Marker Delete Marker Clear Markers Molecular Weight Cursor Position Mal weight Relative Freq Show B and Number Mol Weight Help on these tools Figure 5 2 Molecular Weight Tools Molecular Weight Results Export MARKERS Position Mol Weight 238 14 00 256 52 00 zol 43 00 314 24 00 QUERIES BS ESTERI 240 18 22 260 50 50 203 41 32 3l H A Figure 5 3 Molecular Weight Data Box At the top of the data box is a bar with two menus Marker and Query The area below the menu bar is divided into two sections Molecular weight marker data is displayed in the upper section and the calculated or query data is displayed in the lower section 115 Entering Known Molecular Weights for Markers An unlimited number of molecular weight standards can be defined either all in one lane or in d lanes To m markers i file the Marker menu and select Add LUGI m Notice that the cursor flashes and has a short horizontal line associated with it Move the cursor to the lane containing the molecular weight standards Beginning at the top of the lane position the cursor so the horizontal line is aligned with the band If the SNAP TO PEAK function has been activated the cursor jumps to the highest pixel value in the band as the cursor appro
229. t 1993 20049 All rights reserved With Alphalnnobtech com Alpha iInnotech Figure 3 34 AlphaView About Help Dialog Box To close the box click on the OK button 91 Chapter 4 THE IMAGE ENHANCEMENT TOOLS Image enhancement tools are contained within the Tool Box as indicated This tool set allows the user to zoom the image rotate flip the image show the image histogram perform automatic image enhancement annotate on the image display false colors apply software filters and activate the Movie function Many image enhancement tools do not function with multichannel images Enhancement Tools Analysis Tools The Zoom Tool Factor _ O Ss i i mee lo ee M Tg Figure 4 1 The Zoom Tools The Zoom tool is found in Tool Box Enhancement Tools This function magnifies an image making details easier to see and allows movement around the magnified image 92 An image can be displayed x 2s 1x 2x 4x or 8x larger than the original display by clicking on the appropriate buttons To return to the original magnification click on the 1X button When an image is magnified only part of it can be displayed on the screen at any one time To see different parts of the magnified image use the green Pan Control box The outer box shows a thumbnail of the entire image while the inner green box represents the portion currently displayed on the screen To view different regions of a magnified image
230. t combination Press Ok to overwrite all existing master images or Cancel to stop Flat Field creation operation Cancel Info screen 8 Select OK Flat field calibration is now complete AlphaView 4 new set of Flat Field master images was created Info screen E4 APPENDIX F DATA INTERPRETATION Overview In a typical Western blot experiment the expression levels of one or more experimental proteins are analyzed to determine the effects of various treatments or conditions The appropriate controls must be included on westerns blots as a number of factors can influence the proper interpretation of the signal levels extracted from multicolor images especially when comparing signal levels from different color channels A loading control is a protein that is unaffected by the treatments and whose signal level can be used to normalize the signal levels of the experimental proteins for differences in the amounts of sample loaded in each lane An untreated sample should also be included to provide a negative untreated control for each experimental protein Alternatively a positive or reference control sample may be included to provide a control Consider the case in which the loading control protein is labeled with Cy2 Blue channel and two experimental proteins are labeled with Cy3 Green channel and Cy5 Red channel respectively An untreated sample is included with a number of treated samples The objective is to determine the e
