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Ideas Version 6 Manual

1. 200 Understanding the Shape Features Elongatedness Feature Elongatedness is the ratio of the Height over Width of the object s bounding box See also Width Feature See also Aspect Ratio Feature and Shape Ratio Feature for other shape ratios Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightWidth Thickness min Length ss max l ess min inor Axis ajor Axis Application Examples Measure object shape properties to differentiate between long and narrow versus short and thick objects Quantify the roundness of the morphology mask Identify single cells and doublets Cell classification based on shape change Identify recently divided cells in mitosis 201 Chapter 4 Lobe Count Feature The Lobe Count feature counts the number of lobes in a cell It is determined based on the maxima of the weighted Symmetry features The feature reports the values 1 2 3 or 4 If an object does not have a high value for Symmetry 2 Symmetry 3 or Symmetry 4 it is reported as 1 for no lobes An example is shown below See also Symmetry 2 3 4 Features Symmetry Low Low High Low High Single Focused Cells aaieisisiaisix Application Example Used in cell classification studies Also used to differentiate small round cells from small square cells of similar
2. Application Examples Counting parasites Counting phagocytosed particles FISH spot counting Counting punctate spots in images Used in the Spot Wizard 2014s Understanding the Texture Features Std Dev Feature The Std Dev feature describes the overall distribution of pixel intensities The Std Dev is the standard deviation of the pixel intensity values in the mask The Std Dev value provides an indication of the texture or complexity of an object The following illustrates that apoptotic cells AnxnV positive exhibit higher Std Dev values in the darkfield channel scatter and higher brightfield Modulation values than non apoptotic cells AnxnV negative Brightfield Scatter AnxnV_7AAD Live Cells with low Scatter Std Dev and BF_Modulation Application Example Quantify intensity variation within a mask Distinguish apoptotic and necrotic cells 215 Chapter 4 Understanding the Signal Strength Features Signal Strength features include the following Bkgd Mean and Bkgd StdDev features describe the background of the image Intensity and Raw Intensity features quantify the intensities in the region of interest Raw Max Raw Min Raw Mean and Raw Median Pixel report single pixel values in an image Max Min Mean and Median Pixel report background subtracted single pixel values in an image Saturation Count and Saturation Percent quantify the saturated pixels
3. Understanding the Size Features Height Feature Using the bounding rectangle Height is the number of microns of the longer side and Width the shorter side See also Elongatedness Feature Application Example These features can be used to separate rectangular shaped objects For curved objects measurement is more accurately obtained using the thick ness features 173 Chapter 4 Length Feature Length measures the longest part of an object Unlike the Major Axis feature Length can measure the object s length even if it folds to form a cashew banana or dough nut shape where in many of these cases the major or minor axis features would not be able to differentiate these with true circular shaped objects with no hole This feature is based on an input mask and is sensitive to the variation of the input mask shape Selecting an input mask that can accurately capture the object shape is important See the Shape Ratio Feature Table of Base Features by Category and Thickness Max Feature for more information Thickness max Length em a Shape ratio Thickness min Length Thickness min afite Understanding the Size Features Major Axis and Minor Axis Features The Major Axis is the longest dimension of an ellipse of best fit The Minor Axis is the narrowest dimension of the ellipse of best fit See the Aspect Ratio Feature for more information inor Axis ajor Axis Applicatio
4. 279 Index Chapter Appendix A compactness 200 contrast 208 create combined 117 create multiple 115 create new 114 delete 118 diameter 172 elongatedness 201 Feature Finder Wizard 30 find a feature 118 flow speed 245 gradient max 210 gradient RMS 211 height 173 181 intensity 219 internalization 238 lobe count 202 major and minor axis intensity 176 major axis and minor axis 175 max contour position 190 min pixel 224 modulation 213 object number 246 object per mL 247 object per sec 248 overview 159 perimeter 177 280 Index raw intensity 225 raw max pixel 226 raw mean pixel 227 raw median pixel 228 raw min pixel 229 saturation count 230 saturation percent 231 shape ratio 203 similarity 239 similarity texture R3 235 size 170 spot area min 178 spot count 214 spot distance min 192 spot intensity min 232 std dev 215 symmetry 2 204 table alphabetical list 162 table by category 164 thickness max 179 time 249 valley x andy 193 viewing 114 width and height 173 181 without QI 169 File name extensions 5 281 Chapter Appendix A Fluorochromes table for IS100 61 table for ISX 12 channel system 61 table for ISX 6 channel system 61 Focus building block 47 graphs copy and paste 102 Graphs apply or remove region 102 creating 95 creating regions 99 defaults 9 135 legend 98 moving 99 printing 156 resizing regions 101 Statistics 97 zoom
5. MC It is computed using the normalized cross correlation between the two input images Application Examples Used as amask independent measure of similarity between two images JOA Chapter 4 Understanding the System Features The system features do not require a mask 242 Understanding the System Features Camera Line Number Feature The Camera Line Number feature returns the camera line number values This fea ture is obtained from INSPIRE Application Example Used in objects per mL feature eda Chapter 4 Camera Timer Feature The Camera Timer feature returns the camera timer values that are in ticks This fea ture is obtained from INSPIRE Application Example Used in Time feature 244 Camera Timer Feature Flow Speed Feature The Flow Speed is the calculated flow speed in mm sec of the object The Flow Speed feature is the speed of flow of the cells It is obtained from INSPIRE It should be very consistent across all cells ina file Application Example Determine consistency of flow AG Chapter 4 Object Number Feature The Object Number feature denotes the serial number of a cell in a file Application Example Reference an object ina file 246 Camera Timer Feature Objects ml Feature The Objects per mL feature returns the object concentration with respect to local vol ume Application Example Monitor t
6. Next Step Gate mitotic cells The condensed DNA cells scatter plot of the brightfield Contrast versus the Area of the thresholded nucleus is added to the analysis area Gate on mitotic events with low brightfield Contrast The final 3 plots are shown below Gi QA RSE Fz pEr OBa 83 QAR CANNE 150 annel 6 8 FA R amp Area _Threshold MO6 Char The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu a36 Guided Analysis Co localization Wizard The co localization wizard will guide you through the process of creating the features and graphs to measure the co localization of two probes in any population of cells you identify Co4ocalization To begin double click on Co localization Follow the instructions to analyze your file Step 1 Select the co localization image channels From the drop down menus pick the two image channels that contain the co local izing probes Step 2 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and con
7. leftmost down arrow button on the toolbar Use the Boolean AND or OR operator m Use the AND operator to include only the pixels that are in both of the original masks Use the OR operator to include the pixels that are in either one of the original masks Select all pixels that Use the Boolean NOT operator are not in the original co The NOT operator specifies which mask will not be mask used Affect the order of Use the parentheses toolbar buttons operations c gt Remove an item Click the left arrow button on the toolbar from the end of the definition 4 Add masks and Boolean logic to the definition as needed 5 Click OK to add the definition to the Masks list 6 Click Close Viewing and Editing a Mask To view a mask definition 1 Select Analysis gt Masks The Mask Manager window appears K Mask Manager Masks Name 01 M05 Definition M06 M01 New a 2 Click a mask in the Masks list to view the definition in the Definition area 3 Click Close To edit a mask function 29 Chapter 3 5 In the Mask Manager window select the mask that contains the function you want to edit Click Edit Remove the definition for the combined mask using the back arrow tool as needed r Or click the Function button on the toolbar for a function mask The Define Mask Function window appears Function Click OK when finished E
8. lt Ch2 a j NFkB Sort Order BF O Alphabetical Category DRAQS Select masks KS ES SYS SYS SES KS SES KS ESTES Clear Selected Clear Selected 5 Any list can be cleared by clicking the Clear Selected button 6 When finished click Add Features to add the new features to the list 7 Confirm the features in the next window oot Confirm Feature Creation The following features will be created if they do not already exist Do you want to continue Bright Detail Intensity R3_M04_Ch4 Bright Detail Intensity R 7_MO04_Ch4 Contrast_M04_Ch4 Gradient Max_M04_Ch4 Gradient RMS_M04_Ch4 H Contrast Mean_M04_Ch4_5 H Contrast Std_M04_Ch4_5 H Correlation Mean_M04_Ch4_5 H Correlation Std_M04_Ch4_5 H Energy Mean_M04_Ch4_5 H Energy Std_MO04_Ch4_5 H Entropy Mean_M04_Ch4_5 H Entropy Std_M04_Ch4_5 H Homogeneity Mean_M04_Ch4_5 H Homogeneity Std_M04_Ch4_5 Delete Selected Features 116 Using the Feature Manager 8 Delete any features you do not want to calculate 9 Click OK when finished The new features are added to the list in the feature man ager 10 Close the Add Features window 11 Close the Feature Manager The new features are calculated when the feature manager closes To create a new combined feature A combined feature uses one or more single features created by a mathematical expression 1 Click New in the Feature Manager The right hand area of the Feature Manage
9. Note The tagging mode remains open until you click Close and as long as the Image Gallery is in tagging mode you cannot create resize or move any regions on the graphs 127 Chapter 3 Creating a tagged population from a file of object numbers You can use a comma separated text file of object numbers to create a tagged pop ulation Select Create Tagged Population from File under the Tools menu Create a Tagged Population From a File Select a comma separated text file that contains the object numbers for the population es Create the tagged population Population name Dark Mode Color Light Mode Color Symbol Browse for the file Name the population select the color symbol and click OK 128 Using the Region Manager Using the Region Manager The Region Manager provides a central place for defining the display properties names and positions of existing regions Regions can be deleted in the Region Man ager tool Regions are drawn on graphs to create new populations based on the physical loca tion of objects on a graph and to compute statistics Tools for drawing regions are found on the Analysis Area toolbar See Creating Regions on Graphs for more infor mation To open the Region Manager and view the region definitions 1 Select Analysis gt Regions or right click a graph and select Regions The Region Manager window appears Click on the region in the
10. Pei Guided Analysis Feature Feature name and description Mask used category The number of lobes in a cell Also see Symmetry ooo Shape Ratio Feature Obiect The ratio of Thickness Min Length features J Symmetry 2 3 4 Features These three features measure the tendency of the object to have a OR single axis of elongation a three fold and a four fold variation of the Ee shapes Size basedFeaturesareinmicrons S SiS Area Feature Object Thresh Height Feature Based on a bounding rectangle the Width is the smaller side and Object the Height is the longer side of the rectangle Length Feature Measures the longest part of the mask Major Axis and Minor Axis Features Describes the widest part of the mask and the narrowest part of the Object mask respectively Perimeter Feature Object Thresh Describes the longest width of the mask Describes the shortest width of the mask Width Feature Based on a bounding rectangle the Width is the smaller side and Object the Height is the longer side of the rectangle ato te granuiarty or completi ofthe mage Texture A cate the granularity or complexity of the image Contrast Feature Enumerates changes of pixel values in the image to measure the focus quality of an image H Texture Features Channel mask Measures Haralick texture features Granularity settings 1 5 15 19 Modulation Feature Measures the intensity range of an image normalized between 0 and 1 Spot Count Featu
11. Spot Intensity Min is used when counting spots Note that when the name includes Raw this means that there is no background subtraction 216 Understanding the Signal Strength Features Bkgd Mean Feature The Bkgd Mean feature estimates the average camera background level in an image by taking the mean of the background pixels Application Examples Obtain estimate of the mean camera background level Compute background subtracted pixel values in other feature com putations 217 Chapter 4 Bkgd StdDev Feature The Bkgd Std Dev feature estimates the standard deviation of the camera back ground level in an image computed using the background pixels Application Example Obtain estimate of the camera background noise 218 Understanding the Signal Strength Features Intensity Feature The Intensity feature is the sum of the background subtracted pixel values within the masked area of the image Brightfield Scatter CD45 FITC CD14 PE DRAGS a o amp a za a E gz Intensity_CD45 Application Examples Quantify relative levels of fluorescence between cells and within different regions of the same cell Immunophenotyping Cell cycle analysis Protein expression Protein activation 219 Chapter 4 Max Pixel Feature The Max Pixel feature is the largest value of the background subtracted pixels con tained in th
12. Windows XP operating system and may vary slightly from those created using other operating sys tems The Amnis ImageStream cell analysis system is for research use only and not for use in diagnostic procedures Technical Assistance Amnis Corporation 645 Elliott Ave W Suite 100 Seattle WA Phone 206 374 7000 Toll free 800 730 7147 www amnis com Table of Contents Patents and Trademarks _ 00 00 2 0 cece eec cece cece cce ce eeeceeeeeeeeeeeneeees ii End User License Agreement 002o o oo eee ccc cece cee cec eee ececeecececeececeeeess iii Disclaimers sacar uv aiinera da tids e e aa os pan dened edd anTianlddinmadiis inane vi Technical Assistance So cci6e rite settle a td else a telah Yh hal eG lee vi Table of Contents e neen ro e n E Eeo Aeaaeai vii Chapter 1 Introduction 0 0 aaaaaaaaoaeaeneaeeeaenenerrenrrererrrrenrnerrrereeena 1 Welcome to IDEAS 6 0 sirere iiet raesade paranas g dse 2 How to use this manual pists asst ela htt Se it that atelier an ALB el Ae 2 What s New in IDEAS 6 0 022 0 c ccc ccc c cece eee ecc een eeneeeeeee 3 Setting Up the IDEAS Application 0 00 0 ooo ce cece eee cece 4 Hardware and Software Requirements 0 0 0 0 00 cece cece eee cececececece eee eeeees 4 Installing the IDEAS Application 0 0 0 0 00 002 c cece ec ce cece cece ececcecececececeeeeees 4 Setting Your Computer to Run the IDEAS Application 0 0 0 0 00000000 2 222 5 Vie
13. 1ng 15_1_8 ciF My Recent 0 1ng 30_6_13 ciF Documents 0 1ng 45_11_18 ciF 0 1ng 60_16_23 ciF E 0 1ng 75_21_4 cif 0 1ng 90_26_9 cif 10ng 15_3_10 ciF 10ng 30_8_15 ciF go 10ng 45_13_20 cif 10ng 60_18_1 ciF 10ng 75_23_6 ciF 10ng 90_28_11 cif 33 1000ng 15_5_12 ciF d 1000ng 30_10_17 cif My Computer 1000mg 45_15_22 ciF 1000ng 60_20_3 ciF 1000ng 75_25_8 cif 1000ng 90_30_13 ciF My Documents My Network Places File name 00n 29 Default daf Files of type In the next window you will e Choose a template Name the output file Opening 10ng 60_18_1 cif To use a custom template for analysis Select a template or data analysis file ast daf Name the analysis file to be created Data analysis file 10mg 60_18_1 dat 3 Click the folder next to Select a template or data analysis file ast daf and choose the template to use for analysis If left blank the IDEAS application will use a default template However it is useful to create and save your own tem plates for specific experimental procedures 4 Change the Data analysis file name if necessary The default name matches the name of the cif 54 Opening data files 5 Click OK During the opening of a cif file the IDEAS application calculates the values of the features that are defined in the template you selected The progress is shown by a p
14. Chapter 4 Raw Max Pixel Feature The Raw Max Pixel feature is the largest value of the pixels contained in the input mask RTX FITC CD45 PE Brightfield This image is saturated in the FITC channel but not in the PE channel 1e3 7 800 600 A Raw Max Pixel_Ch3 N Ss 8 200 400 600 800 163 Raw Max Pixel_Ch4 Application Examples Determine the presence of saturated events May also be used to estimate the peak fluorescence activity though the Max Pixel feature is recommended for this application Measure the maximum pixel value within the mask Identify cells that saturate the CCD Saturation Count feature can also be used for this application 226 Understanding the Signal Strength Features Raw Mean Pixel Feature The Raw Mean Pixel feature is the mean of the pixels contained in the input mask This is computed as Raw Intensity number of pixels Application Example Estimate the raw average fluorescence activity This feature is less rel evant that the Mean Pixel feature 227 Chapter 4 Raw Median Pixel Feature The Raw Median Pixel feature is the median of the pixels contained in the input mask Application Example Estimate the raw average fluorescence activity that is robust to outliers This feature is less relevant than the Median Pixel feature 228 Understanding the Signal Strength Features Ra
