Metamorph User Guide
Contents
1. 000 O000000 00C E Intensity Std Dev between i 0 000 0000000 000 be Intensity center X between 0 000 0 000000 00C ke Intensity center Y between 0 00071 OO0000 000 O Radial dispersion between 0 0001 000000 00C H E Shape Position Orientation H E Dimension H 0 Greular H O Gray Level Texture H 0 Optical Density Average intensity Intensity Std Dew Perimeter Length Breadth Oo Reset Accumulated Select Preferences tab choose the options required Select the standard area etc Threshold the image to pick an inclusive threshold containing your signal Click on E Integrated Morphometry Analysis c fst l l Measure The selected area will be green excluded Source NTGLI2_Vim_Ecad_rd1_w3FL A l area will blue O Segment using mask Mask Hone Measurements Preferences View Object data Summary Measurement methods Mouse click interaction Measure all regions Highlight objects on left click Fill holes in objects Add Remove object from m Exclude objects i centroid is not sees se late in the active region Update filter ranges on Shift click C Exclude objects touching edge Object drawing Object standards Draw object borders Standard area a0 E Draw centroid mark Optical density low boundary 3 Draw failed classifier objects Optical density high boundary 1500 Miscellaneous Object mask O Wam when data will be erased Binary intensity of 1 fo2. First copy the image with overlays to paint The software does not export this Click on log data to export to excel There are several data options available to view First click display statistics Then configure and open a log It will go to the excel file E Track Objects Statistics E Track Objects E exes a re Object 1 Object 2 Am Object 3 Source for images Algorithm Template Em sees 25a 18 9797 Stack 22 aoe Sequential Fles Tembam 42750 5 8855 OMDR Someso sme fas s52 E y A ace Jules Pewee 776 mss ats Stack Print Table Summary Log Not Open lt No Joumal gt Select Joumal Log by frame Z Log empty lines T Log Only No Data Display Duplicate Overlay Logged to DDE App a E Track Objects Data Next click on Display Data 2 47 pix min 0 27 pix min 0 24 piemin 0 65 piemin 1 15 piemin 0 50 pic min Pick the type of Data you need 0 3 piemin 0 29 piemin 0 73 pix min 0 18 piemin 2 87 pix min 1 06 piemin 0 22 pix min ae ae 5 Position Deta Xr E Angle Distance Velocity Dist To Orig 5 Interval i Avg Intensity Intensity 5 D Min Intensity Max Intensity Total Intensity 75 3 00 13 75 2450 Delta X pixels Data Type Asis Position Deta XY Time Angle 6 Dista
3. 244 38 453 0 052 143 178 101 955 563 582 8 160 3 445 736 858 000 836 000 87 236 72929 000 24 611 12 905 0 190 65 192 31 174 111 614 7 490 2 360 790 849 000 831 000 83 446 69344 000 23 824 17 951 0 082 74 330 46 824 176 083 4 719 3 315 ie Measure Reset Curent Load State L Create Object Mask Reset Accumulated Save State Close It is difficult for the software to distinguish edges If you need to separate objects use the line tool to draw a line completely bisecting the two parts to be separated Go to the measure menu and use cut objects LineScan This gives the intensity at each point along a line eg if you want to show the relative intensity of signal through a line in a cell Draw a line through the area of interest with a line tool Click on Line scan It will generate a graph and the intensity plot for each of the red green and blue channels E Linescan sz Source Image NTGLU2 Vim_Eead_rd1_w3FL AF568 Rhodanmi Avg CT ma 2 l To on ty 291 Distance I scan Width 5 LockX Y Scale 14Bitimage YValue Average Inactive Window Open Log Configure Header Configure Log Close Kymograph in the stack menu 11 Gives a read of intensity values on a line through a stack either time or Z Requires a 16 bit stack Select a stack Choose the planes to measure Subtract background this removes a steady backgro