231. te resizes it If the lanes of the gel are not perfectly straight click the SKEWING checkbox The template will change to show four handles one at each corner of the template Position each corner of the template until it is positioned appropriately Adjust the template until the border of the template frames the lanes to be scanned and the red lines lie between each of the lanes gt tid te eg qua B ul ta ms LI rmm L sao Doe se Guanio eee w Baag e a so LIE a muss Figure 5 35 Skewed Lane Profile Template Properly Aligned on a Gel The Invert check box should be selected if the image being analyzed is a dark sample on a white background These are usually colorimetric gels such as Coomassie Blue Reversing the image on the Contrast Adjustment window would NOT require the user to select invert 151 Specifying the Scan Width The Scan Width the green line within each lane is the area from which the pixel density is measured The control labeled WIDTH sets the Scan Width The number in the center of this control indicates the current Scan Width setting It can be adjusted by clicking on the right increase or left decrease arrows As the setting is changed the template displayed on the image is updated to reflect the changes The density values for Lane Profile graphs are determined by the average pixel density between the green lines on the template A scan width that is too narrow may n
232. tent ase EAEE E OE EON E N E 53 TRCAO CONAS JALEN e s E DNE Rum A a UU REID OE LIUC 53 TTC TCV TSC BUO tet tu E E A E E CELL MIC OLIM LU CPU OLTRE 53 Ihe Bauart DUU OW cick ETE peas nna aR 54 Making Linear Lo or Equal AdIMWSIEHTS e ed Une foeMa S Sane ettet iduI t n eau NM CIUS 55 Muh OVOR SPA SOILS ION essit Bi ah D cua s eek OMe eke bua eee as tieu bot pale boot huno fa 57 AUTOMATIC ENHANCEMENT oirinn tonshensuat sn smadapanensunis paws dapak past PR Iva pbeses T opBhtuan B ova spbescse Pa ve fusi ss Cd bvifemet 60 TOOL BAR nin a velt uode ipa Va tattidenfa osa feu edem Oo b EE inel ST fatu bed uel E aceite T 61 BED ELIO EET EPIS 64 SLAS DAR LS LM ue EM ELM MM LUE ME EU M M IU E 64 CHAPTER 3 DROP DOWN MENUS Aitina anea Eie Co ode cua g epa sed ee eop ao tao stad eee oio cue ves vod veo Eo Unas e taa 65 DETR Fe TIVE NU rn vcs ease sae ae net loi ots ences Gass Ga ee Rass tence eae Daou ou Se eae ba eas 65 P0 Mete EE 66 SVC OG ANALYSIS r 67 TUTO NG LCS AARON AAA tard oti tte na Desig DE Saeed ellie D NE T NE UL Meee eae tee en eae seta ears Nm DUE seat 67 File Save Save As Save Modified and Save AL ae od vt e o dit etee toe emet e toe in ee bux eese tle Ex eU pet ed deus 67 E141 PM TR 70 1312111881217 Mr a 70 TRO E OU POLIO Gee act testo sd vase E ditor su cupo SEES peri d cse AREAS tb rendu a A oed 71 EPEE DEME N O in ec nna MD es nc M UE M M M M UU ML MOM E M 71
233. the cursor to any of the square handles on the outside of the template Hold down the left mouse button and drag the handles until the template is properly positioned If the image is not perfectly rectangular the template can be skewed by clicking the Skewing checkbox 202 OOOOOO0OOOO0OQ O O O O A Skewed Template Specifying the Areas to be Measured For microtiter plate analysis the toolbox controls labeled Center and Outer specify the size of the inner circle and outer torus scanned to measure each well The number in the center of each of these controls indicates the diameter of the circles Click on an arrow to increase or decrease the diameters As the number is adjusted the objects on the template grow larger or smaller reflecting the changes When specifying the size of the area to be measured exclude shadows or other imaging artifacts Depending upon the lighting conditions when the image was captured there may be a crescent shaped shadow obscuring part of the bottom of the wells especially those near the edge of the microtiter plate We recommend decreasing the size of the sampling area so that these regions are not included To measure the total density within each object reduce the Center value to zero 0 and adjust the Outer value until the objects on the screen are slightly smaller than the areas of interest on the image Reposition the corner objects using the mouse until the objects are properly aligned
