BZ-9000 Software Training
Contents
1. 3 Switch to Monochrome imaging mode Your sample should still be in focus Make sure the red target is still centered on your reference point Click the bottom Compensation Registration button for view gap correction 4 Switch to your second highest magnification lens focus your image and center your original reference point Click the Compensation Registration button Switch to Color imaging mode and click the bottom Compensation Registration button 5 Do the same thing for each Lens When you are finished each registered lens should say Registered under Focus gap View gap power and View gap color fi Objective switch correction The correction information to reduce Focus gap and view gap generated when objective lens is switched are registered Keep in mind that compensation information will be cleared if Objective lens setting is performed STEP 1 Focus gap correction 1 Move stage and adjust Focus 2 Please push a right button and register a compensation value Compensation registration VIEW gap correction 1 Please move sY stage so that the subject used as a mark may be reflected to the center of a screen 2 Please push a right button and register a compensation value _ Compensation registration hh ae 4 similar procedure register by objective lens For all according to every monochrome and color The Focus gap need not be set according to every monochrome and color2. Display pseudo color IT Red 1 Halogen Lamp OFF Fluorescence Excitation Comment Texas Red CH4 Observation method Brightfield bat d Uw white w Halogen Lamp Fluorescence Excitation Comment Used as overlay display White Balance Light quantity Haze Reduction 3 Check the box next to Display pseudo color to set the observation screen to display a pseudo color in monochrome fluorescence A comment can also be entered for each Channel 4 In Channel 4 CH4 check Used as overlay display to use the CH4 window as an overlay merging display while in MultiColor mode 5 Check boxes to specify whether to register common observation conditions Select whether to set the following functions common among all channels Checkbox selected All the channels are observed with the same adjustment value Checkbox not selected Channels are observed with individual adjustment values of their own U Observation Method OFF Brghtfield Fluorescence Phase contrast Yes Can be set No Cannot be set c Focusing Use your mouse scroll bar to fine focus Scrolling down moves the lens turret down and scrolling up moves the lens turret up To scroll 8 times faster medium focus hold down Ctrl as you scroll To scroll 16 times faster hold down both Ctrl and Shift as you scroll rough focus d Navigation To move around within your sample click the sample and drag i
3. BZ 9000 Software Training BZ 9000 Software Training Table of Contents A Image Acquisition Lenses Channel Setting Focusing Navigation Navigation Window Bright field Imaging Fluorescence Imaging Phase Contrast Loading as a Group Capturing a Photo Image stitching Setup Z stack Setup m Quick Full Focus n Binning B Image Modification a Z stack b Image Stitching c Haze Reduction d Image Processing i Black Balance Tool ii Overlay iii White Balance Tool iv Gamma C Image Analysis a Cell Separation and Extraction i Cell Separation ii Brightness Extraction iii Color Extraction b Measurement Tools c Real Time 3D Module HA AO a ie a Section A Image Acquisition Observation mode switching Fluorescent shutter auto OFF function G Artn Multidimensional tasks Mouse wheel setting indication One click image capture Monochrome es Color switching p 6 Quick full focus function p 11 Channel switching Objective lens switching Binning setting Auto focusing Black balance Real time haze reduction Auto calibration scale Navigation function p 8 p 9 iumination ON time User setting storing reading File save setting Electronic Z axis stage operation system Electronic XY stage operation system xY Z axis stage coordinate preset Capture images for image stitch llumination adjusting slider a Lenses Click a lens icon to set y
4. No power Focus gap view gapipower View gapicolor Registered Registered Registered Registered Registered Registered All Clear Registered Registered Registered Registered Registered Registered Registered Registered Registered Registered Registered Registered Close b Channel Setting Register the observation method filter and the observation condition setting for each channel 1 Click the Channel Setting button underneath the Channel switching window Or in the BZ Viewer Menu click Channel setting 2 For each channel CH1 CH4 select the observation method from OFF Brightfield Phase contrast and Fluorescence The halogen lamp and the fluorescence excitation are automatically set according to the selected observation method Phase Observation Method te Fluorescence HalogenLamp Lamp Fluorescence Close Close Open Close Excitation st Channel setting CHI Observation method Fluorescence hi Display pseudo color LE Green w Halogen Lamp OFF Fluorescence Excitation Open Comment FP CH3 Observation method Halogen Lamp Fluorescence Excitation Comment DAPI Common setting is used For all CH Gain Preview speed aE LUT correction Black balance Dynamic Filter Pseudo color is valid in monochrome Fluorescence CH Observation method Fluorescence