4. Metamorph User Guide Contents Page Analysis of cell migration vesicle tracking Metamorph will open most tif files however most analysis needs 16bit tifs We recommend capturing and saving images as 16 bit tifs If not convert them in image J or using the scale command 1 Stack menu Open or build a stack To open a z stack generated in metamorph or another program or create a stack from a series of individual images You will need tif files preferably 16 bit if you want to build a stack the images should all be in one folder and sequentially numbered Most software will export like this If not use InfraView software to Batch rename files To open a tif stack go to File Open To build a stack go to File Open Special Build stack and select build sequentially or user defined as required Scaling an Image This allows you to alter the display range of an image to make it easier to see It does NOT affect the raw data Use the scale bar on the left of the image and move the sliders to give the best picture Scale image command Metamorph makes 16 bit images which won t open in MS Office on in Windows picture viewer They will open in Photoshop however they are greyscale To use your image in MS Office etc and keeping the colour look up table you will need to convert the image to 8 bit 1 Open the scale image menu Select 8 bit copy Press copy Save the image If you want to keep the filename in the 8 bit c
5. a specific region draw the region in Oler a5 percent image A Method Area Average Integrated Select the method of co localization as integrated ii lon Data Log Not Open Configure and open a log for the data This will give you a overlap of the two channels in relation to each other Colocalization coefficient This gives you the Pearsons colocalization coefficient based on the relative intensity of the signal in each channel for a given pixel rather than the simple yes no that the measure colocalisation app uses Use thresholded images Select image A and Image B If there is no co localization the Coefficient 0 complete co localization 1 or 1 Criteria for co localization vary but complete co localization never occurs In this case check your images 17 Review Multidimensional Data This allows you to view and modify the images of data acquired through multidimensional acquisition eg timelapse images and generate movies and stacks for analysis It can do many things The simple things most people require are described here Open the review multidimensional data app Select the base file this should be the file with the ND suffix in your data folder Tick the wavelength box to view 72 74 74 75 76 77 78 79 g0 9182 83 g4 85 86 g7 ae sq 90 91 the image a TIX IX IX IX IX TX TX XTX XTX XXXL XR Right click on the 1 to highlight A all the planes Add or remove individual images by right click
6. ct the image here ill Display result Select the width intensity and significancy start with 5 about 10 percent of your original and work up own 30 as required ae utgrowths Intensity below local background 10 graylevels Click apply the data will be logged and a new image Minimum cell growth to log as significant 10 um 10 pixels with the possible cells and neurites will appear Configure Summary Log Configure Data Log Cells Save this 16 Measuring colocalization See also the using metamorph to measure colocalization user guide for an expanded explanation There are two methods of performing co localization in metamorph the Measure co localization and co localization coefficient apps Neither is perfect and the choice depends on the needs of your experiment Measure colocalization is easier to understand but has a higher chance of false positives E Measure Colocaliz o amp amp You will need background subtracted thresholded 16 bit tiffs of Image A NTGU2 Vim_Ecad rd1_w3F each channel Image E NTGLI Vim_Ecad_rd1_wF Measure Colocalization Region from image A used Open your images Value for all A 7065 00 A Overlapping B 100 00 Select image A Threshold the image with an inclusive threshold to II background pixel seuss kc nn remove all background pixels eet 7065 00 Select image B threshold B Overlapping A LALL E Not Ovedapping A 0 00 If you want to only measure in
7. d g To record move click Save Keep Planes Source Stack A431 EXPT_ Low Range Step Size x Delete Unselected Copy Selected EY Make Movie fo es Source Stack Untitled Play each frame for 3 Movie Fomats 1 30th of a second AVI Check Save Quick Time Low Range Step Size High Range Save ra gt 100 gt I 3 Unselected Select Flanes In Range e Select movie format AVI for windows Quick time for MacOS f Ensure Selected in checked in the Save menu h Select the directory to save to so you can find your video i The Video Compression widow will appear Uncompressed images are large but will open on most systems Best practise is to use uncompressed movies as they will play on almost any computer Not all computers will have the right codecs to play your movies Click OK Configure About Make a montage To make a montage of multiple time points or planes Requires the images to be in a stack Go to Stack menu montage Select the stack to be used the name of the output file the direction the images should be placed How many columns and rows you want and the the zoom in the stack To keep the images in the montage the same size as you took on the microscope select 100 If you want to make sure there is a line around the image or have the number of the image present in the montage select these options click OK top right corner Save