234. ther a box outline to be drawn around the spots or a border outline The box will include more of a background in the drawn area than the border outline User preference and image particulars will determine which outline type is best The sensitivity can also be adjusted by sliding the bars for normal spots lt 98 saturation and for saturated spots gt 98 saturation Auto Spot Options Auto Spot Sensitivity 0 100 Border Type Normal Spots Intensity lt 887 Border Outline Sensitive 4 gu H C2 Box Outline Less Sensitive More Sensitive Saturated Spots Intensity gt 38 Sensitive 4 4 5H Less Sensitive More Sensitive Figure 5 17 Auto Spot Options 133 Bright Spot vs Dark Spot The Bright Spots selection should be checked if the image contains bright spots on a dark background e g ethidium bromide stained fluorescent gels and chemiluminescent blots The Dark Spots selection should be made when the sample contains dark sample on a light background film commassie blue protein gels etc The software should automatically determine this selection but the user may have to override it if the software incorrectly evaluates an image Remember as in all portions of AlohaView software if the image has been reversed negative image using the Reverse selection on the Contrast Adjustment window the Dark Spot selection should not be made Reverse is a visual alteration only and does not ef
235. tment Tool Imaging Display Tools Black Level White Level Gamma Setting with B W G Linear Log and Equalize options 91 To adjust any of these settings place the cursor on the slider Click and hold down the left mouse button while dragging the slider to a new setting As the slider is moved along the scale the image display is updated along with the change in numeric value The arrows above and below the scale bars can also be clicked to change the settings in single unit increments or the user may type in a specific unit Black Level Adjustment The number beneath the Black Level scale corresponds to a gray level There can be 256 4095 or 65 536 possible gray levels depending on the system type For the example below an 8 bit image will be used with 256 total gray scale values When the Black slider is at the very top of the scale the number is 0 As the slider is moved downwards along the scale the number increases and the image becomes progressively darker This is because all pixels at the specified gray level and lower are shown on the screen as black pixels If the slider is set to O all the pixels whose gray levels are at 0 are shown as black If the setting is then changed to 60 all the pixels between 0 and 60 are shown as black and the image appears darker Black Level set at 0 Black Level set at 60 Figure 2 15 Black Level Adjustment example White Level Adjustment The number beneath the White Level scale also corr
236. to all object regions Place a background region in an area of the image representative of the background level for all channels The image contrast display should be adjusted to so that the variations in the background can be seen so that the background region s may be placed in an area best representative of the background Note that more than one background region can be used and the average pixel level of all the background regions is applied The data table is automatically updated with Background Corrected BC values Multi Regional Background Background region applied to a subset of object regions A second or third background region can then be applied to a second or third subset of object regions Each subset of object regions is corrected by the linked background region to account for differences in background level across an image Local Background Background region applied using the average of the 10 lowest pixels in a band region The regions should be slightly larger than the bands to use properly apply this method of background correction The data table is automatically updated with the Background corrected intensity values 137 None No Background If no background calculation method has been selected the background value in the data window BACK is reported as zero 0 Therefore the object s integrated density value will be equal to the sum of all the pixel values within the box ellipse or freehand drawing Recomme