15. Cybernetics Vol SMC 3 1973 pp 610 621 212 Understanding the Texture Features Modulation Feature The Modulation feature measures the intensity range of an image normalized between 0 and 1 The formula is Modulation Max Pixel Min Pixel Max Pixel Min Pixel The following example illustrates Modulation on brightfield images and Intensity of scatter in channel 1 Single focused cells ajig E D T p Intensity_SSC 3 Modulation_BF Low Modulation Low Modulation Application Example Quantify image quality and characterize contrast and texture in cells 213 Chapter 4 Spot Count Feature The Spot Count feature provides the number of connected components in an image The connected component algorithm examines the connectivity of each pixel based on whether this pixel is connected to a particular spot or the background In order to count the number of connected components the mask input is very important See Spot Mask Peak Mask and Range Mask for information on masking spots See also Spot Area Min Feature Spot Distance Min Feature andSpot Intensity Min and Spot Intensity Max Features for more information The following figure illustrates the application of Spot Counting to quantify parasitic infection of Babesia in erythrocytes by staining nuclei with YOYO green Brightfield Babesia YOYO1 Single Parasite Two Parasites Three Parasites
16. ER Select object to export Export to A l Clipboard File OK Cancel Select the object to export in the drop down menu Select to Export to either the Clipboard or File Click OK Paste into desired application 153 Chapter 3 Creating TIFs From Population for Export The IDEAS application allows users to create separate TIF files for channel images for every event in that population The exported TIF files can be opened in image viewing applications that support 8 bit tif format for display or 16 bit tif format for anal ysis To create TIFs From Population for Export 1 ook WN On the Tools menu click Export tif Images The Create TIFs From Population window appears f Create TIFs From Population S Select population a 092011 X101 Unstimulated_12temp cif lik All Select Channels TIF Settings File name prefix Bit Depth 8 it for display 16 bit for analysis Pixel Data padded for display 5 raw for analysis Select the population and channels Type a prefix for the TIF file name Select the bit depth Select padded or raw Click OK ATIF file is created for every selected channel within the selected population 154 Printing Data Printing Data The IDEAS application provides color mapping from the dark mode that you see in the Analysis Area to a light mode that has a white background for the printing and exp
17. Edit Statistic Table opens a Statistics Properties window to enable changes to multiple column statistics 6 Toaddasingle statistic column select Insert Column 111 Chapter 3 7 Select Edit Column to make a change 8 To delete a single column right click on that column and select Delete Column 9 Select Delete All Columns to clear all statistics 10 Order Columns places the columns in default order 11 Copy Statistics copies the selected rows of the table in a text format that can be pasted into other programs such as Excel 12 Copy Statistics Transposed copies the selected rows of the table and trans poses the data so that when pasted into other programs such as Excel the rows become columns 112 Using the Feature Manager Using the Feature Manager This section describes how to create and delete features and to create multiple fea tures by selecting categories The only new feature options for FlowSight basic files without QI are combined features The following subsections cover this information Viewing feature definitions Creating New Features with the Feature Manager Ranking features by discriminating power Overview of the Feature Manager The IDEAS application defines a set of base features that you can use to create fea tures for each object To do so you use the object s mask and or its channel images After a feature has been created and its value calculated for all cells you can plot the fe
18. Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an already existing population Step 2 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 3 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip Next Step Select the spot image channel From the drop down menu choose the image channel for the spot counting 45 Chapter 3 Next Step Assign truth populations From the drop down menus select two truth populations one with high and one with low spot count To create the trut
19. IS100 NFkB Translocation Dose and Time 4 0 ifs 10ng 75_23_ 6 nf C Users sfriend Desktop IS 100 NFkB Translocation Dose and Time 4 0 rifs 10ng 90_28_11 nif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 15_5_12 f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 30_10_17 1f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 45_15_22 rf 1 C Users sfriend Desktop IS 100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 60_20_3 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 75_25_8 if C Users sfriend Desktop IS 100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 90_30_13 nf i Output Files C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 0ng_2_9 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 15_1_8 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 ifs 0 Ing 30_6_13 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 45_11_18 cif i C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 60_16_23 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 Ing 75_21_4 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_26_9 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 ifs 10ng 15_3_10 cif C Users sfriend Desktop
20. IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 30_8_15 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 10ng 45_13_20 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 60_18_1 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 10ng 75_23_6 cif N C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 ifs 10ng 90_28_11 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 15_5_12 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 30_10_17 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 45_15_22 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 60_20_3 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 75_25_8 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 90_30_13 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 0ng_2_9 daf C Users sfriend Desktop S100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 15_1_8 dat C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 30_6_13 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 0 1ng 45_11_18 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 Ing 60_16_23 daf C Users sfriend Desktop IS100 NFkB Translocati
21. Image 1 Channel 1 zj Image 2 Channel 1 3 Select the mask and or image that you want 4 Enter a unique feature name or click Set Default Name The default name is the name of the base feature followed by the name of the mask and name s of the image s 5 Click OK to add the new feature It appears in the Features list on the left side of the Feature Manager 6 Click Close Note When you close the Feature Manager the IDEAS application calculates values for the new features These calculations may take several minutes depending on the number and complexity of the new features and the size of the image file To create multiple features 1 Click Add Multiple Features in the Feature Manager 2 Sor the feature list alphabetically or categorically 3 Select multiple base features and masks 115 Chapter 3 4 Select one image or check the box to create for all channels using default masks and images i Add Features Select base features Select feature inputs S F Texture a C Create for all channels using default masks and images Bright Detail Intensity R3 Bright Detail Intensity R7 Contrast MOT Al Gradient Max M02 Gradient RAMS M03 H Contrast Mean M04 C M05 H Contrast Std MOE H Correlation Mean MC H Correlation Std NMC H Energy Mean None H Energy Std H Entropy Mean H Entropy Std H Homogeneity Mean 3 H Homogeneity Std Select image MJ H Variance Mean ssc TU Viarinnnn Cha
22. Naaienntlddandeaeewbsiebete 91 Example of Creating a Mask ge ccc y pecd ceed oc hea eee Bed idee ele eae aiddek 92 Overview of the Analysis Area _ 2 2 0 0 0 0 0 0o ecco cc ce ccc cece cece cee eeeeceees 94 Analysis Area LOGS 2 has Iva ete 6 92 iad hea sve e 2c en ol ot a Weise aah ce eed dae as 94 Creating Graphs Rea eee ee Ne RA ee i ROA eRe ARI ne eS 95 Creating Regions on CGitapHis a 522 fc ween xs stun arn iitissasbhuesaieedaduh cuts Mlvackhecdiedicern 99 Analyzing Individual mages 240 seh ete blues audi Set cele cies ees 103 Viewing the Object Feature Values a5 a2 alias Se ar 22 dined dda uasbee be eianian ews 108 Adding Text to the Analysis Area 20 2 0 0 00 00 0 0 0 2 cc cece cece sec ecececececececeseeneees 110 Population Statistics 5 x2 bie se ten ehearinedalss en e dl tthe S5 22 sleet Ta cadan ie 2 111 Using the Feature Manager 2 0 00 0 0o ec cc cece cece e ces ec ccc eceececeeeees 113 Overview of the Feature Manager 2 0 0 0 0 0 0 0 ce cece ec ec ec eecececececeseceeeees 113 Viewing feature definitions 2 s1s4 ecn creo seh ees tc ees anatase a eaes cere tt nas 114 Creating New Features with the Feature Manager 0 0 0 0 02022ceeeee ee 114 Ranking features by discriminating power 0 0 0 0 0 0 0ececececececeeeeeeceee 118 Using the Population Manager 0 0 0 0 co cece ec ce ec ee cece cece eee 124 Creating Tagged Populations 2 0
23. Select populations SIE Sit Al 3 N R1 R2 3 0 R3 New Raw Image File rf New Compensated Image File cif In the Select populations list select the populations that you want to include in the new data file s Ctrl click to select multiple populations To create a rif file select the New Raw Image File rif check box the pop ulation name is used as a default You may enter a new name To create a cif file select the New Compensated Image File cif check box the population name is used as a default You may enter a new name Click OK If you created a new cif file you can choose to load it When loading the cif file the application will prompt you for the template six Chapter 3 Viewing Sample Information All of the information associated with an IDEAS file such as the collection infor mation camera settings and corrections is saved within IDEAS and can be viewed in the Sample Information window To open the Sample Information window 1 Goto Tools gt Sample Information to open the window Information for the open data file will be loaded You can browse for a data file by clicking on the folder You can open the Sample Information Window for any of three file types rif cif or daf 2 Select a Tab to see the information for each heading 3 Click Print to print a report of all of the sample information Tip You may click on the folder and browse for a file to view the sa
24. Shape ratio Thickness min Length Thickness min 179 Chapter 4 Thickness Min Feature Thickness Min measures the smallest width of an object This feature is based on an input mask and therefore sensitive to the variation of the input mask shape Select ing an input mask that can accurately capture the object shape is important See also Thickness Max Feature Length Feature and Shape Ratio Feature for more infor mation Thickness max Length CG Ginio i Shape ratio Thickness min Length Thickness min 180 Understanding the Size Features Width Feature Using the bounding rectangle Width is the number of microns of the smaller side and Height the longer side See also Elongatedness Feature Application Example These features can be used to separate rectangular shaped objects For curved objects measurement is more accurately obtained using the thick ness features 181 Chapter 4 Understanding the Location Features Location features include Angle Angle Intensity Centroid X Centroid Y Centroid X Intensity Centroid Y Intensity Delta Centroid X Delta Centroid Y Delta Centroid XY Max Contour position Spot Distance Min Valley X and Valley Y 182 Understanding the Location Features Angle Feature Angle is the angle of the major axis from a horizontal plane in radians Application Example Identify the orientation of an image relative to th
25. Show or Hide Saturation Color respectively One or the other will appear depending on the current state To copy or save images for use in reports 1 Inthe Image Gallery right click an image that you are interested in A menu appears Add Image to Analysis Area Show Masks Color On Show Saturation Color Copy Save Image Copy Save Object Images Copy Save Gallery Column Copy Save Gallery e Tocopy or save the single channel image to the Clipboard choose Copy Save Image To copy or save all of the channel images of one object choose Copy Save Object Images e Tocopy or save the single channel image for all of the displayed images to the Clipboard choose Copy Save Gallery Column e Tocopy or save all the visible images in the Image Gallery choose Copy Save Gallery 87 Chapter 3 Overview of the Mask Manager This section contains the following subsections which describe how to create edit and delete a mask Creating New Masks with the Mask Manager Overview of the Mask Manager A mask defines a specific area of an image to use for feature value calculations The IDEAS application contains a Mask Manager for viewing existing masks and cre ating new ones This option is not available for basic FlowSight files without the Quantitative Imaging QI upgrade When the IDEAS application loads a rif file the application creates a segmentation mask for each channel image and stores the mask along with
26. Spot Intensity Min and Spot Intensity Max Features The raw intensity not background subtracted of the dim mest component spot No No See also Spot Count Feature Spot Distance Min Fea ture and Spot Area Min Feature Com Difference of intensity measurements between parison masks or pixels Bright Detail Similarity R3 Feature Measures the correlation of the bright details between No image pairs Intensity Concentration Ratio Feature Given two masks the ratio of the intensity inone mask No to the total intensity in both masks Internalization Feature The ratio of the intensity inside the cell to the intensity of No the entire cell Similarity Feature The Similarity is a measure of the degree to which two images are linearly correlated pixel by pixel within a masked region XCorr Feature The XCorr is a measure of the degree to which two No images frequencies are cross correlated System features do not require a mask and tend to deal with system wide metrics Camera Line Number Feature No Yes es 167 Feature name The clock rate in KHz This is relative time Flow Speed Feature The calculated flow speed in mm sec Object Number Feature Y Yes The sequence of objects Objects ml Feature A local concentration of all objects per ml Note to get objects per ml of a population use the sta No Yes es tistic Concentration Objects sec Feature A local concentration of number of obje
27. Start Analysis button or by choosing Wizards from the Guided Analysis menu The wizards selection screen will appear once the data file is open If you have an open data file and want to access this wizard choose Wizards from the Guided Analysis menu or click the wand icon Feature Finder To begin double click on Feature Finder Step 1 Gate cells in best focus A histogram of the brightfield channel Gradient RMS values for the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an already existing population Click Next Step 2 Gate single cells A scatter plot of the brightfield Area versus Aspect Ratio for the population chosen in step one has been added to the analysis area Single cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by a light green line and the image next to it in the image gallery is not it s neighbor in the plot The images are pre sented in the order of acquistion You may choose an already existing pop ulation Click Next Step 3 Select subpopulation marker s Choose one or two channels you wish to use to identify popula
28. TOTAL LIABILITY WILL BE LIMITED TO 100 INNO EVENT WILL AMNIS BE LIABLE TO YOU FOR ANY SPECIAL INCIDENTAL EXEMPLARY PUNITIVE OR CONSEQUENTIAL DAMAGES INCLUDING LOSS OF DATA BUSINESS PROFITS OR ABILITY TO EXECUTE OR FOR THE COST OF PROCURING SUBSTITUTE PRODUCTS ARISING OUT OF OR IN CONNECTION WITH THIS AGREEMENT OR THE EXECUTION OR PERFORMANCE OF THE SOFTWARE WHETHER SUCH LIA BILITY ARISES FROM ANY CLAIM BASED UPON CONTRACT WARRANTY TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE AND WHETHER OR NOT AMNIS HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH LOSS OR DAMAGE THE FOREGOING LIMITATIONS WILL SURVIVE AND APPLY EVEN IF ANY LIMITED REMEDY SPECIFIED IN THIS AGREE MENT IS FOUND TO HAVE FAILED OF ITS ESSENTIAL PURPOSE 8 U S Government End Users The Software and Documentation are commercial items as that term is defined in FAR 2 101 consisting of commercial computer software and commercial computer software documentation respectively as such terms are used in FAR 12 212 and DFARS 227 7202 If the Software and Documentation are being acquired by or on behalf of the U S Government then as provided in FAR 12 212 and DFARS 227 7202 1 through 227 7202 4 as applicable the U S Government s rights in the Software and Documentation will be only those specified in this Agreement 9 Export Law You agree to comply fully with all U S export laws and regulations to ensure that neither the Software nor any tech
29. This wizard is automatically run when you use the other analysis wizards or the Open File wizard It is also available to run in any open data file from the Guided Anal ysis menu or from the wizard icon This wizard will set the image display mapping for the channel images you select and create a view of selected images Brightfield and scatter images will be automatically selected To begin select wizards from the Guided Analyis menu or click the wizard icon in the analysis area toolbar oe The Wizards window opens Wizards Select the wizard to use for analysis Name Description Open File Opening ImageStream data files Display Properties Automatically sets image display properties Moonie Creates an analysis template for identifying apoptotic events based poppers on brightfield and nuclear morphology g Cell Cycle Mitosis Creates an analysis template that distinguishes mitotic and apoptotic events Double click on the Display Properties option and follow the instructions The Display Properties adjusts the mapping of the pixel intensities to the display range for optimizing the display and creates a view that includes the chosen chan nels This is for display only and does not effect the pixel values For more infor mation on image display see Setting the Image Gallery Properties EJ Display Properties Wizard Tip Brightfield an Tip2 If you display prope IT Chap