8. duce noise and artefacts and remove background You may also need to calibrate your pixel to micron information for your image or do some basic analysis of regions These commands are found in either the Process or Measure menus Background subtraction Process Menu Background and Shading correction The easiest way to correct your images is just to Flatten the background However here the software makes assumptions about the nature of your background so although it is the fastest and easiest way it may not be the most precise Mee ph SE o nd shading Conecion el Fle Eda Regions Steck Display Process Log Measure Journal Appr Window Help eats x a A ai Eiti Oai Dii E B AriPratE Cirie Shaft oA To correct background Pieet image D Famer Backyard Eaa a Tubed behand Shane Soe Comect shading E fiin backgroud hack Amrut look in the Process menu Biak Fhiri choose Background and Detect Edje e A Morphology Filter Shading correction eon D Arcee Taree fackorcand and Sheckng Corectenn hed ane f D piiki There are several options 70 Diei E 1 uan riy a 6 eed backer Flatten background is the most straight forwards Ratio neces To use Flatten background simply select If your image is fluorescent or transmitted light and pick the size of the smallest object in your image Click Apply Technically the best option is to have taken a separate background image w
9. f 0 5 this is the delay between showing you each step of the track a slight delay makes it easier for you to see Decrease as you get more used to it E Track Objects lo EE wey Source for images Algorithm Template Stack i Sequential Files OMDR Plane 1 aj ali ot a g7 Stack lt No Joumal gt Select Joumal Log by frame E Log empty lines E Log Only No Data Display Duplicate Overlay Logged to DDE App F object not found Al Template Match z _ gorthm Template Select Posti Template Matching uses an image convolution i Quit Object to compare each object s intensities with O Skip Frame the values in the first or preceding image Use velocity for center of next search Delay 0 2 sec object Algorithm Specific Options Template Matching Check update template for each frame Update Template Each Frame and use derivative image Minimum formatch 15 Click OK Use derivative of image recommended for non DIC images Track Objects Interval Options i Next set the track objects Interval options That is the time Table Time Units Minutes between frames type in the rate at which you captured the Time Interval Options Time of Image Creation images e g for timelapse that might be once every 5 mins User Defined Time Interval 5 19 Next set the Track overlay options Tack Pont Cote o OK Highlight display track path and display point on current Point Mar
10. for cell scoring to define settings for each wavelength Open excel and configure the logs Open logs as for cell scoring When configuring logs the summary log gives the simple read out of number of C7 Multi Wavelength Cell Scoring o ee AL pce Adaptive aoe ls Background m p al W Display result image i Segmentation terial Cy Al nuclei o green stuff Name All nuclei W1 Source image No Applicable Images egend color Gray Stained area Nucleus Approximate min width 3 um Approximate max width 15 um Intensity above local background 500 gt graylevels Sonti The All nuclei wavelength is a required stain for all cells cells and number or staining overlap for each channel compared to DAPI and each other Click apply You will get a result image and a data log Save both 14 Cell cycle Requires a 16 bit DAPI nuclei stained image Use to distinguish proportion of cells CY Cell Cycle e s fos x Select the minimum and max width and eee ON Adaptive ale intensity to identify the nuclei Early M 4N G0 G1 2N T I Apoptotic system a2 4N 5 phase Diplayresutimage EF Segmentation DNA content in Source image 1 3 w1FL DAPI ii ee gt ane i moor a 2 3 pigels to um Approximate min width 30 um 30 pixels Using defaui Approximate max width 100 um 100 pixels ratio 1 1 Intensity above local background 100 graylevels Select the intensity range Clas