237. tocol contains all band and background regions created with the loading controls band controls and standard c settings used amp protocol can be saved at any point in the analysis workflow lt lii gt Band analysis results All Channels m a xj Load Protocol Adjust regions after loading a protocol by E View Display Style selecting moving and resizing regions Band BlueSum BlueAverage Blue BC Average Blue Bkad Average Green Sum GreenAverage Green BC Average Green Bkad Average RedSum RedAverag 1 118 094 017 14 415 12 874 1 542 45 540 806 5 553 3 617 1 943 4 463 562 545 2 108 347 064 13 226 11 623 1 598 41 364 218 5 049 3 146 1 303 9 744 097 1 189 3 88 801 405 10 840 9 229 1 612 35 376 321 4 440 2 553 1 882 14 543 474 1 775 4 83 585 521 10 336 9 333 1 603 35 717 885 4 482 2 531 1 952 18 347 077 2313 5 85 923 183 10 489 8 318 1 571 36 074 316 4 404 2 456 1 343 18 321 833 2237 6 82 182 354 10 032 8 500 1 532 35 259 025 4 426 2 427 1 999 15 673 527 1 913 C Help on these tools 7 84 440 555 10 308 8 786 1 522 36 574 588 4 465 2 466 2 000 17 645 765 2 154 8 95 538 570 11 662 10 114 1 549 42 956 527 5 244 3 013 2 225 17 531 983 2 147 3 99 154 899 12 104 10 602 1 502 47 595 698 5 810 3 361 2 450 18 224 833 2 225 RGB 346 2459 1685 Zoom 79 X 849 Y 263 cul Appendix x doc Mi W AlphaInnotech Pot Select the Control T
238. trol e g Red1 Hed 6 Fold Change Fold Change of experimental band relative to control Note If LCN Average experimental band gt LCN Average control band then fold change LCN average experimental LCN average control If LCN Average of the experimental band lt LCN Average of control then fold change 1 1 LCN average experimental LCN average Std Curve Tab p Values of Band region in Std curve in units as defined C 1 Multi Channel Columns RegionTab Z J J 0000000000000 BleeSum Sumoofall Blue channel pixel gray levels in Band Percentage Blue Sum Blue Sum Total of all Blue Sums 100 Blue SD Pixel Standard Deviation of Blue Channel pixel gray levels in Band Levels GrenSum Sum of all Green channel pixel gray levels in Band Green Percentage Percentage Green Sum Green Sum Total of all Green Sums 100 Average Green channel pixel level Green Sum Area Green SD Pixel Standard Deviation of Green Channel pixel gray levels in Band Levels RedSum SumofallRed channel pixel gray levels in Band Standard Deviation of Red Channel pixel gray levels in Band Area Area of Band Region in pixels BackgroundTab a Background Corrected Sum Blue BC Average Area Blue BC Average Background Corrected Average Blue Average Blue Bkgd Average Blue Bkgd Average Average pixel level of Blue Background region Blue Bkgd Sum Bkgd area
239. tum niue Cou innu e aos adeb DUE Du aude tU Tu E ene Od up LU rEE 22 Figure 1 12 5ystetm InferrallOblssricos ieiunus ebd oe a d uso C miu DjU MERE UNDE De ap UNE Lo o iM iei eds 24 Figure 2 1 AlphaView screen showing the image area and display controls sssssse 25 Figure 2 2 Camera setup and preview windOwW eeessssseeseseseee nennen nennen nnne nnnm nnns nnns nen nnn 32 Foure zS Aulo EXDOSQsicisstiskev ee utomeit a velim end cq Leica cin iuc a tu ev bL eso Su uei de nD UR dui iud 33 Figure 2 4 Turbo Modes and Speed Resolution Settings sese 37 Figure 2 5 Zooin ai Mors ee CX 40 Figure 2 5 Movie Mode SelllDJis mi ieterces sete eoa se trete eoa Vetere ase eate eene e oV SL ue see a atte e er babe as 41 Figure 2 7 Movie Mode Load Save SetuP neriie arie NEET E E 43 Figure2 8 Save Load Acquisition Protocol nenasiel 45 Figure 2 9 Save L oad ACQUISITION PFEOLOGCO L itus iod aeneo Iob e fena bestemekold asa ees ndag ces cdd uada 45 Figure 2 10 Autodmage Gapture Al G iranran aa betta eie sasodd asbestos cdtgs etapas se pedido idus 46 Figure Z TFAO Status WINGOW P c 46 Figure 2 12 How to enter Compare ViOW ccccccccsssssseececceaeeseseeceeeeeeaaeeeeeeeeeesseeaesseeceeeeesaeseeeeesessessaaaaeses 48 Figure 2 13 Goripare VIBW tundgueiese sasir a a a ea a iae aa aaaea laa 48 Figure 2 14 Contrast Adjustment TOO iir aniano idn