5. and live cell imaging this function works best 10 j Capturing a Photo You can capture a photo at any time by clicking the Photo button at the bottom of the Observation Menu The photo will open in the BZ Analyzer Click Preview to view the sample again k Image Stitching Setup The purpose of the Image stitching function is to create a wide view magnified image To do this we set maximum coordinates to define our merge points a minimum of 2 coordinates needed Shown below involves creating 4 maximum coordinate merge points 1 Click the Merge button right above the window 2 Now figure out what parts of the image you want in your wide view magnified image Go to the farthest left portion you want in your final image 3 Adjust this area into focus Make sure that Z sync is selected in the Stage position storage box and click Set to save this point Make sure that there are no other saved locations delete if necessary 4 Do the same for the farthest right portion of your point of interest the top portion and bottom portion You have now formed a box around the area you would like to create a wide view magnified image of 5 Now click AutoRangePhoto Select the folder in which you want to save the images and Click OK click Ok again The program will now begin collecting images along the parameters set f Photo method Merge Save Folder Route path d Settings tindreu KominekiDesktanibalmenbe Group
6. background Click OK da Black Balance The black level is adjusted and the object is made ko stand cut fefore processing Note The Black Balance tool can also be used in real time in the BZ Viewer under the Image tab on the left 18 ii Overlay The overlay function enables superimposing fluorescence images 1 Go to Overlay in the BZ Analyzer The window below should appear At the bottom box that says display select up to 4 images you want to overlay Click OK Note In this window you can adjust the pseudocolor brightness and contrast iii White Balance tool White Balance is used to correct the color temperature the ratio of red blue and green light in an image 1 To use the White Balance tool click the Camera Setting tab and check Display push set area in White Balance box A box should appear on your sample move it to the area of the sample you would like to White Balance and click Pushset Initialize co Focus gt Bright field W saturated pixel display Note The White Balance tool can be used in real time in the BZ Viewer in the Camera tah 19 iv Gamma Gamma correction takes place after the image data has been collected from the CCD and it s a function that can be used to correct dramatic differences in light intensity between signals It can be used in Brightfield Phase contrast and Fluorescence but is particularly useful i
7. folder na sasi blank The number of photos of a total photo is 8 piece File The capacity of the disk used is about 4 6MB Format The expectation time until completing photo is 24 sec Prefix Does photo begin Photo option _ High speed photo mode The noise might stand out and the blur is generated Turn off usually gt 11 l Z stack Setup The Z stack fully focuses samples that are thick or have variations in height In one application when you put a bumpy sample in a microscope some portions of it appear in focus and some do not The Z stack function takes image slices along the Z axis up and down and combines focused portions of them to produce a fully focused image To use Z stack follow these steps 1 Select Z stack button from the top middle row of buttons Focus your image on the tallest location on your sample The tallest point on your sample should be focused and the rest should be blurry see below image Tip Scrolling the mouse wheel away from you will focus your tallest highest point Scrolling the mouse wheel towards you will focus your lowest point 1637309 7019500 t ix fe ed fe e pa Toanatyaer ToMems 2 Go to the Stage Control window and click Upper Limit This tells the software your upper limit from which to collect image slices 3 Now focus your image on the shortest location on your sample Scrolling downward moving y