11. fy the areas of interest over background and exclude those too bright for detection and is therefore required for most analysis E Threshold Image Source Image Nuceli Measure Threshold i Nuci Threshold Cip Overay Select the source image men htenstyenge 165k 785535 Set the intensity range 16 bit by preference freee ze 3 Threshold state Options are inclusive all pixels in the range are selected Inclusive _ Exclusive _ Off Or Exclusive pixels outside the range are excluded An orange slider bar appears next to the scale bar on the left of the image Slide the blue arrows to shift the threshold or type values into the boxes Data Log Not Open Region Measurements Use This command is the most commonly used It gives all measurable information about regions of interest selected by user or software distances intensity size etc it also provides a log of the data for export to an excel file Requires a calibrated 16bit tif for maximum efficiency but can give information based on pixel intensity without calibration Select Image Draw select regions using region tools or from your analysis Select include all regions or active region Use the configure tab to select the type of information you want to extract You can label your regions by highlighting the region and typing in the label box Click open log to export the data Logs will continue to record any data you generate after
12. he boxes Intensity above local backgpsamd 1000 graylevels Measure the intensity at background Positiwe Marker and in the lowest signal you want to W2 Source image No eriicable Images detect using region measurements Staingd fea Cytoplasm under measure in the menu bar see Apppaxffnate min width 10 um section 2 Calculate the difference Approximate max width 50 um and use as the intensity above Intensity above local background 800 a ayere background Click preview If it doesn t look good A modify the settings until the correct areas are selected Select the other image for wavelength 2 and whether it is cytoplasmic or nuclear Proceed as for wavelength 1 Click on configure data log select the info you will need Repeat 6r Configure summary log Open Excel Go to the log 7 MetaMorph Offline menu in the File Edit Regierfs Stack Display Process Log Measure Journal Apps Window Help main window Click open log and opepstimmary log Click apply in the Cell scoring Window 13 You will get a read out image and a log in excel Save both Multiple Wavelength Cell scoring Very similar to cell scoring select only if you need 3 or more wavelengths Select the number of wavelengths A tab will appear for each one You can change the name For each wavelength select the source image Choose a colour for each red and green are good as the overlap yellow is very easy to see Proceed as
13. hich you can now subtract from your data x i Background and Shading Correction fo f mE In this case select Source image Untitled g i Subtract background Result image EF Subtract Background ii Select background image Operation iii Set the bit depth Subtract background 6 Statistical comection iv Select the source image as whole stack E Com Hatten background Click Apply if you don t have a background image select None Constant value Statistical Correction T a a pay krk Draw a region in a background area Result bit depth S16 Select average first if the results don t look good try minimum and maximum Description Removes background intensities by subtracting a specified image Click Apply or constant value Result Untitled 1 Correct shading This will correct for uneven illumination It requires a separate image captured on a blank area during your experiment First Background subtract from the blank image Source ij ac Select Correct shading Farm munca ca Hater Erco rna Pea Subir bahya Ta Sosa S Comect shading Platien background Then select the background and shading images which is an image you took on a part of your slide where there only is background no cells or tissue Ennon Aucrecence C Taremeson Obs se J D piii i Diiepian Click Apply Aab Uan Mery Ug b Eia r bra Pei Using Filters to denoise or improve your image for quant