240. ty matrix is a graphical display of the similarity between lanes Exporting and Printing the Similarity Matrix Print and Export are selectable under the File menu Select Print to send the report to a local or network printer Select Export to export the results to the clipboard or to file When exporting to a file there are two file types that can be saved standard text file txt or a comma separated value file csv When exporting to Excel use the csv file type to skip the Excel import wizard or use export to clipboard followed by Paste in Excel Dendrograms The dendrogram window is accessed by clicking the Generate Dendrograms button in the Band Matching dialog Dendrogram Sample FiF File View Options Cua Display Type Distance Matrix Dice Coefficient z Cluster Method Neighbor Joining outgroup Default Root Clusters Distance Matris Method Dice Coefficient Cluster Method Neighbor Joining File Name D Images sS ample tif Metric AdjustedRelatweFrequency Reference Lane 1 Tolerance 1 00 X 188 Distance Matrix Select a method from the Distance Matrix pull down for calculating similarity The choices are Dice Coefficient Jaccard Coefficient Pearson Coefficient and Frequency Similarity These are standard statistical algorithms references can be found in most statistical text books Cluster Method Select a method for calculating the clustering from the Cluster Method
241. uction None The next screen is next filer combination Cy3 Camera Setup and Preview Change interface type cvs CY3 cv Exposure Auto expose You are now previewing your image 1 To have the software automatically calculate your exposure time wait for the status bar to tum green or manually enter exposure time by unchecking Auto Expose and entering a time in the box above 2 Focus on your image If zoom is available zoom in on your image 3 If required repeat step 1 until the image is ready to be acquired 4 To acquire image press Next 28 The user has the same options as with the first filter advanced mode or next filter combination same as done with the previous filter The next screen is the next filter combination Camera Setup and Preview Change interface type osaga CY2 pus Exposure Auto expose e You are now previewing your image 1 To have the software automatically calculate your exposure time wait for the status bar to turn green or manually enter exposure time by unchecking Auto Expose and entering a time in the box above 2 Focus on your image If zoom is available zoom in on your image 3 If required repeat step 1 until the image is ready to be acquired 4 To acquire image press Next The user has the option to use advanced mode back or next to acquire after the acquisition is complete the save protocol UI is generated AlphaMavigator
242. uration checkbox found below the camera control functions allows the user to access the Saturation Palette during image acquisition The Saturation Palette is a modified gray scale palette in which green replaces black gray level 0 and red replaces white gray level 255 4 095 or 65 535 With this palette the image s while areas within the linear range of the CCD chip appear in gray scale During image acquisition it is critical to note the image s regions that appear red or Eliminating red and green in the actual sample area is especially critical for images that will be quantified The saturation view can be turned off by unchecking Show Saturation once the red and green areas have been minimized and or eliminated The other two selections Chemi Display and Auto Contrast act as visualization tools designed to enhance contrast and provide flexibility i iewi Chemi Display utilizes both auto contrast and reverse functions as well as providing gamma adjustments and is intended for use with chemiluminescent samples These options are also available in the Movie Mode NOTE These Contrast Display options are only visualization tools and do not change any image data acquired by the CCD camera 39 Zoom ROI Zoom ROI is a tool designed for use with fast or fixed focal length lenses These lenses t have lower F stop numbers than zoom lenses but do not have zoom capabilities icall However they can also be used f