30. Wizard n abaa ae a a a a a ET mtd dari E 45 Building Blocks 02 55 telethon als 47 Advanced Analysis l a aaa ta alee oe dds heen treed eset Ue crete ets 49 Opening data files l a aaan aaia naianei anaana iaaea aannaaien 50 Saving Data FOS cesteiro i eie aa eaii E 56 Overview of Compensation _ 2 2 0 0 ooo eee cee aaor 12111 58 Merging Data Files anaana aaau naoa oanade aroan aaao irian necetebice 69 Creating New Data Files 2222 0 0 0 0 ooo cc cece ec cece cece 0000100011010 71 Viewing Sample Information _ 0 00 0 2000 0 cece cee cc cece cece cee eec eee eees 72 Batch Processing ni ho ie cae tat A ho st ot els 74 Overview of the Data Analysis Tools 000000000000000000000000000000000000o 78 Overview of the Image GalleryUsing the Image Gallery _ 79 Overview ofthe Image Gallery is 5 cine dle Sends S ea landnn Tugs daaducds Set adnaak conltse eas 79 Setting the Image Gallery Properties 2 2 2 0 0 0 0 0 0 cece cece cece ec eceececececececeeeees 81 Working with Individual Images 0 00000 0 2c ccc eee ce cece cece ceeeecececececeeeeees 86 Overview of the Mask Manager 2 2 0 0 0 0 o coco ec ce cece cece cee eec eee eee 88 Overview of the Mask Managers 4 00520 i eiesent i St ovpn sae Feosdasdecseet ladaanl veedeeent 88 Creating New Masks with the Mask Manager 2 0 0 00 0 0 02 o eee e eee 88 Viewing and Editing a Mask aioe heen ci dentin ety hdec ob
31. Y Features The Shift X or Shift Y feature is the location of the highest cross correlation of a pair of images When two identical images are aligned perfectly the cross correlation is at it s maximum The shift X or Shift Y is the shift required to get the highest cross correlation value for the 2 images This feature is used mainly for troubleshooting cross camera alignment 19 Chapter 4 Spot Distance Min Feature The Spot Distance Min feature provides the shortest distance in microns between two spots connected components in a spot or peak mask This is one of four features that can be used to identify objects with spots that are close together dim bright or small when counting spots in an image To use these features the spots need to be individually masked such as using the Spot or Peak Mask The Spot Area Distance and Spopt Intensity Min or Max features measure properties of different spots in an image and are often used with the Spot Count fea ture under Texture For more information see Spot Area Min Feature Spot Count Feature Spot Intensity Min and Spot Intensity Max Features e Spot Area Min is the Area of spot 1 e Spot Distance Min is distance 2 in microns Spot Intensity Max is the Raw Mean Pixel of spot 2 Spot Intensity Min is the Raw Mean Pixel value of spot 3 Application Example In FISH Spot Counting these features are used to identify ambiguous spots that are located too close tog
32. area Draw a region to include the cells with high internalization The example below shows the internalization of labeled CpG red REU CAE htemsizaton_C_E4_ Cpe Low Internalization High Internalization _ For a more thorough explanation of the Internalization feature seelnternalization Featurelnternalization Feature The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 40 Guided Analysis Nuclear Localization Wizard This wizard will create an analysis template for measuring the nuclear localization of a probe in any population of cells you identify Nuclear Localization To begin double click on Nuclear Localization Follow the instructions to analyze your file Step 1 Select the translocation image channels From the drop down menus pick the nuclear image channel and the translocating probe image channel Step 2 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an already existing population Step
33. be removed during installation To install IDEAS software Download the application Setup file from your account at www amnis com View the contents in My Computer or Windows Explorer Double click Setup exe Follow the instructions until the installation process has completed BR O N Setting Up the IDEAS Application MadCap help viewer is installed and opened during installation or upgrade 6 Select the language and check box to not show this dialog again 7 Anicon appears on the desktop and IDEAS Application appears on the All Pro grams menu Setting Your Computer to Run the IDEAS Application Setting the Screen Resolution Confirm that the screen resolution is adequate for the IDEAS application To be able to view the entire application window you must set the width of the screen res olution to a minimum of 1024 pixels To set the screen resolution 1 From the Start menu select Control Panel and then click Display 2 Click the Settings tab to set the screen resolution Viewing File Name Extensions When loading a file the IDEAS application uses the file name extension to deter mine the file type It will be easier for you to distinguish raw image files com pensated image files and data analysis files from each other if Windows Explorer does not hide the extensions To view file name extensions 1 In Windows Explorer go to Tools gt Folder Options 2 Click the View tab and make sure that the Hid
34. divided by the Major Axis Yes Yes orm lg Aspect Ratio Intensity Feature M01 Ch01 The ratio of the Minor Axis Intensity divided by the Major Yes Yes x M12_Ch1i2 Axis Intensity Circularity Feature No The degree of the mask s deviation from a circle Describes the density of intensities within the object fee Elongatedness Feature The ratio of the Height Width which use the bounding No Yes M01 M12 box Lobe Count Feature Feature rrerunperaTbbes nacol AkoseeSymmetyy PO Shape Ratio Feature Symmetry 2 3 4 Features These three features measure the tendency of the object to have a single axis of elongation a three fold and a No No four fold variation of the shapes See also Lobe Count Feature Texture Texture features measure pixel or regional variation and indicate the granularity or complexity of the 165 Chapter 4 Feature name Bright Detail Intensity R3 and Bright detail Intensity R7 Features for a description of the bright detail image Contrast Feature Enumerates changes of pixel values in the image to measure the focus quality of an image Gradient Max Feature The maximum slope of the pixel value changes in the image to measure focus quality of an image Gradient RMS Feature Enumerates changes of pixel values in the image to measure the focus quality of an image M01_Ch01 M12_Ch12 M01_Ch01 M12_Ch12 M01_Ch01 M12_Ch12 M01_Ch01_ 5 M12_ Ch12_5 Modulation Feature es
35. esseet yep n te a tec in heretic fa Oo ror OSes Conair 273 3 Cae hee ROS SEE RE OES EE a a ES NER OTe PEE WU ERe ERENT ce 275 Chapter 1 Introduction Chapter 1 Welcome to IDEAS 6 0 Welcome to the IDEAS version 6 application documentation for ImageStream and FlowSight data analysis How to use this manual This manual provides instruction for using the Amnis IDEAS application to analyze data files from the Amnis ImageStream and FlowSight cell analysis systems The intuitive user interface of the IDEAS application makes it easy for you to explore and analyze data The application can quantify cellular activity by performing sta tistical analyses on thousands of events and at the same time permit visual con firmation of any individual event Furthermore you can operate the application in a batch processing mode and store specific analysis templates for either repeated use or sharing with colleagues The fastest way to put the IDEAS application to work is to first skim through this manual becoming familiar with the application s structure compensation file types and analysis tools and then use the application wizards on some sample exper imental data to begin exploring the power that the application provides This manual has been integrated into the IDEAS application to provide searchable and context sensitive help Typing F1 while in the application opens the help files What s New in IDEAS 6 0 What s Ne
36. in the experiment e If you want to clear a column double click on the channel heading If needed you can create new scatter plots by using the Analysis Area tool bar For example a 4_Intensity versus 5_Intensity plot may be useful See Creating Graphs for more information The slope of the line on the plot is the coefficient in the matrix If objects in the population exist that are outliers they should most likely be removed from the positive population within the compensation matrix by the fol lowing optional steps Matrix Coefficient Inte mm 3_Positive 80000 70000 3 80000 350000 240000 f 230000 20000 1000 Intensity_MC_Ch03 Coefficient value 9 749 Add Graph to Analysis Area Coefficient error 0 00036 1 1 T j 20000 40000 60000 80000 Close e The slope of the line on the plot is the coefficient in the matrix e If objects in the population exist that are outliers they should most likely be removed from the positive population within the compensation matrix by the following optional steps Option Remove Objects from the Population Within Step 2 of the compensation wizard double click the coefficient to display the intensity plot 64 Overview of Compensation Matrix Coefficient Intensity Plot 12_Positive 21000 15000 MC_ChO on S Intensity _ j 1 I 1E 05 2 4E 05 Intens
37. in IDEAS The following steps find the best features that distinguish changes in actin dis tribution 1 Gate single focused actin positive cells View cells of interest 2 Create the truth populations from within the cells of interest using the tagging tool Note If truth populations are in different files merge them together before begin ning When selecting truth populations choose images that represent the full phe notypic range of each truth In this example case note that the uniform actin truth population contains cells of varying shape and intensity that all have uniform actin distribution Bias introduced during the selection of truth populations will likely also bias the outcome during statistical ranking 119 Chapter 3 The following figure shows the truth populations chosen to find a feature to dis criminate uniform versus polarized actin 2_FITC 2_FITC 3 Create the Morphology and one or moreThreshold masks for the actin image 4 Create features from the Size Shape and Texture categories using the Mor phology Threshold and Default actin channel masks a Choose Features from the Analysis menu and click Add Multiple Features Features Area_Fill Threshold M02 2_Actin 50 a Feature Type Area_M01 Area_M02 Area_M03 Area_M06 Area_M07 Area_M09 Area_M11 Area_MC Area_Morphology M02 2_Actin Area_Object M02 2_Actin Tight Area_Threshold MO02 2_Actin 50 Aspect Rati
38. in the image gallery is not it s neighbor in the plot The images are pre sented in the order of acquistion You may choose an already existing population Step 3 Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot to the analysis area Click Skip if you do not wish to use this step Draw regions in the scatter plot to identify as many pop ulations as you want This step will be repeated until you choose Skip or Finish The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 28 Guided Analysis Feature Finder Wizard The feature finder wizard will guide you through the process of choosing a feature for morphological analysis when a specific application wizard is not appropriate This wizard is available once a data file is open and will guide you through choosing the focused cells then single cells then choosing subsets of fluorescent positive cells for phenotypic analysis before progressing on to choosing a feature for mor phological analysis Open a data file that contains images of both phenotypes you wish to separate Note that it may be necessary to merge two files together if the populations are in sep arate files Open the data file using the
39. legend shows the population and its associated point style and color If the graph is a histogram or overlay histogram the legend shows the population name associated color and line type To move the legend click and drag it You cannot drag the legend past the boundary of the graph panel To order the plots on a graph or change the fill and lines for a histogram 1 Right click anywhere on the graph and choose Plot Order and Properties on the graph context menu that appears The Display Properties dialog appears Moving a Graph e With any graph in the Analysis Area you can move it to another location by clicking in the center of the graph and dragging it Alternatively select the graph right click in the a blank space in the analysis area choose cut and then right click where you would like to move the graph and choose past Creating Regions on Graphs Regions may be drawn on graphs to create new populations based on the physical location of objects on a graph and to compute statistics Tools for drawing regions are found on the Analysis Area toolbar A line region may be drawn only ona his togram All other types of regions may be drawn only ona scatter plot A region can be copied to another graph in the same file or other open files Regions may also be copied from one instance of the IDEAS application to another When you draw a region on a histogram or scatter plot you create a population of objects defined by the reg
40. name for the compensation matrix file ctm and click Save 66 Overview of Compensation Save As Compensation Matrix ctm File Save in O compensation E compmatrixJune20 ctm ees HE compmatrixJune25 ctm My Recent Documents a Desktop My Network File name Places Save as type compensation matrix files ctm Note The matrix is saved as a compensation matrix file ctm file This file con tains the compensation values and can be opened later for editing To provide the values for fluorescence compensation you select a ctm file when opening a rif file See Opening a rif file for more information Preview and edit a compensation matrix A compensation matrix can be applied to a population or rif file in a preview mode for editing a matrix To open a compensation Matrix 1 Select View Edit Compensation Matrix from the Compensation menu to view edit or preview the matrix on image data Select the data file by clicking on the folder and then click Open The matrix values are displayed in a table and may be edited 67 Chapter 3 Compensation Matrix Select a compensation matrix PO_647_APCCy7 ctm ChOT ChO2 Ch03 ChO6 ChO7 ChO9 ChiO Chit Chi2 GO 0 051 0 084 0 026 o 0 002 0 017 1 oz 0 036 0 008 0 129 0 212 1 0 015 0 006 0 075 0 078 0 512 0 012 0 005 0 063 0 018 0113 0 005 0 011 0 03 0 055 01 0 005 0 004 0 069 0 009 0 019 1 0 05
41. necessary by refining creating new truth populations NOTES ON EVALUATING THE FEATURES Consider the features that produce the highest Rd If there are any intensity based features make sure that the staining was not uneven due to technical issues If it is a size feature does it make sense with what you know about the cells and biology of your experimental system Since the feature value ranges vary between features this is an approximate comparison and the result should be validated by viewing images across the feature range from the whole population 123 Chapter 3 Using the Population Manager A population is a group of objects You create populations by drawing regions on graphs by hand selecting tagging objects in the Image Gallery or on plots or by combining existing populations After a population has been defined you can view it in the Image Gallery or on a plot and you can use it to calculate statistics The Population Manager provides a central place for maintaining the display prop erties of existing populations and for creating new combined populations To open the Population Manager and view the population definitions 1 Select Analysis gt Populations or right click a graph and select Populations The Population Manager window appears Z Population Manager Populations Properties 011 X101 unstimulated_tt cif Name Al Dark Mode Color L Simple Dot gt Light Mode Color E Symbo
42. population of hand picked objects For more Tagging Tool information see Creating Tagged Populations lti New Histogram Tool Creates a new histogram New Scatter Plot Tool Creates a new scatter plot Refer to 2 Populations Statistics Creates a table to display population statistics table Object Feature Values Creates a table to display selected object feature values table A Allows user to add text notes to the Analysis Area Refer to Adding Text Tool Text to the Analysis Area Draws a horizontal line on a histogram to define a region E Line Region Tool b Rectangle Region Tool Draws a rectangular region on a scatter plot Oval Region Tool Draws an oval region on a scatter plot Draws a polygon region on a scatter plot graph Each click starts a new segment in the polygon until the entire image is double clicked to complete the region a Wizards Tool Short cut to using Wizards for guided analysis e Short cut to using Building Blocks for guided analysis ia Polygon Region Tool Building Blocks Tool Select All Tool Selects all panels in the analysis area g Tiles graphs in the analysis area after changing the size of the anal Tile Graphs Tool ysis area to fit all graphs to the new space oO Layout Tools Switches the layout of the image gallery and analysis area B Changes the background of the graphs to black or white Graph Bkgd Tool a 3 LPP y ga ga Graph sizing _ Changes the si
43. single color control files Open an exper imental rif file or from the Compensation menu choose Create New Matrix 2 A cif and daf file are automatically created Analyze the experimental file using data analysis tools in the daf file to create an analysis template 3 Create a statistics report table within the daf file and save the data file as an anlaysis template Note this is usually done on the positive and negative controls to create the appro priate analysis and then applied to the rest of the experimental files in the next step 4 Perform batch processing applying compensation and template files created above S17 Chapter 2 Overview of the Data File Types Data from the Amnis cell analysis systems are collected and managed using three types of data files raw image file rif compensated image file cif and data anal ysis file daf This section describes each file type and the table summarizes the features of each file Raw Image File rif The INSPIRE application saves the digital image data pixel intensities and location that were acquired by the instrument to a rif file A rif file contains e Pixel intensity data counts and location collected for each object that the instrument detected e Instrument settings that were used for data collection e Calibration values from ASSIST Compensation matri x if used while acquiring data Compensated Image File cif The IDEAS applica