8. manually adjust an image used for Cell Separation and Extraction Display details Set the color and thickness of the outline of the counted cells the label color and extraction color Save the image button Save adjusted images with the Save As dialog box You can enter and save Comment and save images in JPEG format with the Image quality settings 26 b Measurement The measurement module allows users to easily quantify the results of our observation Click Measurement Module in the BZ analyzer to open Radius Tool The radius tool allows the user to approximate the radius of any points of interest like cells 1 Click Radius 2 Click 3 points on the outside of the circle to set the circumference of the circle The radius of the circle should be displayed in the Measurement result box To see radius in microns check Calibration EW Overlay HR Haze Reduction1 tif 24bit a J Z 1 Radius 143 xi Z Q Between Radius Centers Count 2 points Aj al 2 Angle 1 Angle 2 Perpendicular Parallel a O a xl Segment Freehand Garbage Delete line line box All Measuring line Measurement line Yelow _ Measurement point Yelow Y Display details Label white _ O Thin Thick Measurement result Display O Non Display Measurement result No Mea Result 1 Radius 143 Centers Tool The Centers Tool calculates the distance a between two cell centers Bet
9. micrometer 1 micrometer 1 micrometer 5 micrometer 2 micrometer 32 Additional information can be found in the official BZ 9000 User s Manual and the BZ IT Analyzer Reference Manual 33
10. 5 a Z Stack Follow these steps to create a fully focused image from a folder of captured Z stack images When using the Z stack tool for fluorescence images apply the haze reduction tool first 1 Open up the BZ Analyzer and click Load as a group Find your folder Click Load You should now have a window open with a scroll bar you can scroll down to view the images captured 2 Click the Full Focus button at the top of the menu Note When setting the Z stack upper and lower set points in multipoint time lapse mode keep in mind that these upper and lower limits are the same for all points b Image Stitching gt DN Follow these steps to create a wide view magnified os image from a folder of captured images 1 Open the BZ Analyzer and click Load as a group Find your folder Click Load 2 An Image Merge window should appear with a group of image files selected Click Confirm position swe 3 The captured image slices should be lined up Now click Merge start to merge them If your final picture has stitching lines an image can be used for correction Select a blank background image next to the radio dial labeled Using image for correction and then click Merge start 16 T Image merge ed Image merge Images of BZ series are merged in high speed and high accuracy Select File Positional result confirmation Merge start Image characteristic Brightfield PhaseContra
11. adius Centers Count PPE Angle 2 Perpendicular Parallel E a x Freehand Garbage Delete box Measurement line Violet E v Measurement point Green M v Display details Label a Yelow v 1hin O Thick i Mesero eni fesuk ea Display O Non Display Freehand line a Calibration rmber of des 0 Fake aee 29 c Real Time 3D Module 3D imaging is a function that enables observation of specimen structure and documentation of observation results It s fast and easy to get an impressive 3D image If you re trying to view a fluorescence specimen in 3D it s helpful to apply the Haze Reduction function first to remove fluorescence blurring 5 Haze reduction the tiu wheres to hucrescence is removed at a high speed without artifact Ongina moge alti tig quaty Muorescerce bha renova a Tip Typically the highest Haze Reduction setting is ideal for 3D imaging Now click the Realtime 3D analysis button nG Realtime 3D analysis The window below should pop up oi erro oy There are many options for 3D analysis available In the Realtime adjustment box we can separate the red green and blue emission light to enable counting of fluorescent signals EW HR2_pic_CH4 Z stack 3D analysis 24bit Display setting Orthogonal display Realtime adjustment GREEN Brightness lt Contrast lt Gamma lt Motion picture deta
12. ay details Outline White C O Thin Thick 1 2 r 3 Count Count result Label Green E v Font Size 9 Extraction z 41 a SB Violet Hi Permeability J 41 8 Save this image Exit iii Color extraction The Detail button for the Color Extraction sets the color for extraction the location and sets the tolerance and permeability yong stay 3 Signal mage binary image a bids are BT Ging wine C Thin Ce Thik H Ligupip Libre C TES en minekin aE ai Dex Labas uman MF Fo fie 4 si mm vem E e Poma J bail 24 The options on the right side of the cell counting software can be used with the Cell counting modalities for additional analysis if the Binary image or Mask image radio dials are selected Below is a definition of the options available ml eee a F kaan cell count Untitled tif Switch display Extraction method selection a Original image Binary image Mask image Ho 5 Correction Cell Brightness Color separation extraction extraction Einne particles Fil holes Expansion Shrinkage z oe circle Merge regions Remove selection Image correction a Outline extraction Reverse 1 5 O e5 Count Display details ga Outline White C v Thin CO Thick Label Green LC Font Size 9 Extraction r S Count result aaa violet L Permeability i f i Save this image Count button Automa