14. ing with the mouse Only those marked with an x will be loaded Stage Position i L se ect the stage position you DMSO 1 e noe a 41 A DD gt Timepoi lt 1 Elo s7 4 D gt S F ah Enable Montage of Thumbnails If the images are multicolour both the wavelengths and the Selections s1 Display Event marks display wavelengths and colour Red Green Blue Gray DE Coor Composte composite should be checked In the selections xs click load images and a new image will load Save this image as a stack for further analysis such as tracking or counting or making a movie Tracking objects 18 Requires a stack of tifs as a movie ie where each plane is a time point These can be generated using Review multidimensional acquisition see above You should have a set of criteria before you begin eg stop tracking if a cell dies divides goes off the screen Use to track a migrating cell or moving granule etc within time and 2 D space This app requires more input from you while it is running Open your stack Go to apps Track objects Select the planes in your image avery long video might be better analysed in chunks Open excel Click Config Log to pick the measurements you require Click log data e There are several menu options you will need to set First select the Search options a new window will open Select the algorithm template match Start with a delay o
15. itative analysis Filters are a useful way to tidy up your image for analysis or presentation and remove background artefacts Some analysis processes such as co localization and cell counting require image modification to remove artefacts which interfere with the algorithms used Filters can also be used to emphasis fine detail or remove haze from z stacks Background and noise elimination The best way to eliminate noise is to apply a median filter these replace each pixel with the median of the surrounding pixels This works best in 16 bit tifs but will work in lower resolution images E Basic Filters Source image MNuceli E Result image Ef Median Fiter Operation fe Go to Process basic filters Pegm A dialogue box will appear Select Median filter Select the filter width and height for 16 bit tifs start at 5 and work S e ee el ESSE Median 0 Sharpen down if too much is lost Rank Unsham mask Filter width 5 Fitter height Sub sample ratio Median filters can also be used to remove out of focus light from PRES piels qf F widefield images To do this make a median filtered image with large filter settings try 32x32 for 16 bit tifs Then subtract this image from the original Desain subtracts out of focus blur estimated by applying a low pass filter Other filters Low pass blurs an image Close Sharpen defined edges of object
16. ker Type Coss pame Point Marker Size 5 Point Marker Display Mode Next set the origin Options first point in track Display all Points _ Fill Circle Markers Display paint on current plane Display Track Path Display Track Numbers Display Track Pattem Tack Paton Coor Now you need to select the objects to track We suggest only selecting 3 5 objects at a time as you have to be able to follow each track as its being laid Click Track A window opens define the size of the region and the area in which the software should search for that object to have moved to These can be adjusted again later E dmsol tracking 68 1 97 yis WITH CONTROL PRESSED DOWN click an object to select it In the example below we ve selected the cell nuclei 20 Once you have selected your objects Click OK Tracking will begin Keep observing the tracks If it looks as though the software has made a mistake use the escape key This pauses the tracking and gives you some more options First use the skip back to go to the point before the mistake occurred You can now Poa s repositioning the target region either reposition the region correctly stop the tracking of that object or skip that Overlay point n OF This Object T Lock Region Sizes Continue tracking Update Template And Continue Data extraction
17. nce Plane Velocity 9 Dist To Orig 9 Interval 5 Avg Intensity 9 Intensity D O Min Intensity Max Intensity Total Intensity J C Graph Single Object Check that all of the data you need has been logged to excel exported BEFORE you start the next set of tracks 0 34 piemin nm rh