44. spectrum of the probe extends beyond the channel 3 spectral band width The light from a second fluorochrome may appear primarily in channel 4 but unless you subtract the light emitted by the first fluorochrome into channel 4 you cannot generate images that accurately represent the distribution of the second fluor ochrome Emmission Spectra for two fluorochromes Channel 3 Channel 4 Percent Emission 475 500 525 550 575 600 625 650 675 700 725 Wavelength in nm Below is an example of cells stained with two fluorochromes independently and run together as one sample Intensity scatter plots and images are shown uncom pensated and compensated Image compensation is performed on a pixel by pixel basis 58 Overview of Compensation Un compensated Compensated _ 1664 165 a 5 1653 2 q zod ae Bad 5 al pai S 1e43 I le4 J T 3 oh 10004 TE TETN J ig 04 Ip aanak 1000 Doo T r 1000 T Oon a 1000 1e4 1e5 1000 oO 1000 164 1e5 3 Intensity 3_Intensity Uncompensated Compensated ssc Brightfield FITC PE PE Alexa610 Draq 5 PE Alexa610 Draq 5 Channel 1 Channel 2 Channel 3 Channel 4 Channel S Channel 6 Channel S Channel 6 The IDEAS application builds a matrix of compensation values by using one or more control files A control file contains cells stained with one fluorochrome Because it is critical that matrix values be calculated from intensities derived from a sole source of ligh
45. the analysis area Gate on the apoptotic cells with low nuclear area and high brightfield contrast Sete rae x w Cortrad_Bnghttelt Non apoptotic The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 34 Guided Analysis Cell Cycle Mitosis Wizard The cell cycle mitosis wizard will guide you through the process of creating the fea tures and graphs to analyze the cell cycle and identify mitotic events using the images of a nuclear dye Cell Cycle Mitosis To begin double click on Cell Cycle Mitosis Follow the instructions to analyze your file Step 1 Select the nuclear image channel From the drop down menu pick the nuclear channel image Step 2 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an already existing population Step 3 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area
46. the plot You may choose an already existing population Step 3 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 4 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip 333 Chapter 3 Next Step Optional Select additional subpopulation marker s OR Gate nucleated cells A histogram of the nuclear channel Intensity is added to the analysis area Gate on the positive events Next Step Gate apoptotic cells The nucleated cells scatter plot of the brightfield Contrast versus the Area of the thresholded nucleus is added to
47. value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 4 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip 35 Chapter 3 Next Step Optional Select additional subpopulation marker s OR Gate G2 M cells A histogram of the nuclear channel Intensity is added to the analysis area Gate on the G2 M population with 2n DNA Next Step Gate cells with condensed DNA The G2 M cells scatter plot of the threshold Area versus Bright Detail Intensity of the nuclear image is added to the analysis area Gate on the cells with condensed nuclear that have low nuclear area and high Bright Detail Intensity values These will include apoptotic cells which you can remove in the next step
48. white background you can change the colors when using the light mode To use light background graphs in the Analysis Area Click the graph background tool to switch between light or dark mode Py To map the dark mode colors to light mode colors 1 See the Application Defaults Colors tab Directories Populations Masks Statistics Graphs image Export Colors Map population colors for light and dark graph backgrounds Select a dark mode color a light mode color Ene Eee EEE EEE gt NENNEN Lt NT i EERE EERE TT ft ET l Update Populations in Open Files ox Cancel _ 2 Click Update Populations in Open Files when done 3 Click OK to save the changes or Cancel to exit Copy full or partial screens To copy the entire screen to the Clipboard 144 Reporting Images and Graphs e Press CTRL PRINT SCREEN It is then available for pasting into a third party application To copy a window to the Clipboard e Select the window and then press ALT PRINT SCREEN It is then available for pasting into a third party application Print the analysis area or image gallery directly To print the Analysis Area data Select Reports gt Print Analysis Area The IDEAS application prints all the graphs statistics text panels and images that are displayed in the Analysis Area To print the Image Gallery data e Select Reports gt Print Image Gallery The IDEAS appli
49. 0 0 o ooo ce eee ccc cece cee cece cece eee 127 Using the Region Manager 20 2 cece cece cece ec eee ececcececeeeeee 129 Creating Reports and Exporting Data 0 0 20 ee cece 131 Viewing and Changing the Application Defaults 0 0 0 00 000000 0e cece eee eee 132 Setting the Image Gallery Properties 0 0000 00 cc cece cece cece cee ecececeeeececeees 138 Reporting Images and Graphs 2 2 2 0 o ooo eee ec e ccc cece ee cece eee 144 Prepare the image gallery and analysis area for reporting 0 0000 2 2 2 2 144 Copy full or partial Screens en 3 ad no Sx sheerg idcaten aden ad cba acsonaeecaucenasnted ec 144 Print the analysis area or image gallery directly 2 0 0 0 0 0 0c cece cece ec ee eee 145 Copy images to the clipboard or save images to files 0 0000 0 2 c ccc ee eee eeee 145 Copy graphs to the clipboard or save graphs to a file 0 0 0 0 0 146 Reporting Statistics 2 3 40 4cccn selec edie eee tien toes sdb esse fame 11111 148 Define a Statistics Report i 2 scecsccceceee elt cc tes cee eee cath scenes sete oe 148 Generating a Statistics Report using daf Files 0 0 00 0000000 2 cece ce cece eee eee 150 Reporting Statistics from a Single Graph or Statistics Table 0 0000000000000000000 151 Exporting Data gcc soe e a rs oe ea ed 152 Creating TIFs From Population for Export 0 0 0 0 00 0 2000 cece eee 154 Printi
50. 0 4095 At each intensity on the X Axis of the graph the gray histogram shows the number of pixels in the image This histogram provides you with a general sense of the range of pixel intensities in the image The dotted green line maps the pixel intensities to the display intensities which are in the 0 255 range Manual setting is done by Click dragging the vertical green line on the left side crossing the X Axis at 0 allows you to set the display pixel intensity to 0 for all intensities that appear to the left of that line Doing so removes background noise from the image Click dragging the vertical green line on the right side allows you to set the dis play pixel intensity to 255 for all intensities that appear to the right of that line From the Image Gallery window select the object to use for setting the mapping It appears in the Image Gallery Properties window Tip You might need to select different objects for different channels because an object might not fluoresce in all channels To adjust the pixel mapping for display click drag the vertical green line by click ing near it but not near the yellow cross Tip For fluorescence channels set the vertical green line that appears on the left side to the right of the large peak of background pixel intensities as shown above and set the right vertical green line to the right of the brightest pixel intensities Click Set Linear Curve to make the transformation linear For the brigh
51. 051 1 0 212 0 078 0 018 0 055 0 009 0 044 0 008 0 004 0 002 0 004 1 0 0 0 0 0 0 0 0 0 0 0 O Means Positive Control Populations Step 2 Validate the compensation matrix None 2_Positive 3_Positive 4 Positive 5 Positive None HOW Ch07 Ch08 Ch093 Ch10 Ch11 Chi2 Double click each matrix coefficient to validate the fit of the positive control population The resulting graphs can be added to the analysis area to tefine the positive control populations ojoj o jojojojojojojojo Restore Matrix 7_Positive E 8_Positive X None ha None 11_Positive R1 amp 12_Positive S 021810101 THP1 merged_comp_control matri All O 2 Positive O 3 Positive O 4 Positive 5 Positive O 7 Positive O 8 Positive 11_Positive B 12 Positive D None 5 The coefficient value is automatically recalculated when a new population is selelcted 6 Repeat these steps as required to redefine the coefficients 7 Click Preview Images to view individual objects with corrections applied Double click on an image to add it to the preview window Note the corrections are only applied to on camera channels For example if the object is brightest in channel 3 on the first camera only channels 1 6 are shown corrected for that object 8 When the matrix appears satisfactory click Finish 9 Enter a
52. 1 0 076 0 045 0 081 0 359 0 05 0 4 0 008 0 174 0 061 0 045 0 033 0 004 0 08 0 026 0 086 0 035 0 002 0 021 0 01 1 0113 0 004 0 027 0 086 0 267 1 o ojololojloj ojojojlojo Preview a file with this matrix applied Select an existing rif file V Overwrite preview files Preview 2 To preview the matrix on image data browse for a file or select a population from the current file to preview and click Preview Note It is recommended that you first create a small tagged pop ulation to preview compensation changes because previewing large populations requires a lot of memory and may be slow 3 You may repeat editing the matrix and previewing until satisfied 4 When done click OK and save the matrix 68 Merging Data Files Merging Data Files Merging Raw Image Files You can merge rif files together for analysis This option is not available for basic FlowSight files without the Quantitative Imaging QI upgrade To merge rif files 1 Onthe Tools menu click Merge rif Files The Merge Raw Image Files window appears Z Merge Raw Image Files Select raw image files for merging A sf file will be created containing the objects from each file and the comection information from the first rif Note during the loading of the merged if the apply cell classifiers option needs to be set manually 2 To select the rif files to merge click Add Files The rif file
53. 1000ng 45_15_22 daF 1000mg 60_20_3 daf 5 1000ng 75_25_8 daf My Network Places File name Files of type Data analysis files daf jaf Data analysis files daf The progress is shown by a progress bar The state of the IDEAS application is restored to what it was when the daf file was saved 55 Chapter 3 Saving Data Files Data files are saved at several stages of analysis Raw image files are saved during data acquisition by merging multiple rif files or by creating new files from pop ulations Compensated image files and Data analysis files are saved when opening tif files merging multiple cif files or when running a batch analysis The IDEAS application also saves other types of files that are used for data correction and pre sentation Template files ast save the structure of an analysis and compensation matrix files ctm save the compensation matrices Application Defaults are set that direct the files into specific folders and can be viewed or changed by the user See Viewing and Changing the Application Defaults for more information Saving a Data Analysis File daf A daf file contains a snapshot of an analysis as described in Overview of the Data File Types Saving the analysis as a daf file allows you to recall that analysis simply by opening the file When you quit the IDEAS application you are always prompted to save changes to a daf file You can also sa
54. 101 Guided Analysis 23 Hardware requirements 4 Histogram tool 95 282 IDEAS getting started 22 Image copy 87 exporting defaults 9 136 individual image 86 Image Display intensity mapping 83 139 Image Gallery channel view 80 composites 85 142 overview 79 population 80 printing 145 155 properties 81 82 138 properties tool 79 resize 81 show hide color 81 show hide masks 80 tools 79 using 79 views 84 141 Image panel size change in Image Gallery 82 139 Individual image display properties 106 283 Index Chapter Appendix A manipulating 86 measurement tool 104 pixel intensities 104 show hide mask 108 Internalization using a wizard 39 Laser information 73 Line Region Tool 95 Mask Manager overview 88 tools 91 Masks about 250 combining 91 creating new 88 default color 8 134 dilate 252 edit 91 erode 253 examples 92 fill 254 intensity 256 list of 89 morphology 258 284 Index object 259 peak 260 range 261 show in Image Gallery 79 skeleton 262 spot 263 system 265 threshold 266 valley 267 viewing definitions 91 Merging cif files 70 Merging raw images files 69 Mitosis using a wizard 35 Name and Color Image Gallery 82 139 Nuclear Localization using a wizard 41 Nuclear translocation using a wizard 41 Object Data 108 Object Feature Values Table 95 One color histogram building block 47 285 Chapter Appendix A Ope
55. 3 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 4 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken 41 Chapter 3 Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip Next Step Gate double positives A scatter plot of the last gated or selected population of the Intensity values for the nuclear image and the translocating probe image is added to the analysis area Draw a region around the double positive cells Next Step Gate translocated events A histogram of Similarity of the double positive cells is added to the analysis area Draw a region to include the cells wit
56. Axis labels 12 Region names Default export settings to Graph 5 Defaults Graph Options E Legend Show sample name in title E Cursor Statistics Font Size Title f2 Values Henden The Image Export tab contains the default settings for image export when copying and pasting from IDEAS for reporting into other programs Chapter 1 Directories Populations Masks Graph Display Graph Export Options 7 Show channel names Text Color 7 Show object number v Show scale bar Background Black White Transparent Channel names f2 Seseto HL The Colors tab contains the mapping of dark and light mode colors ee Directories Populations Masks Statistics Graphs Image Export Colors Map population colors for light and dark graph backgrounds Select a dark mode color a light mode color Eat ee Eee BETTEN 1 NENNEN EEEN ot EERE EERE Tt EEE EEE Update Populations in Open Files ok cna 10 Setting Up the IDEAS Application 11 Chapter 2 Overview of IDEAS Chapter 2 Overview of IDEAS 13 Chapter 2 Overview of the IDEAS Application This chapter provides an overview of the IDEAS application user interface the files that the IDEAS application creates and uses the recommended director
57. Batch1 Template C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 analyzed cif and daf files 0 0ng_2_9 _4 Analyzed daf Compensation CAUsers sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 analyzed cif and daf files FITC DRAQ ctm Corrections Alignment Camera Background Brightfield Gain EDF Flow Speed Output Options Cell classifiers applied Single objects separated Clipped objects removed Non4ramed objects erased Input Files C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 0ng_2_9 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 Ing 15_1_8 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 30_6_13 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 45_11_18 nf C Users sfriend Desktop IS 100 NFkB Translocation Dose and Time 4 0ifs 0 1ng 60_16_23 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 75_21_4 rf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 90_26_9 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 153 10 f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 10ng 30_8_15 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 45_13_20 rf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 60_18_1 nf C Users sfriend Desktop
58. Bright Detail Similarity R3 for the double positive population is added to the analysis area Draw a region to gate on co localized events mares frequen Bap Dotai Servtarty COW a CPGB Low co localization High co localization For a more thorough explanation of the Bright Detail Similarity feature see Bright Detail Similarity R3 Feature The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 38 Guided Analysis Internalization Wizard This wizard will create an analysis template for measuring the internalization of a probe in any population of cells you identify Intemalization To begin double click on Internalization Follow the instructions to analyze your file Step 1 Select the internalization image channels From the drop down menus pick the cell image the channel that defines the cell sur face and the internalizing probe channel Step 2 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an alread
59. ERREEL QQ RSIS AI ARs Intensity_MC_ChO8 Intensity_MC_Ch10 325 605 206 4e8 6e6 Intensity_MC_ChO7 Intensity_MC_ChO9 Intensity_MC_Ch11 1 1 3e5 6e5 Se5 1 2e6 6 InStep 3 choose one of two methods for calculating the coefficients The Best Fit method is used for objects such as cells where intensities vary e The Means method is used for objects such as beads that have only slight variations in intensity 62 Overview of Compensation For each fluorochrome the application automatically identifies a positive con trol population excluding the brightest and dimmest objects and assigns it to the proper channel Inspect the matrix values in the table of coefficients Coefficients should always be less than 1 and decrease from the assigned chan nel In other words leakage should be greater in the channel nearest to the assigned channel Fluorescence always extends in the long wavelength direction from the exciting light Verify that no coefficients are larger than 1 Verify that ina column corresponding to a fluorochrome the coefficients decrease from the assigned channel Verify that the coefficient is greater in the channel below the 1 in the table than the value above the 1 in the table Verify that these coefficients also decrease in subsequent channels below the 1 Verify that there are no changes from the identity matrix in the columns where there are no fluorochromes including
60. Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an already existing population Step 3 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 4 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken SAG Chapter 3 Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip Next Step Gate fluorescence positives A histogram of the last gated or selected population of the Intensity value for the cell image is added to the analysis area Draw a region around the double positive ce