13. full focused image 1 Find the lowest point of the image that remains in focus by re scrolling down with your mouse 2 Click the Quick Full Focus button at the bottom of the screen in the BZ Viewer 3 Now scroll up without using the Ctrl button As you scroll you should notice your image slowly come into focus Tip You can also start from the highest point of the sample and scroll down n Binning Binning is a process that combines a cluster of pixels into a single pixel For example in 2x2 binning an array of 16 pixels become 4 pixels reducing the overall number of pixels Binning increases the frame rate but it also lowers the resolution Since binning increases the sensitivity of the camera it 1s an important function to use during live cell imaging to prevent photo bleaching samples To change the binning settings click the Camera Setting tab and adjust the type of binning in the Binning ROI box shown below Preview mode High speed va Gain Ode AE Average we Note Binning is only used in SEEN rae Fullscreen Monochrome camera mode pe NAY AL di Cd Binning ROI OFF Cannot use in color camera Shooting conditions Full size pixel count 1360x1024 wt 1360x1024 or less in multidimensional photo ROL Bining is Fixed ko 1360x1024 Multicolor amp 2 stack order Multicolor gt stack 2 stack Multicolor 14 Section B Image Modification 1
14. iled setting gt Play E lt C Start C mid C End step angle v 10 degree Play methods Loop play O Sequential play YZ XZ Rotation axis X 10 ie Basic plane X ction Save motion picture Save still image Close 3 0 To navigate your 3D image 1 Drag with the left mouse button 2 Zoom with your mouse wheel and 3 Drag with your mouse right button to visualize sections El HR2_pic_CH4 Z stack 3D analysis 24bit Display setting Orthogonal display Realtime adjustment GREEN Brightness lt Contrast lt Gamma lt Motion picture detailed setting P Play E Stop lt C Start C Mid Set Set step angle v 10 degree Play methods Loop play Sequential play Basic plane RY YZ XZ Rotation axis A Y Z io ia a ase Wheel for zoom Up Down Right drag to cross sectic Save motion picture Save still image You can replay the movements made with your mouse by setting Start Mid and End settings in the Motion picture detailed setting box and then clicking Play 31 Recommended pitch for Z stack Tool Lens CFI Plan Apo 2x CFI Plan Apo 4x CFI Plan Apo 10x CFI Plan Apo 20x CFI Plan Apo 40x Plan Apo Oil Immersion 60x Plan Apo Oil Immersion 100x Phase contrast ELWD 20x Phase contrast ELWD 40x Minimum Pitch 60 micrometer 20 micrometer 5 micrometer 3 micrometer 2
15. n fluorescence when an image has large variations in emission intensity 1 In the BZ analyzer click on the menu bottom at the top labeled Image Processing Click Brightness Contrast Gamma and the window below pops up Increasing the Gamma below brightens the image below the contrast has also been increased Brightness Contrast Gamma Before processing After processing Brightness Contrast Gamma J ma Save Conditions Load Conditions Clear conditions 20 LS Image Analysi Section C cI I Man O a 5 CF ae 21 a Cell Separation and Extraction Since the BZ is used primarily for biological applications it makes sense that a Cell Separation and Extraction tool would be a necessary function It s important to know that the BZ software has the capability to separate and extract cells based on brightness a ee sii The er ge bhar lee iwa Pe ee Foe ram alar Cell separation level can be changed using the slide bar Kataga Ci Et Dami che Tab Ts Lin Kaban kar he E tomes Ca conn r Dynamic Cell Count Histogram na AN eee Aree Data sachon 10 Count 400 300 200 1887 3113 5660 Area Resut output Close 22 1 First open your image in the BZ Analyzer 2 Click the Cell Separation icon A window should pop up that shows three methods of cell extraction each Cell Separation method is defined below i Cell Sepa