18. opening Region Measurements Peace es NTGL2_Vim_Ecad_rd1_w3FL Open Log Include all Regions Cloze Measurements Graph Configure Labels Region Label Image Name Image Flane Polygaril NT GLe Vir Ecad_rd _wsFL 4F5638 Ahodamine 1 Polygone NT GL Vim Ecad_rd1_w3FL 4F566 Ahodamine 1 Polwlines NT GL Vir Ecad rd wsFL 4F563 Ahodamine 1 Image Name Image Plane To transfer a region from one image to another go to the Edit menu transfer region Integrated Morphometry Analysis Use to obtain data based on the morphometry of your samples ie size shape area intensity locale Requires 16 bit background subtracted noise reduced filtered images Software is never as good as the human eye at detecting edges so the better your images the better your results Select the IMA tab Click on select measurements There are a lot of possibilities Pick what you need C7 Integrated Morphometry Analysis ee ee Select the measurements from the tree below that you j want to appear in the table in the setup dialog E Segment using mask Mask None H P Area Measurements Preferences View Object data Summary ol Intensity 5 ns ie fy Average intensity Measurement parameters and filters select Measurements i 2 Total intensity Parameter Display Filter Comparison Llmti Limit 2 0 Minimum intensity Area y 99996 000 F000 000 g Maximum intensity between 0
19. opy check the box if you want to copy the entire stack check this box A Basic Image handling YY Scale Image Image dmsol racking Range Settings Auto scale Low 0 63 l l High 0 Scale within the active region Gray level minimum and maximum values Curent plane 587 2714 Entire stack 550 3233 A me g f i ra ps a Lae F fa 7 _alculate Stack Min Wax Copy to amp bit image m Cony C Transfer filename when copying Copy entire stack Modifying a stack For any of these processes always check your Source stack and destination stack names Stack menu here you can select an individual plane add or remove planes The most useful command is keep planes This allows you to select the planes you wish to keep and save them as a new tiff stack to work on analyse This is particularly helpful when you want to analyse large files Select keep planes Set the destination folder and copy selected Set the range you want every 1 3 etc Set the first and last Set the destination folder and copy selected Click select planes in range Click Apply Making a Movie Under stack menu click make movie a Select the source stack b Select the frame rate c Select the planes required in menu and then click Select Planes in Range d Double check you have the planes you want by scrolling through the Check Save window If not click clear all and repeat steps c and
20. r each object E Log summary data on single line 16 bit intensity matches object number E Use legacy parameter order 10 To see the results click on the object data tab Click on the configure log and open log Highlight the region It appears yellow in the image 5 Integrated Morphometry Analysis Source siControlOmin 3_MIP tf green e Segment using mask Mask None E siControlOmin 3_MIP tif green 100 Measurements Preferences View Object data Summary SAC Cr meme go T Display mode Epe PA ET E Lg af z ER 7 A wos hehe et th y Current Open Log _ Configure Log of N EA x OTF ce ARTE Accumulated Object Log Not Open Object Total area Area Average Total Intensity Std Radial Shape factor Length Breadth Fiber length Fiber breadth Texture 1 3002 000 2870 000 82 406 236505 000 31 205 26 783 0 051 116 503 85 186 423 374 6 779 2 931 3 141093 000 91987 000 80 826 7434935 000 20 906 210 767 0 016 686 997 658 798 5185 294 17 740 2 349 282 2557 000 2262 000 70 055 158465 000 10 661 48 454 0 028 194 551 52 690 529 276 4 274 5 097 317 10253 000 8739 000 80 304 701779 000 22 265 77 743 0 025 274 213 169 872 1122 587 7 785 3 079 441 3394 000 3115 000 81 650 254340 000 22 234 30 002 0 106 144 506 59 793 306 596 10 160 2 416 622 1462 000 1396 000 73 822 103055 000 15 034 19 830 0 072 84 214 40 687 246 267 5 669 3 530 650 5370 000 4599 000 75 549 347449 000 18
21. s useful for separating two close cells or objects Unsharp mask removes haze and sharpens an image shifts the greyscale range to emphasise weaker objects When using filters it is important to record the pixel number and or kernel type used as this should be included in your methods for a paper Simple Analysis Using the Measure menu These provide statistical distance and morphometric analysis of objects or regions Calibrating Images Calibrate distances Use This command will calibrate pixel number to distance and is required for any size tracking or counting analysis Requires 16 bit tiffs and the type of objective used A Calibrate Distances To 2 as ici NTGLI2_Vim_Ecad_rd1_w3FL AFS6S image Calibration 1 peepee Click on Calibrate Distances pixel pixel Calibration Last loaded saved calibration file Name x Y Units Im Select the objective used click apply None 1 0000 1 0000 Pixels x 5x Obiective 20383 2 0383 um X 1 0182 1 0182 um xl Liew 20k Objective 0 5102 0 5102 um gt e 25x Objective 0 4008 0 4008 urn gt Calibration 7 0 9300 0 9300 urn gt 40 oil 0 2567 0 2567 um 3 Calibrate by Region Load from File Save to File Apply To Al Open images Close Calibrate Greyscale Used to calibrate the greyscale intensity to a known value such as ion concentration for calcium imaging Thresholding Thresholding is required to identi