61. IDEAS Image Data Exploration and Analysis Software User s Manual Version 6 0 March 2013 amnis part of EMD Millipore Amnis part of EMD Millipore 645 Elliott Ave West Suite 100 Seattle WA Phone 206 374 7000 Toll free 800 730 7147 Patents and Trademarks Amnis technologies are protected under one or more of the following U S Patent Numbers 6211955 6249341 6473176 6507391 6532061 6563583 6580504 6583865 6608680 6608682 6618140 6671044 6707551 6 763 149 6778263 6875973 6906792 6934408 6947128 6947136 6975400 7006710 7009651 7057732 7079708 7087877 7190832 7221457 7286719 7315357 7450229 752275 7567695 7610942 7634125 7634126 7719598 Additional U S and corresponding foreign patent applications are pending Amnis the Amnis logo INSPIRE IDEAS and ImageStream and FlowSight are registered or pending trademarks of Merck KGaA All other trademarks are acknowledged End User License Agreement AMNIS CORPORATION SOFTWARE LICENSE AGREEMENT PLEASE READ THE FOLLOWING TERMS AND CONDITIONS CAREFULLY BEFORE DOWNLOADING INSTALLING OR USING THE SOFTWARE OR ANY ACCOMPANYING DOCUMENTATION COLLECTIVELY THE SOFTWARE THE TERMS AND CONDITIONS OF THIS SOFTWARE LICENSE AGREEMENT AGREEMENT GOVERN USE OF THE SOFTWARE UNLESS YOU AND AMNIS CORPORATION AMNIS HAVE EXECUTED A SEPARATE AGREE MENT GOVERNING USE OF THE SOFTWARE Amnis is willing to license the Software to you
62. M05 Graph Options E Legend F Show sample name in ttle 7 White background E Statistics Font Size The 12x Values 8 Headers 9 Save as Default Settings Save to File Copy to Clipboard Close To export graph statistics to the Clipboard Right click a graph and then click Export Statistics To Clipboard They are then available for pasting into a third party application To copy population statistics from a Statistics Table Right click the table and then click Copy Statistics or Copy Statistics transposed They are then available for pasting into a third party application e147 lt Chapter 3 Reporting Statistics Define a Statistics Report A statistics report definition can be saved in a daf file or an ast template file It allows users to select specific statistics within a daf file and open the data in Excel A statistics report can be generated during batching if it is part of the template used It may also be applied to pre existing daf files from the Reporting menu In this case the rest of the template is not processed only the report The statistics report definition allows you to specify population percentages and feature statistics and the layout of the report is accessed from the reporting menu To create a Statistics Report Definition 1 Select Reports gt Define Statistics Report The Statistics Report Definition appears Report ti
63. Name the output files with a new name if necessary 6 You may change the number of objects to load in the box under Enter the number of objects to process The default value is the number of objects in the file 25x Opening data files Tip you can select a smaller number than the maximum if you have a large number of objects to load This helps save time for creating a template file The IDEAS application randomly loads the specified number of objects within the file 7 Click OK The application then creates the cif and daf files and the daf file is loaded into the Image Analysis area Note Most often the defaults will be adequate For some older data files you may need to provide control files for certain settings For assistance call Amnis appli cation support e To view the corrections that will be applied to the rif file click Advanced within the Opening a rif file window The Opening file window appears Opening file NFkB Fitc Dq5 No LPS_2 rif SS WZ Apply Bight Feld Crosstakc Compensation 7 Perform corection Comected during acquistion Fluorescence compensation matrix sf cf daf or ctm Chol Cho2 C03 Cho Ch05 Ch06 1 0 0 0 0 o o 1 0 0 o 0 o 0 1 o o o o 0 0 1 0 o o 0 0 0 1 0 Minimum 2099 Maximum 23 33 4 Change Corection Offsets __Select Change Compensation Matrix Choose EDF Kemels Reference Camera 1 7 7 Perfo comection Reference Camea 2 3 en Kende oe ee a Ene A Off
64. Select a template file ast or daf Leave blank to use the Default template Set the output files parameters If the template contains a Statistics Report template click on the Preview Sta tistics Report button Order the files as you wish them to be reported by select ing a file with a left click then right click the desired position and select move here See Reporting Statistics for more information Click OK The Define a Batch window closes The batch appears in the Batches window TS Chapter 3 a zm M S Eal Batches to Run Edit Batch Remove Batch Submit Batches Processed Batches a Batch 6 29 2011 11 05 33 AM a Batch 5 3 2011 10 38 34 AM a Batch 5 3 2011 9 51 14 AM a Batch 5 3 2011 9 41 07 AM a Batch 4 5 2011 11 28 23 AM H E Batch 3 24 2011 11 47 57 AM G9 Batch 3 23 2011 2 29 21 PM 429 Batch 3 23 2011 2 00 37 PM Batch 3 2 2011 8 02 21 AM H E Batch 3 1 2011 1 17 04 PM H Batch 2 9 2011 11 49 07 AM m 9 The Batches window offers the following options e Add Batch If you want to create another batch to add to the list e Remove Batch If you want to remove a batch from the Batches to Run list Edit Batch If you want to edit a batch in the Batches to Run list 10 When you are satisfied with the Batches to Run list click Submit Batches The files to process are listed and the progress is displayed in the Processing Batch window Onc
65. There are some differences in the available features and anal ysis that can be done The table below outlines these differences Data comparison Pe FlowSight basic FlowSight QI ImageStream eee tea aha IDEAS default fea IDEAS default fea Default template and acquisition anal i ture set analysis __ ture set no analysis ysis Default ti 7 n compensation INSPIRE matrix o compensation No compensation matrix New feature calculation ees features U limited Unlimited INSPIRE mask cal Default Object Default Object mask computed in sition Open File Display Properties and Begin Analysis ll Merging files Y es es Yes Intensity and All features and All features and Images only Images Images Requires version Requires version Can open in 4 0 or 5 0 x or later 5 0 x or later later 15 Chapter 2 Understanding the Data Analysis Workflow Data analysis in IDEAS begins with opening a raw image file rif that was collected and saved using INSPIRE Then an existing compensation matrix or a new com pensation matrix is applied to the rif file and two additional files are created the cif compensated image file and daf data analysis file A compensation matrix performs fluorescence compensation which removes flu orescence that leaks into other channels See Overview of Compensation for more information about compensation A compensated image can accurately depict the correct amount of fl
66. Y es es es es es es o es es es es es H Texture Features Measures Haralick texture features Measures the intensity range of an image normalized between 0 and 1 Spot Count Feature Enumerates the number of spots See also Spot Distance Min Feature Spot Area Min Fea No ture and Spot Intensity Min and Spot Intensity Max Fea tures M12_Ch12 M01_Ch01 M12_Ch12 Describes the overall distribution of pixel intensities Signal Signal Strength Features are measured in pixel Strength __jvalues The average intensity of the camera background Bkgd StdDev Feature Intensity Feature Ch01 Ch12 Ch01 Ch12 MC_Ch01 The sum of the pixel intensities in the mask background MC Ch12 subtracted Max Pixel Feature MC Ch01 The largest pixel value within the mask background sub tracted MG eile Mean Pixel Feature The average pixel value within the mask background MOT Cnr M12_Ch12 subtracted Median Pixel Feature ly ly M01_Ch01 The median pixel value within the mask background sub ES Es M12_Ch12 166 N Y Y Dg es Table of Base Features by Category Raw Median Pixel Feature The median pixel intensity Raw Min Pixel Feature Yes Ye MC_Ch01 The lowest pixel value within the mask MC_Ch12 Saturation Count Feature Yes Yes M01_Ch01 The number of pixels in the mask that are saturated M12_Ch12 Saturation Percent Features Wes Wes The Percentage of pixels in the mask that are saturated
67. You can build custom viewing modes as shown in this example For more infor mation see Setting the Image Gallery Properties IDEAS OVA DFSv04 A 3 050 O O File Guided Analysis Analysis Compensation Tools Options Reports Windows Help e Population 2n single T cell amp double amp Focus amp conjugates View Custom x BF Actin ADAP Drags ADAP ActiniT BF Actin ADAP Drags ADAP ActinT Image Gallery Tools Table 1 Image Gallery Tools Allows you to create a population of hand Xo Tagging Mode Tool picked objects See Creating Tagged Pop ulations m Provides custom display features See Set Image Gallery Properties Tool Iting the Image Gallery Properties Displays masks on the imagery See Over Show Hide Mask Tool view of the Image Gallery Using the Image Gallery 79 Chapter 3 Sets the Image Gallery color See Over El Show Hide Color Tool view of the Image GalleryUsing the Image Gallery Click on the tool and it will show any sat Show Hide Saturated Pixels urated pixels will turn red See Overview of Tool the Image GalleryUsing the Image Gallery Zoom Tools Zoom in or out and reset zoom on the image QQQ gallery See Overview of the Image Gal leryUsing the Image Gallery To view the imagery for a population E 1 Inthe Population drop down menu of the Image Gallery click the population that you want The list includes
68. _M02 Aspect Ratio_MO06 _AddFies _ RemoveFiles a edge gd Mean_ Bkgd Mean_Ch06 Bkad StdDev_Ch01 Select a population All Sort features by a S a a aj Export to Order by Clipboard Object O Text File O Feature Export all used features _ Export all features 2 Addfiles to the list on the left to export values for multiple files 3 Inthe Select a population drop down menu select the population that you want If you haven t defined any populations All is the default To make a new pop ulation refer to Creating Tagged Populations 4 Inthe Select feature values to export area select features by clicking items in the list or hold down the Ctrl while clicking to select multiple items 5 Select the Export to option that you want Note that data exported to the Clip board can be pasted directly into a spreadsheet program 6 Select the Order by option that you want Note that ordering by object causes the values to be listed in a column whereas ordering by feature causes the values to be listed in a row 7 Click OK 152 Exporting Data Exporting Pixel Data Exporting pixel data is useful when importing the data into third party programs where you would need to graph the individual pixels To export pixel data 1 of ON On the Tools menu click Export Image Pixel Values The Export Image Pixel Values window appears Export Image Pixel Values
69. a Width Feature Based on a bo bounding rectangle the Width is the smaller Yes M01 M12 side and the Height is the longer side of the rectangle an ets Location A an origin in the upper left corner pixels or contour Angle Feature a angle of the major axis from a horizontal plane in radi ans Angle Feature The angle of the major axis intensity from a horizontal plane in radians Centroid Features central tendency of the pixels along the X Axis and No fres porn 1 164 Table of Base Features by Category Feature name Y Axis respectively PT Centroid Features M01_Ch01 The central tendency of the pixels along the X Axis and No M12 Ch12 Y Axis respectively with the pixel intensities weighted Centroid Features The distance between the X or Y Centroids of two No No images Eae ff The distance between the Centroids of two images Max Contour Position Feature The location of the contour in the cell that has the high lest intensity concentration Spot Distance Min Feature The shortest distance between two components spots See also Spot Area Min Feature Spot Intensity Min and Spot Intensity Max Features and Spot Count Feature Valley X and Valley Y Features The X Y coordinates of the minimum intensity within the skeletal lines that are used when creating the Valley Shape Features define the mask shape and have units that vary with the feature Aspect Ratio Feature The ratio of the Minor Axis
70. age to include in the composite Tip As you make the changes the image in the Object box updates accordingly If you want to preview any new changes in the Image Gallery return to the Image Gallery and select the View drop down menu to your new view Then return to the Image Gallery Properties window and click Preview Changes in Gallery Continue to add images as desired To remove and image from the composite Click Remove Image The composite is automatically added to the list on the left A composite can be removed from the list by clicking Delete Continue customizing the Image Gallery display properties with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes 2443 Chapter 3 Reporting Images and Graphs The IDEAS application allows users to copy and print images and graphs export sta tistics feature data pixel data or TIF files for separate analyses Prepare the image gallery and analysis area for reporting 1 Before you print or copy images see Setting the Image Gallery Properties to opti mize the image display 2 In addition to formatting the graphs and statistics in the Analysis area see Over view of the Analysis Area the IDEAS application provides color mapping from the dark mode that you see in the Analysis Area to a light mode that has a white background for the printing and exporting of data Because the population colors might not show on a
71. all the populations as well as the currently selected bin from a histogram To create a population refer to Creating Tagged Pop ulations 2 Toselect an individual image click on it A thin green frame indicates the selected object e The object s feature values are displayed in a table if an object is selected and a table is added to the Analysis Area e The selected object is identified in each scatter plot graph with a green cross The image can be placed in the Analysis area by right click gt Add Image to Analysis Area Tip Conversely in any scatter plot in the analysis area clicking a graphical point causes the Image Gallery to highlight and display the corresponding object Note that the objects are presented in the Image Gallery in the order of acqui sition and are not necessarily near each other in a scatter plot To change the viewing mode Inthe View drop down menu of the Image Gallery select a specific view The imagery display changes according to the new view To make a new view use the Image Properties tool To show or hide masks Click the Show Hide Masks toolbar button to toggle between showing and hiding the selected masks for all images in the Image Gallery a The mask is shown as a transparent layer over each image The mask dis played is selected in the Image Gallery Properties View tab The color of the overlayed mask can be changed in the Applications Defaults under the Options menu Ch
72. annel 6 80 Overview of the Image GalleryUsing the Image Gallery Tip To hide the mask for a specific channel only set the individual channel mask to None in the view tab of the Image Properties dialog To show or hide color e Click the Show Hide Color toolbar button to toggle between showing and hid ing the colors for all images in the Image Gallery See Setting the Image Gal lery Properties for more information To zoom on the image gallery e Click the Zoom In toolbar button to view the images in the gallery closer and the Zoom Out or Reset Zoom to reverse the zoom e zoomin f zoom out o reset zoom To show saturation e Click the Show Hide Saturated Pixels toolbar button Saturated pixels in images if any appear in red Setting the Image Gallery Properties When a new data file opens in the default template you might find it difficult to clearly see cell morphology because the Image Gallery display properties have not yet been properly adjusted for the data set To optimize the display you may use the wizard Display Properties Wizard to set the pixel intensity mapping to the display range Manual adjustment and other settings are described below Clicking the Image Gallery Properties toolbar button opens the Image Gallery Prop erties window which contains the following tabs e Display Properties Allows you to define the name color and display inten sity mapping for each image Allows adj
73. apoptotic cells single cells Q raved b gt Fst Eat Sl x Frequency High Bright D val 206 Understanding the Texture Features Application Example Identify cells that have bright specks such as Apoptotic cells Used in the Cell Cycle Mitosis Wizard 207 Chapter 4 Contrast Feature The Contrast feature measures the sharpness quality of an image by detecting large changes of pixel values in the image and is useful for the selection of focused objects or apoptotic brightfield images For every pixel the slopes of the pixel inten sities are computed using the 3x3 block around the pixel This is similar to the Gra dient RMS calculation with different weighted assignments to the pixel arrays with no background subtraction Example images are shown in the figure below nD a Contrast_BF Gradient RMS_BF Application Examples Find apoptotic images with high contrast in brightfield imagery Determine overall focus quality of images Use with Gradient RMS to determine focus quality Characterize texture See also Gradient Max Feature and Gradient RMS Feature 208 Understanding the Texture Features Ensquared Energy Feature The Ensquared Energy feature is a measure of image quality Computes the inten sity of the square block around the brightest pixel using the diameter input as the side for the square divided by the intensity of the total intensi
74. apter 4 Understanding the IDEAS Features and Masks This section contains the following subsections which describe the features that the IDEAS application uses for data analysis See the following lists of base features Table of Base Features Alphabetical Table of Base Features by Category Table of Basic Features available for FlowSight without QI Understanding the Size Features Understanding the Location Features Understanding the Shape Features Understanding the Texture Features Understanding the Signal Strength Features Understanding the System Features Understanding the Comparison Features About Masks Mask Functions 158 Understanding the IDEAS Features and Masks Overview of the IDEAS Features and Masks Objects passing through an Amnis cell analysis system are illuminated in different directions by lasers and or brightfield LEDs Light emitted from the object is focused through an objective lens and relayed to a spectral decomposition element which divides the light into six spectral bands located side by side across a charge cou pled detector CCD as shown in the following diagram Therefore each object has six images that can be individually analyzed or because they are in spatial register with respect to one another reconstructed Each of the separate bands is called a channel Below is an example of collecting 6 images The IlmageStreamx system has a second camera option which enables collec