16. nt whet O focus Ovcom start Previously stitched images through Image Merge function can also be used as a navigation image Click Open in the Navigation window In the dialog box displayed select the image to be registered to the Navigation window Click Open to set image as a Navigation image f Bright field Imaging In Bright field imaging a sample is imaged against a bright background This is the mode we would use for most non fluorescing images To view images in Bright field mode follow these steps 1 Switch your channel to Bright field Channel 4 if you followed the channel setup in Part b This utilizes the channel that contains NO filter cube since we do not need to separate wavelengths of light in this mode 2 Select Color camera mode 3 In the top left corner make sure that Single window mode is selected 4 Place your sample on the stage 5 Select the lens you want to use by clicking its lens icon 6 Focus your sample by scrolling your mouse g Fluorescence Imaging In Fluorescence imaging light of a specific wavelength excites the specimen causing it to fluoresce The mode used for this is Monochrome mode To view images in Monochrome mode follow these steps 1 Switch your channel to one of the designated fluorescence channels 2 Switch camera mode to Monochrome 3 Place your sample on the stage 4 Select the lens you want to use by clicking its lens icon 5 Focus
17. ocuments My Computer 05 C i Z stack DELL Upper 1 Documents and Settings Administrator All Users Andrew Kominek 5 Application Data O Cookies 5 Desktop Allada Boule ab miwi vasa slidel BZ Demo I demo vavigaion Now i at Goldberg 3 Image Stitching Function Pitch 0 0 um Brain 1 1 num gt Cell oma CO Heart O ips O Petri O Retina 0 Tissue ips JH OGD 09 20 2010 Jones Lab Examples CQ keyence Time lapse Loyola D New Folder ba NORTHWESTERN CORE Start EO Now i Ohalloran Interval 00 00 00 1 1 num Rana solomon Merge U of Chicago uchicago 3G uluc columns 9 xrows 12 num Prev P Next N Univ of Wisconsin UWAT Zhang lab VY Favorites gt Local Settings My Documents LO My Recent Documents O NetHood CO PrintHood SendTo 5 Start Menu Templates Reset R Load L When opening Z stack or Z stack merged images there are two focus options at the bottom of the window Full Focus and Best Focus The Full Focus option works by sampling the locations with the best focus at each coordinate in grouped images that were captured with a combination of the Z stack and Merge functions and merges them The Best Focus option automatically selects and merges grouped images with the best focus at each coordinate captured with a combination of the Z stack and Merge functions For time lapse
18. our desired magnification Tip Start at low magnification to image samples and switch to higher magnification oa ai ff Z Corr Reg Registering the Objective Lenses It s important to register each objective lens before imaging to apply the View gap and Focus gap corrections when switching lenses Click Z_Corr_Reg in the lower right corner of the Lens window to open the Registration window 1 At your highest lens magnification in Color mode select a small reference point on a sample in focus It should be small enough that your lens correction 1s done correctly but large enough that you can still see the point of reference in your lowest magnification Select this point by double clicking on it to center 2 Click both Compensation Registration buttons in series cp Sabit kda la Mago es a Keepin mind thet compensation information will be cleaned if Objecther lens setting k performed Eu 1 Mova T Gage od aa a 2 Plaats jaah a ght agom ahd epee a Count PUTA 1 ogee mone Xf shage a that the mask nang be nefletied bo Bs camber of a binoan 2 Please push a night button are regster chia Zz a amle procedure segisher by obecn lens for all according is aas Ely Diet Bordi Blete ug aaa ngpa naalala E J AUTO a SS Fagggg efhp cc pP Brighit feid Cf Fuorescence OFF HI I D Sey if i ftes fia Faf h pale z eah Oath E enai
19. our lens turret down it should be your last point to come into focus Your tall points on the sample should be out of focus NAP Merge MuttiPomne 4 Go to the Stage sew nee mel mam om Control window and er 2G LEY ak click Lower Limit T ik This tells the software your lower limit from which to collect image slices fe le j gt fae pai fad anog a kapa Ts preview iy Promo Ep Str reorap Photo mode rape corrector Kaba bk Color 1360x1004 Hai speed OB 5 Now notice that we have not told the software how many image slices to collect We do this by adjusting the pitch which denotes the micron level spacing between image slices Increasing the pitch tells the program to increase the distance between image slices i e decrease the number of image slices 6 Lastly Click Photo Z stack will prompt you to save in a folder and will then begin collecting image slices bai ed had kei Fed o LI CN Puti foj w Famo ji jaman Kage pester toa 1 gt 163 SEMIS ee ee ey eer W soo ew wiki moo Bag san crama gon Mako moe mage Corrector eaha Garet mod Orias DOi Otome Tom stack inon magset oa m Quick Full Focus The Quick Full focus tool has a similar function as the Z stack tool but it works even faster It works by utilizing the focused points in images captured by scrolling your mouse and combining them to create a