243. using the dilution series in the red channel as a standard curve The Color Control is constructed using equi molar concentrations of a reference protein that has been separately labeled with the GE DIGE minimal dye labeling kit using CY2 CY3 and CY5 dyes Therefore the Color Control has a known quantity of material in all three color channels in this case the relative ratios are 1 1 1 This Color Control permits direct comparison of the red green and blue channel data when the corresponding signals are normalized to the Color Control signals this process is termed Color Control Normalization or CCN Following the steps in the above Example 1 launch AlphaView and open the image Quant tif Then open the Multiplex Band Analysis module and load the protocol quant sda G8 Y Alphalnnotech AlphaViewQ File Edit Image Setup Overlay Utilities View Window Help Og oOo eU eo i EJ Main 2 Quant tif 4bx Gamma Linear Log _ C Show Grid Black OO BLA Whte 7 9 9L s Gamma 9 9 9 1 00 mmi Display composite Auto Enhance Level LeeLee Enhancement Tools Analysis Toos _MoleculaWedt 00000000000 _MultiplexBandAnaysis Bkgmd Control l Std Curve Protocol Repor gt Save Protocol tocol contains all band and reat Load Protocol Band analysis results All Channels n x X A T E
244. vean Subuxe hdd cunda Dixon 121 Figure 5 5 Molecular VelghteTOOlS usssosiciee sene uaria aa n und auo vod od bero dM IS oni o xo fac So Reod oai 122 Figure 5 6 Example of Point to Point and Least Squares fit graph ccccecccsseeeeeeeeeeeeeeeeseeeeeeeeeeaeeeees 122 FIgUre 5 7 COlOMY GOURD TOO Sssdscd d ihtcenita aom osea uae tino db od roD ood ab ores anco cu dde aves eoe eee oa resa aer 123 Figure 5 8 Sample with Two Types of Objects sseeeeeesssssseeeeeennnneenennnnn nnne nnne 123 Figure 5 9 Colony COUNT TOOS siad quascso diac cet i nxadk ae ocu ea pesa Rude SE Rc 124 Figure 5 10 Colony Count Sample Results for an AOI eeessssesesessessseeseennn nennen 126 Figure 5 11 Results of Colony Count After Manual Addition of Three Spots ssseeesuuus 127 Figure 5 12 Colony Count Data Window Showing AOI Summary Data eseeeseeeeeeee 128 Figure 5 13 Selecting the Colony Count Data Window to Show Individual Spot Details 128 Figure 5 14 Magic Wand and AutoSpot Tools cccccccccccceeeceeeseeeceeceeeeeeeeeeeeeeeeesaeeeeeeeeseessuaaeeeeeeeeeeeaas 131 Figure 5 15 Magic Wand Parameter Window sssesesssssssesesese enne enne nnne nnne nnns nnns 131 Figures T6 AUIO ODOLD aid coc sette sets cit iuso ases e ud esa delka dinde see A a ec cea ree 133 Figure S T Auto SDOLODHIODNS s acutius orbe imu eu isset er neis G du E
245. ved including interference that may cause undesired operation This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations Cet appareil num rique de la classe A respecte toutes les exigences du Reglement sur le mat riel brouilleur du Canada Compliance Safety Electromagnetic Compliance Emissions FCC CFR 47 Part 15 Class A IECS 003 Issue 3 Class A VCCI V 3 2006 04 EN 61326 1997 A1 1998 A2 2001 A3 2003 Safety UL 61010 1 2004 2nd Edition CAN CSA C22 2 No 61010 1 2004 2nd Edition EN 61010 1 2001 2nd Edition Power Requirements AC Input voltage rating AC 100 120 V 50 60 Hz 3A AC 200 240V 50 60 Hz 3A Environmental Requirements Example Operating temperature 0 to 40 C Non operating temperature 20 to 65 C Operating altitude Sea level to 10 000 feet Operating relative humidity 1096 to 9096 non condensing Non operating relative humidity 596 to 9596 non condensing H1 A English CAUTION The power supply cord is used as the main disconnect device ensure that the socketoutlet is located installed near the equipment and is easily accessible German ACHTUNG Zur sicheren Trennung des Ger tes vom Netz ist der Netzstecker zu ziehen Vergewissern Sie sich da die Steckdose leicht zug nglich ist French ATTENTION Le cordon d alimentation est utilis comme interrupteur g n ral La prise decourant