75. ar button to begin Xo The Tagged Population window appears Z Tagged Populations oes Tagged Populations Update existing Create new Image viewing mode Nuclear Localization_Ch01_Ch07_Ch v Objects Tagged 0 Ose 2 Select either Update existing or Create New To Create New double click images within the Image Gallery and select Save Enter a new population name Each population is given a new color and the symbol solid diamond for ease of viewing in plots 3 If you selected Update existing choose a population to update in the drop down menu 4 Inthe Image viewing mode list choose the mode that you want from the drop down menu See Setting the Image Gallery Properties for more information 5 To add or remove an image from the tagged population double click either the image in the Image Gallery or a dot in a bivariate plot The selected channel image for each tagged cell is displayed in the viewing area of the Tagged Populations window In the Image Gallery a small smiley face icon appears on the left side of each tagged image Each tagged object is also dis played as a yellow star in a graph in the Analysis Area The number of objects in the tagged population is updated in the bottom left corner 6 If you are updating an existing population click the Update button in the Tagged Populations window 7 When you are finished updating click Close in the Tagged Populations window
76. area 202 Understanding the Shape Features Shape Ratio Feature The Shape Ratio is Thickness Min divided by Length The Shape Ratio feature is based on an input mask and is sensitive to the variation of the input mask shape Selecting an input mask that can accurately capture the object shape is important Shape ratio Thickness min Length Thickness min See also Aspect Ratio Feature and Elongatedness Feature for other shape ratios Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightWidth Thickness min Length SS Max ess min Application Example Measure object s elongatedness to provide shape classification 203 Chapter 4 Symmetry 2 3 4 Features The Symmetry 2 feature measures the tendency of the object to have a single axis of elongation and therefore 2 lobes The Symmetry 3 feature measures the tendency of the object to have a three fold axis of symmetry and likewise Symmetry 4 a four fold axis The absolute value of these features are dependent on the number of lobes For example an image that has high 4 lobe symmetry will also have high 2 lobe symmetry See the Lobe Count Feature for more information 4 lobe symmetry o X Single Focused Cells o X Single Focused Cells Qi sl PAA E x 10 18 15 Single Focused Cells 9 Frequency D Frequency N W 9 6 3 0 1 0 1 1 10 20 Symmetry 4_Ch6 30 40 50 S
77. at you selected Tip Before you click OK you can click Cancel or you can click the Pointer but ton on the Analysis Area toolbar if you decide not to create the region D Draw a region on a Histogram On the Analysis Area toolbar click the Line Region tool E Drag the line across the histogram 100 Overview of the Analysis Area Q Qi oS a 3 ab a gt Oo Cc g 2 u 2 wo is N w E o z 2e4 4e4 6e4 8e4 1e5 1 2e5 Intensity_MC_Ch05 To move or resize a region on a graph 1 Click the Move Resize Region toolbar button on the graph panel toolbar Click the region that you would like to move or resize When the region is selected squares that can be moved appear at the vertices and the label The first time that you drag the region the entire region and label move Dragging a specific vertex or label moves only that vertex or label To finish moving or resizing the regions on the graph click the Move Resize Region toolbar button again to deactivate the tool lt p The populations and statistics are updated and the Move Resize Region toolbar button is deactivated Note The recalculation of statistics and populations may take a moment if the data file is large or if many populations are dependent on the regions that are being moved or resized To zoom in on the scale of a graph 1 Click the Scaling toolbar button on the graph panel toolbar Al Click and drag to define a r
78. atedness Feature exture low Speed Feature ystem Gradient Max Feature exture Gradient RMS Feature exture eight Feature ize Texture Features exture ntensity Concentration Ratio Feature omparison ntensity Feature ntemalization Feature omparison ength Feature ize obe Count Feature hape Major Axis and Minor Axis Features ize Major Axis Intensity and Minor Axis Intensity Features ize Max Contour Position Feature Location Signal Max Pixel Feature Strength 162 Table of Base Features Alphabetical eature Name Mean Pixel Feature Median Pixel Feature Min Pixel Feature Minor Axis see Major Axis Intensity and Minor Axis Intensity Fea tures Modulation Feature Object Number Feature Objects ml Feature Objects sec Feature erimeter Feature pot Intensity Min and Spot Intensity Max Feature Raw Intensity Feature Raw Max Pixel Feature Raw Mean Pixel Feature Raw Min Pixel Feature Raw Median Pixel Feature Saturation Count Feature Saturation Percent Features hape Ratio Feature imilarity Feature imilarity Feature pot Area Min Feature ot Count Feature ot Distance Min Feature Spot Intensity Min and Spot Intensity Max Feature td Dev Feature mmetry 2 3 4 Features hickness Max Feature able of Base Features by Catego ime Feature alley X and Valley Y Features Width Feature Corr Feature 163 Category Signal Strength Signal Strength Signal Strength Size Texture ystem ystem ystem i
79. ation calculates values for the new features These calculations may take several minutes depending on the number and complexity of the new features and the size of the image file To delete a feature 1 Select one or more features in the Features list by clicking them To select more than one feature use the Ctrl key 2 Click Delete A warning message will confirm or cancel deletion Note Deleting a feature also deletes any populations that are dependent on that feature Your feature list may become large and unwieldy You can narrow down the list without deletions by sorting the list See Using the Feature Manager for more information Ranking features by discriminating power With the IDEAS application you are able to create an unlimited set of features by using the Mask manager to define location and the Feature manager to choose a mathematical expression that uses the image pixel data and or the mask This can make it difficult to choose a feature that provides good statistical separation of pop ulations of cells that have different appearances from each other The following pro cedure describes the manual process to find features that separate two populations of cells from each other with minimal knowledge of the feature set A general descrip tion of the steps is followed by a specific example Note IDEAS 6 0 includes the Feature Finder wizard which replaces this manual process This process may still be useful for making feat
80. ature values or view them as statistics for any population For descrip tions of all the base features see Understanding the IDEAS Features and Masks When the IDEAS application opens a cif or rif file the application calculates the values of features as defined by the selected template You can refine your template so that it includes only those features of interest for your experiment You use the Feature Manager to examine existing features and to define new ones To gain access to the Feature Manager select Analysis gt Features or select it from one of the context menus that are available in the histogram and scatter plot panels with a right click While the Feature Manager is open all calculations for creating graphs and statistics are disabled However you may view images and change the population and channel views When you close the Feature Manager any changes to feature names definitions and values are reflected in any currently displayed graphs and statistics The values of newly created features are also calculated at this time You can create single features and combined features You create a single feature by selecting a base feature such as Area or Intensity along with a mask and or an image This option is not available for basic FlowSight files without the Quantitative Imaging QI upgrade You can create a combined feature by defining a mathematical expression that includes one or more single features that exist in th
81. atures Median Pixel Feature The Median Pixel feature is the median of the background subtracted pixels con tained in the input mask It is more robust than the mean as an estimate of the aver age fluorescence since it is less influenced by outliers The relationship of Max Mean Median and Min Pixel is shown in the figure below Cell B paxil sj ma co ae mz Application Example Estimate the average fluorescence activity This feature is preferred over the Raw Median Pixel feature 223 Chapter 4 Min Pixel Feature The Min Pixel feature is the smallest value of the background subtracted pixels con tained in the input mask There will be some negative numbers due to the back ground subtraction therefore the Raw Min Pixel feature is preferred cene mare ss E Meanie i ts O D aT Application Examples Obtain the minimum value in an image after background subtraction Very likely to be negative in brightfield imagery Quantify spectral absorbance using the brightfield image Identify over compensated images 3994 Understanding the Signal Strength Features Raw Intensity Feature The Raw Intensity feature is the sum of the pixel values within the mask including camera background Application Example Estimate raw fluorescence activity This feature is less relevant than the Intensity feature because it includes camera background intensity 225
82. bjects Population AddFies Remove Files Compensated image file cif Select a template or data analysis file ast af Data analysis file daf ok Cancel _ 2 Toselect the cif files to merge click Add Files The cif file names appear in the list If you want to remove a file from the list select it and then click Remove File Type a unique name for the output files Select a template Click OK The merged files are created and the new daf file is loaded with a population created from each file NO 1 fF W 70 Creating New Data Files Creating New Data Files Creating new data files from populations To further analyze a population or merge it with other data when working in a daf you can save it to a new data file This course of action is useful if your data file con tains a large number of objects that are not pertinent to your experiment Decreasing the data file size results in better performance by the IDEAS application as described in Creating Regions on Graphs Note that you cannot create a new cif or rif when multiple data files are open This option is not available for basic FlowSight files without the Quantitative Imaging QI upgrade To create data files from populations 1 On the Tools menu click Create Data File from Populations The Create cif and or rif From Populations window appears r Create cif and or rif From Populations
83. cation prints all the images that are visible in the Image Gal lery Copy images to the clipboard or save images to files To copy or save Images 1 Right click anywhere in the Image Gallery and then choose one of the following options e Copy Save Image for a single image e Copy Save Object Images for the row of images for one object e Copy Save Gallery Column for the images of one channel in the current gallery e Copy Save Gallery for all images in the current gallery 2 A preview of the image s is shown Changes from the default settings may be made to the following Show Channel names e Show Object number e Show Scale bar e Text color for channel names Background Number of rows and columns e Size and DPI settings Font sizes and clipboard format 145 Chapter 3 IA Copy Save Image s nnn Sox Options W Show Channel Names Object number Columns 2 Width 30 020 in v DPI 300 Font Sizes Clipboard Format Metafile Ry Channel Names ae Scale bar 12 Object number 10 Save as Defaut Settings Load Defaut Setings L Saveto Fie _ Copyto Clipboard Close 3 The settings may be Saved to the Default settings or Default set tings may be loaded 4 Click Save to File or Copy to Clipboard when done Files may be saved as png bmp or tif formats 5 If Copy to Clipboard was chosen paste into third party applicatio
84. cc 20 480 40 nn _860 388nm 95 S0rm 890 74Snm 748 8000 PE Tex S Be a Orange 5 Background and spatial offset corrections are performed the imagery is dis played bivariate plots of adjacent channels Intensity are added to the analysis area and the compensation matrix values are computed and displayed in a table 61 Chapter 3 Example 5 D Create Compensation Matrix dme Step 3 Validate the compensation matrix Double click each matrix coefficient to validate the fit of the postive control population The resulting graphs can be added to the analysis area to refine the positive control populations 0 olo o o o o o o o ojojoj ojojojojojo ojo ojojojojojojojojo Best Fit Means Preview Images Restore Matrix Positive Control Populations Ch01 None 7_Positive Ch02 2 Positive 8_Positive Ch03 3_Positive None Ch04 4 Positive None 5 Positive 11_Positive HHH None 12_Positive aage ga gege Previous l The Positive Control Populations are shown in the graphs below RE ENEE QQ Ge asx Al er Spina Ed 2 Y g Ed g g m g ta MC_Ch02 E D E3 a amp Intensity_MC_ChO4 3 es Intensity_ Intensity_MC_ChO6 o o o 1 1 1 i 4e5 6e5 8e5 1e6 1 1 165 265 1 1 j 5e5 168 1 566 Intensity_MC_ChO1 Intensity_MC_ChO3 Intensity_MC_ChO5 ai EEN
85. cept to the extent such activities are expressly permitted by law notwithstanding this pro hibition 3 Ownership The copy of the Software is licensed not sold You own the media on which the Software is recorded but Amnis retains ownership of the copy of the Software itself including all intellectual property rights therein The Software is protected by United States copyright law and international treaties You will not delete or in any manner alter the copyright trademark and other proprietary rights notices or markings appearing on the Software as delivered to you 4 Term The license granted under this Agreement remains in effect for a period of 75 years unless earlier terminated in accordance with this Agreement You may terminate the license at any time by destroying all copies of the Software in your possession or control The license granted under this Agreement will automatically terminate with or without notice from Amnis if you breach any term of this Agreement Upon termination you must at Amnis option either promptly destroy or return to Amnis all copies of the Software in your possession or control 5 Limited Warranty Amnis warrants that for thirty 30 days following the date of pur chase or delivery if Amnis has made the Software available to you without charge the Software will perform in all material respects in accordance with the Documentation As your sole and exclusive remedy and Amnis entire liability fo
86. cluded during acquisition of the file with their associated default masks This mask may be changed for the default view however the images in this view cannot be changed The list of existing views is shown on the left 84 10 11 Overview of the Image GalleryUsing the Image Gallery z Image Gallery Properties Display Properties Views Composites Views View Definition 2 w BF Name All Channels Ch2 t Ch4 t DRAQS i NFkB S SSC All Channels SSC mask M01 Ch2 mask M02 H NFkB mask M03 Ch4 mask M04 BF mask M05 i DRAQ5 mask M06 To create a new view Click New The new view is automatically added to the list onthe left In the right section of the window type in a name for the view Click Add Column Define the column by selecting an image and a mask or a composite from the dropdown menu Repeat the previous 2 steps until finished adding columns to the view A column will be added under the column currently selected To insert a column click on the image above insertion point Columns may be removed by clicking on Remove Column A view may be edited at any time by selecting the view and following the same procedures If you want to delete a view click the view to select it and then click Delete A confirmation window appears If you want to preview any new changes in the Image Gallery return to the Image Gallery and choose your new view in the View drop down menu Th
87. color button The Graph Displaytab contains the default list of statistics shown for a graph Check the box next to the statistic to have it show below the graphs when statistics are shown for a graph The default graph size and font size for the graphs in the anal ysis area may be changed in this tab ia Z Application Defuts TT k E Directories Populations Masks Default Graph Statistics Graph Display Graph Export Image Export Colors v v Count Total Gated Plotted F Objects mL Mean Median Std Dev MAD CV F Mini Maximum Geo Mean Mode Variance T NaN Update Graphs in Analysis Area Graph Size Font Sizes Statistics Default Graph Size Small Medium D Large Default Graph Font Sizes me 2 Axis Labels Tick Mark Labels 10 Region Names 10 _ Apply to All Setting Up the IDEAS Application The Graph Export tab contains the default settings for printing and exporting graphs when copying and pasting from IDEAS for reporting into other programs Directories Populations Masks Graph Display V Graph Size 300 DPI Width 1 027 Height 0947 fE in Lock aspect ratio Font Size Tile 12 Tick mark labels