22. sification ey easton intensity 1000 IUT TATTO 5 phase G2 4N 0 q 2000 s 00 H e finty a ert Mitotic classification Click preview modity the settings Mitotic specific staining DNA average intensity Source image DNA content image tn 1 3 w1FL DAPI Minimum average intensity 100 vevel Preview Configure the logs So graylewels Apoptotic classification Click apply feel Cell Count 5000 10000 15000 20000 25000 30000 Integrated Intensity x1000 15 Count nuclei The same as the first stage of cell scoring E Count Nuclei Source image No Applicable Images Adaptive Background Display result image No Applicable Images ial Parameters Approximate min width 25 Approximate max width 100 Intensity above local background 4000 Neurite Outgrowth Works best with a nuclear stain image but can be used without Requires a stack of tiffs where each plane is a timepoint The images should be calibrated TY Neurite Outgrowth keea Neurite image dmso 1 racking calibration of j j i piels to pm Select the stack and illumination types Z Display resut image EE Neurite i es ratio 1 1 Illumination C Auorescence Transmission f Cell bodies Define the approximate width and grey scale of your ae ET m 30 pice cells using region tools as for cell scoring onn a 20 ierd Minimum area 15 pm 15 pixels Nuclear stain optional If you have a nuclear stain sele
23. the image i d Montage Planes 100 Width 1392 Montage Width 12568 Height 1040 Montage Height 12535 Fill Order Horizontal Vertical Rows O ZigZag Horizontal O ZigZag Vertical Columns Zoom Stitch images Width of separator line 5 lt Color for blank frames in the montage Draw separator line between frames Draw separator line around image Draw sequence number Don t use the stitch command here unless you are stitching together images Stitching If you have used an automated stage to acquire images you can stich them together here Simply check the stich images button and select the correct image overlap It s important to remember which way the stage moved when you were collecting your tile because you will need to ensure that either ZigZag Horizontal or Zig Zag vertical are selected If you are stitching you don t want separator lines etc so deselect these boxes C7 Montage Stack Planes 100 Width 1392 Montage Width 12566 Height 1040 Montage Height 12535 Fill Order Horizontal O ZigZag Horizontal O ZigZag Vertical Columns Stitch images Width of separator line 5 Color for blank frames in the montage Draw separator line between frames Draw separator line around image Draw sequence number 2 Preparing an image for analysis Make sure your files are 16 bit tiffs Before most forms of analysis you will need to re
24. und point such as a dust mark Click create It will create an image where each plane is represented as a line Kymograph Image Ef Untitled Source Stack dmso tracking 1 Average Maimum Minimum Background Subtraction None Average Minimum Median Result Kymograph Size is 1363 36 12 3 Complex Analysis Using Apps Metamorph contains several apps which put together several steps to make more complex measurements They are all under the apps menu and are very simple to use Journal Apps Window Help Cell scoring Multi Wavelength cell scoring Requires 16 bit tifs of tissue or cells stained with multiple wavelengths ONE OF WHICH MUST BE A COMPLETE NUCLEI STAIN SUCH AS DAPI Tifs should be background subtracted and may be calibrated if size or area is required Use To count cells with multiple stains eg total cells in a section and those which are c fos positive Choose cell scoring for 2 wavelengths in or multiple wavelength cell scoring for 3 or more Open your images E Cell Scoring oo E zz Select the image with the nuclear stain All nuclei as Wavelength 1 WStatte image No Applicable Images Adaptive Background a Display result image Ef Segmentation Correction Measure the minimum and maximu n ee syster width of your nuclei using the line tool Approximate min with Hm and add slightly higher and lower Approximate max width 25 um values in t
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