246. when it is Superimposed To select a color option click on the color bar until the desired color is shown e The last column in the dialog box specifies the pattern of the line when the graph is Superimposed To select a pattern click on the current pattern until the desired pattern is shown It is especially important to choose different patterns if the graphs will be printed as different colors cannot be distinguished using a gray scale printer 164 When the selections have been made click on the DONE button to dismiss the dialog box and superimpose the selected graph s If no graphs have been selected the dialog box is simply dismissed Three Graphs Overlaid To turn all graph superimposing off click on the ALLOFF button at the bottom of the dialog box Molecular Weight Mass and Band Scoring integrated into Lane Profile A new menu item has been added to Lane Profile Under this menu the user now has the option of calculating Molecular Weight and or Mass The user can also access the Band Scoring feature from this menu This unifies four important analysis tools into one area of the software and reports Mass Gel Smiling and MW on one report scoring Band Matching Protocol Report Tools Result Gel smiling Mal wit Mass Std l Figure 5 41 Molecular Weight Mass and Band Scoring integrated into Lane Profile 165 Molecular Weight and Mass integration AlphaView supports Mass calculations based on the users M
247. y Chemi Display Show Saturation Auto Contrast Checkbox for selecting Turbo d Mode Exposure Auto expose r L a gt gt gt mx AE comp 0 1 8 0 0 6a Cabinet Excitation E mission Preview Advanced Channel assignment Tab labek Speed Resolution Noise reduction Acquire Buttons for selecting Speed Resolution setting Figure 2 4 Turbo Modes and Speed Resolution Settings 37 The camera modes are selected by the Turbo Mode check box When the Turbo Mode checkbox is unchecked the camera operates in Normal mode Turbo mode checkbox is checked the camera operates in Turbo Mode Both Normal and Turbo mode can be used with each of five binning modes and auto expose functions to optimize image acquisition speed and for fluorescence and chemiluminescence applications Speed Resolution Settings for this mode captures the image using the CCD sensors full 2048x2048 pixel resolution to generate highest quality vibrant images that are ready for quantitative analysis and publication this mode captures images with a 2x2 pixel bin to decrease the required exposure time while maintaining a high resolution 1024x1024 image This mode decreases exposure times from full resolution images by approximately a factor of four this mode captures images with a 3x3 pixel bin 683x683 pixels with decreased image acquisition time and medium reso
248. y 3 tif coomassie16 tif tiny 1 gray tif 3 Crop tif tinycolor tif Desktop CukSample tif Eroded FC tif peaks tif rotated ti J SA Noise tif REENEN 54_5harpen tif pu 5 Samplez tif PE 5 Sample tif D 5 Spotter 3d tif hu Computer File name Mu Network Files of type Tiff Files tif v Tiff Files tt Windows bitmaps bmp JPEG format 4p Da Figure 3 3 File Open Dialog Box Using the left mouse button click on the name of the file to be loaded That name is then highlighted in the list and appears in the text box below the File Name prompt Alternate disk drives can be accessed using the Look In dialog box Once the file has been selected click on the OPEN button to load the file Alternatively double click on the file name The dialog box disappears and the selected image appears in the image window on the screen To dismiss the dialog box without loading an image click on the Cancel button 66 Save Load Analysis You can use Save Load analysis feature from file drop down menu to save all your work and then load it back for later use Note The load analysis menu item can only be accessed when the user opens any the analysis tools If the image opened has an analysis saved the item will be activated mi m Edi Image Setup Overlay Utilities View dex iex ess ai 3 ld Save Analysis p Load Analysis Cr Ctrl F4 Figure 3 4 Save Load analysis feature File Clos