88. control before beginning step 1 5 Refine the analysis and save the template 6 Perform batch analysis on all data files in the experiment using the compensation matrix and analysis template ie Guided Analysis Guided Analysis Data analysis always begins with opening a data file The Start Analysis button will step you through opening a file setting the image display mapping and choosing an analysis wizard Application wizards are available to guide you through an analysis The wizards can be accessed from the Guided Analysis menu or the wizard tool A k at the end of the Start Analysis routine The following wizards are available a Wizards Select the wizard to use for analysis Open File Creates a template to facilitate analysis Display Properties Automatically sets image display properties Begin Analysis Identifies single focused fluorescent positive cells Assists the user in picking relevant features for separating populations The file must contain members of each population Apoptosis Creates an analysis template for identifying apoptotic events based on brightfield and nuclear morphology Cell Cycle Mitosis Creates an analysis template that distinguishes mitotic and apoptotic events Co4ocalization Creates an analysis template for measuring the co4ocalization of two probes on in or between cells in your sample Intemalization Creates an analysis template for measuring the i
89. cts per second Note to get objects per ml of a population use the sta tistic Concentration Time Feature Yes Y The camera timer feature converted to seconds ombined Any combined feature will be listed under Combined 168 Table of Basic Features available for FlowSight without QI Table of Basic Features available for FlowSight without QI The default masks used for FlowSight Basic non Ql files is the INSPIRE mask FlowSight Basic Features Mask_Channel Brief definition Ara Mol GM MC The size of the mask in square microns The ratio of the Minor Axis divided by Bked Mean ChO1 Ch12 The average intensity of the camera background iation of th k Bked idivey Ch01 Ch12 The standard devia ion of the bac ground intensities INumber Enumerates changes of pixel values in the image to measure the focus quality of an image MC_Ch01 MC _ The sum of the pixel intensities in the mask background subtracted M01 M12 Describes the narrowest part of the mask Object Number none fhe sequence of objects Raw Centroid X The central tendency of the pixels along the X Axis and Y axis respec Raw Centroid Y tively Uncompensated mask background subtracted no com Intensity pensation applied 169 Chapter 4 Understanding the Size Features Size features are in microns and include Area Diameter Length Major Axis Minor Asix Major Axis Intensity Minor Axis Intensity Perimet
90. d Symmetry 2 3 4 The Texture features determine local intensity variations in images and include Bright Detail Intensity R3 and Bright Detail Intensity R7 Contrast Gradient Max Gradient RMS H Texture H Contrast H Correlation H Energy H Entropy H Homogeneity and H Variance Modulation Spot Count and Std Dev Contrast Gradient Max and Gradient RMS are generally used to determine best focus The Comparison features describe the difference of intensity measurements between masks or pixels in different images or the same image with different masks These include Bright Detail Similarity R3 Intensity Concentration Ratio Internalization and Similarity The system features do not require a mask 161 Chapter 4 Table of Base Features Alphabetical Delete this text and replace it with your own content eature Name Category ngle Feature ocation ngle Feature ocation rea Feature ize Aspect Ratio Feature hape spect Ratio Intensity Feature hape Signal Strength Signal Strength Signal Strength right Detail Similarity R3 Feature Comparison amera Line Number Feature ystem amera Timer Feature ystem entroid Features ocation entroid Features ocation ircularity Feature hape Bkgd Mean Feature Bkgd StdDev Feature Bright Detail Intensity R3 and Bright detail Intensity R7 Features hape ontrast Feature exture entroid Features ocation entroid Features ocation iameter Feature ize longatedness Feature hape long
91. d must reside in the same directory as the corresponding cif file The daf file contains 18 Overview of the Data File Types e Feature definitions e Population definitions e Calculated feature values e Image display settings e Definitions for graphs and statistics Loading a daf file restores the application to the same state it was in when the file was saved i e with the same views graphs and populations In IDEAS versions 3 0 or later a daf file may be used as a template Note When a daf file is opened the cif file must be located in the same directory as the daf file since the daf file points to images used for analysis that are stored in the associated cif file Template ast The IDEAS application saves the set of instructions for an analysis session in a daf file to a template ast file Note that a template contains no data it simply contains the structure for the analysis This structure includes definitions for e Features e Graphs e Regions e Populations The ast also contains settings for e Image viewing Image names Statistics The templates subdirectory under the directory where the IDEAS application was installed contains the default template named defaulttemplate ast Because a template is required for loading a cif file you must use the default template to create the first daf file After you save a custom template you can use it for any sub sequent loads of cif
92. d pixels in an image Pixel intensities are measured on the camera pixels from 0 to 1023 10 bit and therefore become saturated and cannot be quantified after 1023 See also Sat uration Count Feature An object with saturated pixels shown in red Application Example Measure the validity of the experiment setup Saturated data may not produce useful information 231 Chapter 4 Spot Intensity Min and Spot Intensity Max Features Spot Intensity Min provides the smallest Raw Mean Pixel value not background sub tracted of the dimmest spot connected component The Raw Mean Pixel values for each spot is computed and the smallest value is reported Spot Intensity Max provides the largest Raw Mean Pixel value not background sub tracted of the brightest spot connected component The Raw Mean Pixel values for each spot is computed and the largest value is reported These are two of four features that can be used to identify objects with spots that are close together dim bright or small when counting spots in an image To use these features the spots need to be individually masked such as using the Spot or Peak Mask The Spot Area Distance and Intensity Min or Max features measure prop erties of different spots in an image and are often used with the Spot Count feature under Texture Spot Area Min Size provides the area of the smallest spot Spot Distance Min Location provides the shortest distance between t
93. difference in the means divided by the sum of the standard deviations for the two populations The larger the RD value the better the separation afforded by the feature A scatter plot is added to the analysis area of the truth populations for the top two features It may be necessary to refine your results Visually verify the morphology and sep aration for the features listed Additional features may be quickly plotted by selecting them in the list and clicking Plot Features To return to the truth population assign ment step click Refine populations To choose different channels or categories click Change Category Table 1 List of additional features beyond default that are created and explored in the Feature Finder wizard rere Feature name and description category Location Features are in X Y pixel coordinates from an origin in the upper left corner pixels or contour Delta Centroid XY Feature The distance between the Centroids of the intensity weighted and channel mask non intensity weighted image Shape Features define the mask shape and have units that vary with the feature The ratio of the Minor Axis divided by the Major Axis Circularity Feature Obiect The degree of the mask s deviation from a circle J Compactness Feature bce Describes the density of intensities within the object J Elongatedness Feature Obiect The ratio of the Height Width which use the bounding box J Lobe Count Feature Object
94. e 12 v Tick mark labels Axis labels f2 Region names Default export settings to Graph Defaults Graph Options E Legend Show sample name in title E Cursor The Image Export tab contains the default settings for image export when copying and pasting from IDEAS for reporting into other programs 136 Creating Reports and Exporting Data Directories Populations Masks Graph Display Graph Export Options V Show channel names Text Color Show object number V Show scale bar Background Black White Transparent Font Sizes Channel names f2 Seset 12 Obec nner The Colors tab contains the mapping of dark and light mode colors 2 Aon te E Directories Populations Masks Statistics Graphs image Export Colors Map population colors for light and dark graph backgrounds Select a dark mode color a light mode color EES m DICIT Seer n L EEEE EEEN EENEN LLL EENEN EENEN Update Populations in Open Files Lo cna 137 Chapter 3 Setting the Image Gallery Properties When a new data file opens in the default template you might find it difficult to clearly see cell morphology because the Image Gallery display properties have not yet been properly adjusted for the data set To optimize the display you
95. e extensions for known file types check box is not selected 3 Click OK Copying the Example Data Files Copy data files to a single directory on your hard drive Sample data files are avail able on your workstation or at www amnis com login for customers with an account To copy the example data files Copy the data files Right click the directory that contains the data files and click Properties Clear the Read only check box Click OK WD Viewing and Changing the Application Defaults To view or change these defaults chooseApplication Defaults from the Options menu Each tab allows you to view or change the default settings Chapter 1 4 ee M te Directories Populations Masks Graph Display Graph Export image Export Colors Default Data Files Directory C Users sfriend Desktop NFKB Translocation FS v Update automatically when file is selected Default Template Files Directory C Users sfriend App Data Roaming Amnis Corporation templates Update automatically when file is selected v Use default data directory Default Batch Report Files Directory C Users sfriend AppData Roaming VAmnis Corporation batches E Update automatically when file is selected Default Compensation Matrix Files Directory C Users sfriend App Data Roaming Amnis Corporation compensation Update automatically when file is selected v Use default data directo
96. e feature list FlowSight files without the QI upgrade can utilize the combined feature option Some features such as Area depend on the boundary of a cell These features require you to select a mask that defines the portion of the image to use for the cal culation Other features such as Max Pixel depend on pixel intensity meas urements and require you to select an image Other features require you to select a mask and one or more images You can add and remove features from the feature list The feature definitions are stored in templates so the definitions are available when you analyze multiple data 113 Chapter 3 files The default template used for ImageStream data or QI FlowSight data includes most of the base features for each channel image and channel mask that the feature list contains Certain features such as Similarity and Spot require extensive cal culations so the default template does not include them The reason is to save time when you load files However you can add these features to the feature list Viewing feature definitions To view existing features 1 Click Analysis gt Features or select Features from a graph panel context menu The Feature Manager window appears Z Feature Manager 092011 X101 unstimulated _1 daf Lo je f Features Area _M01 a Feature Type Area _M02 Area _M03 Aspect Ratio Intensit Area_M04 I Area _M05 Area _M06 Area _M07 Area_M08 Area_M09 A
97. e graph on the right side split the population because the change from a linear to a logarithmic scale occurred in the middle of the pop ulation In general the gt X value should be 1000 for 40 and 60X data and 100 for 20X data REP ei ENa e P ATSE 12 Normalized Frequency 1 TE 1 1 EU O R 1 titi DA A D AODAINN AONE 1 TOAN J3 2e3 1e3 O 1e3 203 363 4e3 1e3500 0 5001e3 Se2e3 1e4 1e2 10010 e2 e4 5 Intensity 3400 5_Intensty 3400 5_Intensity 3400 OE i mnt e Scaling Scaling Sealing Auto C Manual G Auto Manual Auto Manual X Asis X Axis XAxis Minimum Minimum Minimum Maximum Maximum Linear Linear Linear Clo xol one x gt 1000 log X gt 10 og 12 The font sizes can be changed for an individual graph 13 Click OK Tip After you have created a graph you can change its properties by right click ing the graph and selecting Graph Properties The same window that you used to create the graph will reappear and you can then make any changes that you want Graph Properties Statistics Show Hide Legend Plot Order and Properties k roll BIB A Apply Remove Regions Clear Selected Bin Copy Region to Clipboard Paste Region from Clipboard Features Masks Populations Regions Export Statistics To Clipboard Copy Save Graph and or Statistics Print Graph 20 40 60 80 100 Gradient RMS_M05_Ch05 To show selected statistic
98. e image frame Angle Intensity Feature Angle Intensity is the angle of the major axis intensity from a horizontal plane in radi ans Brightfield 7AAD Composite Major Axis Major Axis Angle 7AAD 1 2 Angle 7AAD 0 83 Angle Intensity 7AAD 1 2 Angle Intensity 7AAD 0 81 Application Example Identify the orientation of an image relative to the image frame 183 Chapter 4 Centroid Features Centroid X and Centroid Y Features Centroid X is the number of pixels in the horizontal axis from the upper left corner of the image to the center of the mask Centroid Y is the number of pixels in the vertical axis from the upper left corner of the image to the center of the mask In this example the Centroid X 54 and the Centroid Y 32 Brightfield RTX AF488 Application Examples Identify the center of the mask Calculate the Delta Centroid or the distance between two fluorescent markers Used by IDEAS to calculate the Delta Centroid X Y or XY Centroid X Intensity and Centroid Y Intensity Features Centroid X Intensity is the intensity weighted X centroid and is shifted from the center of the mask toward the center of fluorescence The Centroid Y Intensity is the intensity weighted Y centroid X and Y pixel coordinates are calculated from an origin in the upper left corner 184 Understanding the Location Features Centroid X Y i Centroid X Y PE freatwe Pme Pee m
99. e input mask An example plot is shown below that demonstrates the advantage of using this feature over the Intensity feature for identifying true positive events For a concentrated signal Max Pixel is more sensitive than Intensity as shown in the figure below The relationship of Max Mean Median and Min Pixel is shown in the figure below single focused celle Intensity M4_CD71 T 1 00 600 Max Piel M4_CD71 FITC CellA CellB a azn Application Examples Used to estimate the true peak fluorescence activity Is preferred over the Raw Max Pixel for this application 220 Understanding the Signal Strength Features Max Pixel to Mean Pixel ratio identifies bright punctate staining vs uni form staining 221 Chapter 4 Mean Pixel Feature The Mean Pixel feature is the mean of the background subtracted pixels contained in the input mask This is computed as Intensity number of pixels The relationship of Max Mean Median and Min Pixel is shown in the figure below fMaxPixet srs ess fin Pixet soos fo Application Examples Estimate the average fluorescence activity This feature is preferred over the Raw Mean Pixel feature Quantify relative levels of mean fluorescence between cells Identify bright punctate spots by calculating the max to mean pixel ratio Track intemalization of surface bound antibodies 222 Understanding the Signal Strength Fe
100. e mask for a single channel image e Click the Mask button on the image panel toolbar or right click the image and then click Show Hide Mask on the image context menu T cell a spacey The mask appears as a transparent cyan overlay on the image To turn the color on or off e Click the Color button on the image panel toolbar or right click the image and then click Color Off or Color On T cell a s gace Viewing the Object Feature Values The Object Feature Values table which is shown in the following figure displays a selected set of feature values for selected objects For each feature the name value and description are shown 108 Overview of the Analysis Area Current To view and customize the features shown in the Object Data table 1 Click the Object Feature Values tool O 2 Right click anywhere in the table area to open the menu Select Features Delete Feature Add Current Object Delete Object Row Copy Feature Values to Clipboard 3 Choose Select Features The Select Object Features window appears 109 Chapter 3 amp Select Object Features 2s Features Area_M01 Area_M02 Area_M03 Area _M04 Area _M05 Area _M06 Area _M07 Area _M08 Area _M09 Area_M10 Area_M11 Area_M12 Area_MC Aspect Ratio Intensity_M01_Ch01 Aspect Ratio Intensity_M02_Ch02 i Sort featuresby A B Le a 2 4 Select the features to view Mul
101. e you have started processing batches it may use up a fair amount of your computer s processing power Processing Batch Batch1 Unprocessed yy Processing Processed rf File 0 0ng_2_9 rf O 1ng 15_1_8rf 0 1ng 30_6_13 1f 0 1ng 45_11_18 f i m r cf File 0 0ng_2_9 cf 0 1ng 15_1_8 c 0 1ng 30_6_13 cf O 1ng 45_11_18 cf O Ninn 6 16 23 cif lt gt m r daf File Total elapsed time 0 minutes Cancel Batch 4 Tip To cancel the batch processing at any time click Cancel Batch The IDEAS application will confirm cancellation and complete the file it is working on When the batch processing is complete the IDEAS application saves the rif cif and daf files in the batch results directory In the Batches window a list of 76 Batch Processing processed batches appears in the Processed Batches list If a batch did not suc cessfully complete it will appear in red Tip To display the error that occurred during processing double click the batch 11 If you want a batch report double click the batch in the Processed Batches list of the Batches window The Batch Results window appears 12 Inthe Batch Results window click Print 13 Inthe Batch Results window click Close 14 In the Batches window click Close Batch Report IC Users sfriend App Data Roaming Amnis Corporation batches Batch 2 1 2011 11 53 28 AM Batch