20. ration Separates and counts cells based on an edge counting algorithm ii Brightness Extraction Extracts and counts cells based on variations in cell brightness iii Color extraction Extracts and counts cells based on a user selected color hue I ynamic cell count Cell Count BMP SS x ce Sree Dae Ome Hi ri Cell Brightness Color separation extraction extraction EE z k Undo All Display details Outline violet LE C0 Thin aus Label Green E Font Size E Count result 43 ma white L Fermeability T t Save this image Each method can be selected and used to count by pressing Count Details for each of these methods are different and can be found by clicking Detail i Cell Separation F a The Detail button for the Cell Separation method enables changing the cell counting threshold extraction of color and separation level Papi obrint 23 ii Brightness Extraction The Detail button for the Brightness Extraction tool bring up a histogram and enables setting the brightness range and color for extraction El Untitled tif 50 00 24bit BK K 3 D oa m ley re O a x O Originalimage C Binary image Mask image Correction Cell Brightness Color separation extraction extraction Detail Brightness 12 amp 255 8 range fie 5 Display details Extraction violet v Permeability color Displ
21. st Fluorescence MultiColor Monochrome O Fluorescence G O Fluorescence B O Fluorescence R Shading correction Auto O Using image For correction Select File OorF y Right click to copy position information and paste into BZ series Confirm position Save k c Haze Reduction The Haze Reduction function can be used to remove blurring from captured fluorescence images It can be used 1n real time in the BZ Viewer or in the BZ Analyzer as shown below n 1 Open the BZ Analyzer and load your captured fluorescence image 2 Click Haze Reduction button at the top of the Analyzer A window should pop up that has different settings of Haze Reduction Select your desired setting from Preview 1 5 The box next to Noise removal may be checked to remove minute noise from the image 3 Click OK 17 ke Haze reduction me bir inherent w fucrexence ts removed ac a highspeed wihara IYA Dral mage Hary hee blur area Note The Haze Reduction tool can also save conditions and load them for processing future images d Image Processing i Black Balance Tool The Black Balance tool creates a uniformly black background for fluorescence images which enables fluorescence signals to stand out 1 Click Black Balance in the BZ Analyzer The window below should appear On the left original image move the small white square to the black background The improved image should have a crisp black
22. t It is just like Google Maps Double click a point of interest to center it Right click can change the Zoom and Exposure time both of which are adjusted with the mouse wheel Or if you prefer you can use the slide bar below the image window to adjust the exposure Underneath the Lens controls window in the Microscope tab are the Stage controls Here you can move your lens turret right left up or down to adjust the field of view of the sample Points of interest can be memorized where Stage position storage is by clicking the Set button Make sure to click Z sync to save the Z axis coordinates as well Stage position storage E Te SET Gt tel Plate Image merge photo e Navigation Window The Navigation System enables navigating around a specimen at high magnification by using a low magnification reference image 1 Image your sample at low magnification Click Navigation under Stage Control and a Navigation window should pop up Now you can switch to a higher magnification and click a location on the Navigation image to move to that location Be tao Merge MuPoint Time apse Tip If you are using the Navigation Window and get an error message that reads Outside of Nabis Range click Image Registration button to reset the Navigation window This error message indicates that you are outside the field of view set in low magnification gt YE Tore Alab Curre