249. y Aa NN T ET 15 POULT AUS IVI eR RE 13 QUESTIONS Or C OMMES ar r a E E E ET 13 Sarine AIMAN EW M SOWAT e EE E 14 FLUORCHEM IMAGING SYSTEM SBTUP ue a XR N ia I a ls N 15 SVS FETC ONHIDOROCHLTS Qu eM ORO UE TUE tea AS OA EAE AEROS 15 VRAYIZ ION ITI 22 12 tT 15 Cable CONTEC ONS es it SERERE PLUR est ene t ase On bene ue Lala Pee eR Eau O Sau bass N un M ene 15 HARDWARE INSTALLATION 5 5 deett iU eee ra RESP NC Ede IO SRI Ue de enti ab Un U sale eU EQUUM gues oa e EAM Ced e us ORDRE CVs petu RHCUd 16 Power SMED Surge Protel OT SCH i EE ied seoeeu eae ue edens Ute SERM Hu RU Ru OU else d Utm dieu dunes E Ue Iu med du usa dura a Rue 16 Computer Monitor Mouse and Keyboard Setup nirre nieni neira en NO AE nnne eere sensa annees sens 16 Cabinet Installatton INSU ONS o oe site ear eraniecied iv etu iae i ena nud edo E NCTE EOE Oa irae Uu dum Vd equa 17 CameraXzomponentscana ThstalldftONi ue cdeo cese eue E eit i voe Uu dean NEU NCT e d av ERU ne ede NE Num Oa d equ DR 21 GCONNCCHIVG TNE PINION E 22 POWOI UD SQUE socis at devi MR apnea E meus ACER ANDA DM asec A E LUE L R CD SOL C chu dE 23 Starting AlphaView FC Q System with AlphaView software eeeeseeeeeeeeeeeeeee nennen enhn nennen 23 SYSTEM INFORMA TION dis Na diteit te ue ida ta Dust rts pulos timuisse ee tsim dot um eue Dot 24 CHAPTER 2 GETTING STARTED BASIC IMAGING FUNCTIONS ccce eere e neue eene noun 25 BASIC IMAG
250. y3 goat a hamster IgG Cy2 ico b i d b ud Cys G o amp cys Cy3 a n Cys Cy2 amp gt e Cy 9 Frimary antibody 1 Frimary antibody 2 Frimary antibody 3 Rabbit a Protein A Mouse a Protein B Hamster a Protein C om4omomomHmAHONHONHONH NNMON Figure 1 Typical multicolor fluorescence labeling anti body combination The advantage of using multicolor fluorescence labeling protocols is that the final signal levels for each protein can be balanced for accurate quantitative analysis of each labeled protein For example the concentrations of the secondary reagents can be adjusted while maintaining the optimal concentrations of primary antibody for each protein This is especially useful when the proteins vary significantly in abundance Often the loading controls are much more abundant that the experimental proteins It is also feasible to actually monitor the fluorescence or immunostaining procedures to determine the optimum processing time by placing the container with the blot immersed in the staining solution directly into the FluorChem Q and acquiring an image After processing dry blots often produce images with superior contrast to images obtained from wet blots for the CY dyes Some fluorophores such as FITC fade significantly upon drying Auto fluorescence levels may be high for some nitrocellulose or PVDF polyvinyl fluoride membranes especially under blue light illumination Immobilon FL Millipore Corporati
251. zoom factor to fit image in available screen area The Image Drag icon is useful for to pan with a zoomed image To activate this function click on the icon and move the mouse cursor to the image The cursor will have changed to a small hand Click the left mouse button and drag to move the image When you are done you can click the Image Drag icon again to deactivate it Note Image Drag is only active when the image is zoomed in beyond 1X greater than 10096 The icon is grayed out in other zoom modes The Saturation icon allows for a quick image display of saturation Completel This is a useful tool to check for linearity of an image before analysis occurs Saturation is a feature that is most important during the acquisition stages and is thoroughly detailed in the acquisition features of the system manuals The Image overlay creates an RGB image from three grayscale images Select the images for the Red Green and Blue channels in the selection window The Extract Channels icon is available when a Multichannel image is active Extract channels produces a separate image for each channel The Channel Viewer icon is available when a Multichannel image is active 62 Averages Hed 5 385 Greer 5 951 Blue 25 478 The Channel Viewer opens a window that displays the multicolor image and each corresponding channel separately The window may be moved around the image by simply dragging the window The average Red Gr

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