23. tically counts the number of cells extracted using the different extraction methods Count result Displays the number of cells counted Size button Lists the area circle length radius XY major axis minor axis and integrated value of the various cells measured in the Dynamic cell count measurement dialog box Statistics button Lists the minimum value maximum value range average value standard deviation total and sample number for the various measurements including area circle length radius XY major axis minor axis and integrated value in the Dynamic cell count stats dialog box Histogram Displays a histogram in the Measurement results histogram dialog box for the various measurements including area circle length radius XY major axis minor axis and integrated value as well as the data section 23 Switch display Selects the method of displaying the image Select from the following three types Original image Displays the original image Binary image Displays a binary black and white image Mask image Displays an image with extraction color set in Display details Correction button icons Adjust the cells for counting with 9 different correction methods Eliminate particles Fill holes Expansion Shrinkage Separate circle Merge regions Remove selection Image correction and Outline extraction Note The Image correction function is a powerful tool that gives the user the ability to
24. tig 5 n i 7 e j Ng igo y gt j gt s 3 ng a s Co i q nS 5 lo E a in ni A oo 1 A if i PW so is a as s gt s a ie mig A J wy ova oe gt pa s p Sh a ko 2 4 ex F s s oi H f ET e r aT a Wer feat TIBA Mg t Pagi w a a 4 7 4 gt ka e y pf bla f nA dys vr 7 n O 5 S Angle 1 Angle 2 Perpendicular Parallel MI Segment Freehand Garbage Delete line line box Measuring line Measurement line Black HD Measurement point Black ww Display details N Label Yellow v O Thin Thick Measurement result Display O Non Display Result 216 um No Measure Between 2 points 28 Calibration Setting number of decimals 0 Result output Freehand Line The freehand line tool is an incredible tool because it can calculate the length of a curved line which makes it ideal for measuring the length of the mouse dendrites below 1 Click Freehand Line 2 Click your start point to set it and then click your end point to finish measuring EW FF 20050415204254 tif Z stack Full focus 24bit pa ATO a ma AA GT a AA AG x Pig ma ng AN ii a nA p r Ti Dog PA a me 1 a AN EERE ip ora Main Measurement FF 200 2 be Freehand line po UM Sie FAR ES a a r a 5 gin BEA re Pann ex Sat een Se ee E er e O wt G F Ke irt aye Kk AG i iin 5 8 ss a ke ve pr pus Bet R
25. ween centers 54 Hl lt G 1 a Blg L 1 Click Centers 2 Click 3 points on the outside of the first cell to set the circumference of the first cell An approximation of the shape of the circle should appear around it Do the same for the second cell The distance between the centers of the two cells is automatically ieee KA tama determined Ea ska Zi Angle Tool The angle tool enables approximation of angles 1 Click Angle 1 2 Click the 3 points that define an angle with the second click corresponding to the angle center The software automatically calculates the angle EW Cell Count BMP 200 00 24bit aaa f NG pis 5 is z j Main Measurement Cell Cou x Z al e A ajaj 4ngle1 Angle2 Perpendicular Parallel s o a x Segment Freehand Garbage Delete line line box All Measuring line Measurement line White E Measurement point white C Display details Label Yelow E O Thin Thick Measurement result Display Non Display an uaaal Measurement result Cell Count BMP No Meas Result 1 Angle 1 37 degree C Calibration Setting number of decimals 0 2 Result output Between 2 points Between 2 points calculates the distance between two points 1 Click Between 2 points 2 Click point 1 then click point 2 The software automatically calculates the distance between the two points Radius Centers Count oa y Rot
26. your sample by scrolling your mouse 6 Click MultiColor to overlay channels on Channel 4 7 Click each Channel window to image samples The final channel window displays an overlay of the 3 fluorescence channels h Phase Contrast Phase contrast is an excellent method for enhancing the contrast of thin transparent specimens without a loss of resolution 1 In the Channel Setting window switch the observation CH4 method to Phase contrast Observation method Phase contrast 1V 2 Switch to a Phase Contrast objective lens 20x or 40x BES Halogen Lamp ON Fluorescence Excitation Close Note Phase Contrast and fluorescence images can be asese Used as overlay display overlaid in both Multicolor viewing mode and in the BZ Analyzer I Loading as a Group When the BZ viewer captures a group of images that are linked together for the purpose of combining them for future analysis such as images captured from the Z stack function image stitching time lapse or a combination of these functions sain a they can be accessed in the BZ Analyzer by clicking Loading as a group a group This opens up the window below Notice that Merge is selected when I select the Fat folder on the left the software automatically detects whether the images need to be Z stacked or Merged or Time lapsed To open the file click Load in the bottom right corner ng Loading as a group i My D
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