102. eamX These masks are stored in the cif file and cannot be changed by the user Conversion note Versions of IDEAS prior to 3 0 were using the System function mask with a weight of 5 for the default masks which was more permissive and resulted in larger masks Below is an example of the difference between the default masks oe ae Inspire mask no mask System mask Default Object mask 2 Combined masks are created using Boolean logic to combine and subtract masks For example the cytoplasmic mask is created by taking the brightfield mask and not the morphology mask of the nuclear image You can use the Mask Manager to combine masks of different regions or images The IDEAS application default template provides a combined mask named MC that is the union of the pixels from all channel masks and a NMC mask that is everything outside of MC The following illustration shows two channel masks 250 About Masks that are combined into one mask which is shown in the right most panel innn O Dilate 1 pixel Erode 3 pixels A And Not B 3 Function masks are created with user input There are fourteen types of function masks Dilate Erode Fill Inspire Intensity Interface Morphology Threshold Spot System Object Peak Range Skeleton and Valley Each of the functions masks are defined here Refer to Creating New Masks with the Mask Manager for m
103. eckleness of cells Separate connected spots in a Spot Mask into individual components 260 About Masks Range Mask The Range mask provides a capability to select components in an image within a selected size and or aspect ratio by setting a minimum and maximum area and mini mum and maximum aspect ratio To select pixels within a range of intensity values see Intensity Mask lt i Selecting small aspect ratio object 3 object 4 Selecting small size object 1 object 3 D 2 Selecting small intensity object 1 object 4 FISH IS Masking a cell with one spot using the spot and range masks N 44 Range_Area gt 2 Range_AR gt 0 4 Application Examples Usewitha Spot Mask to constrain the Spot Count feature to round spots Use on any other mask that has multiple components to define unwanted objects such as debris objects that are too small or whose shapes are not circular 261 Chapter 4 Skeleton Mask The skeleton mask provides the barebone structure of the object from the starting mask Two options are available thin or thick skeletons The thin option produces the condensed shape of the object and typically takes a form of 1 pixel wide skeletal line The thick option is intensity weighted The thin option is dependent on the shape of starting mask thick uses the pixel intensities and is less sensitive to the shape of the starting mask The user will need to pay careful attention t
104. ect Graph Properties ee 235 x Chapter 3 Open File Wizard This wizard will guide you through the opening of a data file and setting the image dis play mapping Use this wizard to open a file if you are not using one of the appli cation specific wizards To begin double click on Open File Follow the instructions to open your file Tip You can limit the view to specific file types daf cif or rif by using the drop down menu Files of type in the Select Data File window A daf file will open directly without further input a cif file will require a template and a rif file will require a template and a compensation matrix If the template or com pensation matrix boxes are left blank the default template and or matrix will be applied For more information on opening data files see Opening data files amp Open File Wizard Step 1 Select the data file you wish to open Step Progress 1 Select data file to open This wizard will take you through the steps involved in opening ImageStream data files There are 3 types of data files that can be opened in IDEAS Raw Image File rif uncompensated data from the instrument Compensated Image File cif compensated data Data Analysis File daf analyzed data Click the folder button to select the file to open Once a data file is open you may begin analysis 26 Guided Analysis Display Properties Wizard
105. ectangular region for rescaling The Zoom Out Scaling toolbar button appears in the graph panel toolbar next to the Scaling toolbar button a Click the Zoom Out Scaling toolbar button to automatically scale the graph The Zoom Out Scaling toolbar button is removed from the graph panel toolbar To resize a graph 101 Chapter 3 e Select the graph s to be resized and then click the sizing button tool small medium or large ey gu go Agraph may be resized by dragging the right bottom or lower right corner Tip Select multiple graphs to make them all the same size To copy and paste a region to another graph 1 Right click anywhere on a graph and click Copy Region to Clipboard on the graph context menu that appears The Copy a Region to the Clipboard window appears Click the region to copy in the list and click OK Right click on the graph where you want to paste the region and click Paste Region from Clipboard on the graph context menu that appears If the region already exists in other words you are copying it within the same instance of the application the Create a Region window appears Rename the region and set the display properties for the resulting new pop ulation and click OK Note When you copy a region the scale is copied and is no longer associated with the feature from which it was originally drawn Therefore the region might not fit on the new graph To Apply or Remove a regi
106. ed by the starting mask The user chooses the starting mask when creating the Threshold mask See also Intensity Mask In the example below cell 10678 is bright and cell 11992 is dim The 50 Thresh old mask is similar for both images whereas the Intensity mask 250 is quite dif ferent since only a few pixels in the dim image are greater than 250 counts while most of the metaphase plates in the bright image are masked gt ae a Threshold 50 Intensity 250 1023 Application Example Used with the Area feature to define apoptotic cells in the Apoptosis Wiz ard 266 About Masks Valley Mask The Valley mask is a rectangular mask that sits between two bright regions in a start ing mask such as between two nuclei It is constructed by finding the minimum intensity along the skeletal line between these two bright regions The skeletal line is obtained internally using the skeleton thin masking as described in Skeleton Mask This minimum intensity identifies the intersection between the two objects The mask is drawn perpendicular to this skeletal like The length of the valley mask rec tangle is equal to the minor axis of the object and the width of the mask is defined by the user in pixels Application example Quantify the intensity of a probe in an immune synapse 267 Appendix Troubleshooting Appendix Troubleshooting Chapter Appendix A Troubleshooting This chapter co
107. ell The spatial offset spatial offset is measured during calibration and the values are saved to the image database The table used by the compensation matrix to place the detected light Table of Coefficients that is displayed in each image into the proper channels on a pixel by pixel basis A file that saves the set of instructions for an analysis session Note that a template contains no data it simply contains the structure for the analysis This structure includes definitions of features graphs regions and populations image viewing settings channel names and statistics settings soe Index Index Acquisition information 72 Advanced Analysis 49 Analysis Area adding an image panel 103 adding text 110 overview 94 printing 145 155 tools 94 Apoptosis wizard 33 Application defaults 5 132 Area 171 ast about 19 Batch processing 74 Brightfield information 73 Building Blocks 47 Fluorescence Positives one color 47 Fluorescence Positives two color 47 Focus 47 Single Cell 47 Single Cell Default 47 275 Chapter Appendix A Size SSC 47 Tool 95 Camera settings information 73 Cell Classifiers 73 Cell Cycle using a wizard 35 Channels collected 73 cif merging 70 opening 53 saving 56 cif about 18 Co localization using a wizard 37 Color mapping dark light mode defaults 10 137 Show in Image Gallery 80 Compare FlowSight FlowSight QI ImageStream 15 Compensation adjusting compensation popula
108. en return to the Image Gallery Properties window and click Preview Changes in Gallery Continue customizing the Image Gallery display properties with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes To create a composite 1 Within the Image Gallery Properties window click the Composites tab The list of existing composites is shown on the left 85 Chapter 3 CON OO A z Image Gallery Properties Display Properties Views Composites NFkB DRAQ5 E Translocation Name NFkB DRAGS Object 0 NFkB DRAQS NFkB 100 Image DRAQS 100 NFkB F Percent 100 In the right section of the window type a name for the composite or leave blank to allow the name to be built automatically from the image names added to the composite Click Add Image The selected image appears in the Object box Change the Percent if desired The percent specifies the percentage of of the image to include in the composite Tip As you make the changes the image in the Object box updates accordingly If you want to preview any new changes in the Image Gallery return to the Image Gallery and select the View drop down menu to your new view Then return to the Image Gallery Properties window and click Preview Changes in Gallery Continue to add images as desired To remove and image from the composite Click Remove Image The composite is automatically added to the l
109. enssa f ts E esl ia Application Examples Identify the center of peak fluorescence Calculate the distance between two fluorescent markers Used by IDEAS to calculate the intensity weighted Delta Centroid X Y or XY Delta Centroid X and Delta Centroid Y Features Both the Delta Centroid X and Y features measure the distance between the Cen troids X or Centroids Y respectively of two images using the user provided masks for each image Either one or both the centroids of the images may be intensity weighted X and Y pixel coordinates are calculated from an origin in the upper left corner to obtain the centroid positions and the distance between the centroids is con verted to microns 185 Chapter 4 An example is shown below PE Podo Composite gt O Cc Q O PE Podo Delta Centroid Y Delta Centroid X Centroid X Delta Centroid XY v Delta Centroid X Delta Centroid Y The graph below illustrates using the Delta Centroid X versus Delta Centroid Y to identify cells with a variation of location of a protein with respect to the nucleus Cells with no spatial shift of signal between the nuclear stain Ch6 and protein of interest Ch4 have a low Delta Centroid X and Y and are found in the lower left corner Cells with a large shift between the images in both the X and Y direction are found in the upper right section and those with a large shift in X but not Y are found in
110. eported directly from an open daf from the graph or statistics tables in the analysis area To export graph statistics to the Clipboard e Right click a graph and then click Export Statistics To Clipboard They are then available for pasting into a third party application To copy population statistics from a Statistics Table e Right click the table and then click Copy Statistics or Copy Statistics transposed They are then available for pasting into a third party application 151 Chapter 3 Exporting Data You can export feature values for a population to the Clipboard a text file or a Flow Cytometry Standard FCS file You can export pixel intensity values for an object to the Clipboard or a text file Later you can open or paste the FCS file into a spread sheet or other programs that uses the FCS file format Keep in mind however that limitations might exist on the number of values that these programs can import Exporting Feature Data Exporting feature data is useful if you want to create an fcs file or graph the feature data in a third party graphing application To export feature data 1 Onthe Tools menu click Export Feature Values The Export Feature Data window appears S Export Feature Data Select daf files to process Select features to export Area_M02 Area_MO6 Area MC Aspect Ratio Intensity_M01_Ch01 Aspect Ratio Intensity_M02_Ch02 Aspect Ratio Intensity_MO6_Ch06 Aspect Ratio_M01 Aspect Ratio
111. equency and change the bin count The default font sizes are used you may change them by clicking Font Sizes Assign colors by Population default for dot plots or by Density for density plots Set the scaling for each axis of the graph The default is Auto which allows the application to automatically scale the graph To set minimum and maximum values for an axis select Manual Select Linear or Log and enter Maximum and Minimum limits If you selected Log enter the X gt value Note You can scale the X Axis of a graph or the Y Axis of a scatter plot in one of two modes Linear or Log The Linear mode is the default The Log mode allows you to logarithmically scale a section of the graph or scatter plot Selecting this mode causes the IDEAS application to perform bi exponential plotting The gt X value defines the linear portion of the graph as X through X The application plots the values outside of these limits on a log 96 Overview of the Analysis Area arithmic scale You can plot negative values as well as positive ones on a log arithmic scale by adjusting the limits Take care not to split a population such that it appears to be two separate pop ulations This splitting is especially likely when negative values exist due to com pensation or corrections on the imagery The graph on the left side was plotted on a linear scale the ones in the center and on the right side were plotted on log arithmic scales Th
112. er Thickness Max and Min Spot Area Min Width and Height 170 Understanding the Size Features Area Feature The number of microns squared in a mask is equal to the Area In the following fig ure a1 symbolizes whether the area is included jn the mask The number of pixels is converted to ym Note that 1 pixel 0 25 um As an es acellwitha mask that includes 2000 pixels is therefore equal to 500 m Brightfield ae ie al P f bf Channel 5 PI DNA Application Examples Quantify and compare cell size Identify single cells Calculate the radius diameter and volume of the cell Identify apoptosis using the Area of the 30 threshold mask of a nuclear dye Create a pseudo FSC va SSC plot for comparing with flow cytometry 171 Chapter 4 Diameter Feature Wormadzed Frequency Diamekr_BrighWen The Diameter feature provides the diameter of the circle that has the same area as the object The accuracy of the diameter is highly dependent on a close fitting mask and roundness of the cell Area T Diameter 2x The images below depicts beads with a uniform diameter of 9 microns In the next figure note that images with longer shapes that have the same area will have the same diameter value Focus Brightfield Brightfield Brightfield Brightfield Brightfield Brightfield Application Example Used to obtain approximate size of the cell 172
113. erview of Compensation Creating a New Compensation Matrix File Viewing Sample Information Merging Data Files Creating new data files from populations Batch Processing 49 Chapter 3 Opening data files Use the File menu which is shown in the following figure to open save and close image and analysis files and to quit the IDEAS application Alternatively you may open a data file by drag and drop into an open IDEAS window Muliple data files can be open in one instance of the IDEAS application File Opening a rif file A rif file is opened when there is new data and the IDEAS application needs to apply corrections When opening a rif file the IDEAS application corrects each image for the spatial alignment between channels camera background normalization flow speed and brightfield gain normalization If you want fluorescence compensation to correct for spectral overlap you must create or choose a compensation matrix at this time by using the control files that were collected for a particular experiment If a FlowSight data file was acquired with a compensation matrix that matrix will be used by default For more information on compensation see Creating a New Com pensation Matrix File The application performs the corrections by using calibration information that was saved to the rif file during acquisition To open a rif file To use a wizard to do this see Open File Wizard otherwise 1 From t
114. es and can be used to quantify the co local ization of two probes in a defined region such as that of endosomes The Bright Detail Similarity R3 feature is the log transformed Pearson s correlation coefficient of the localized bright spots with a radius of 3 pixels or less within the masked area in the two input images Since the bright spots in the two images are either cor related in the same spatial location or uncorrelated in different spatial locations the correlation coefficient varies between 0 uncorrelated and 1 perfect correlation and does not assume negative values The coefficient is log transformed to increase the dynamic range between 0 inf The following figure shows the Bright Detail Similarity R3 graph of two populations one that has colocalization and one that has no colocalization Double Positive Not Colocalized Colocalized 0 i i 0 3 6 Bright Detail Similarity R3_C3 amp C4 The figure below illustrates the process of obtaining the localized bright spots The bright areas are eroded from the original image and the detail eroded image is sub tracted from the original image resulting in the bright detail image 235 Chapter 4 Original Image Detail Eroded Image Bright Detail Image The figure below shows the correlation analysis between an image pair Non specifically Localized Cell ADC Image Endosomes image Codocalized Cell RTX HBAS ADC Image AF 488 RTX End
115. es with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes To customize the Image Gallery views images and masks 1 Within the Image Gallery Properties window click the Views tab Note The Image Gallery view can be customized to view any combination of channel images or composites The default view All Channels is a view that displays all image channels that were included during acquisition of the file with their associated default masks This mask may be changed for the default view however the images in this view cannot be changed The list of existing views is shown on the left i Image Gallery Properties Display Properties Views Composites Views View Definition EH All Channels H BF Name All Channels Ch2 E Ch4 H All Channels DRAQS SSC mask M01 Ch2 mask M02 NFkB mask M03 Ch4 mask M04 BF mask M05 DRAQ5 mask M06 NFkB i SSC 141 Chapter 3 2 Tocreate a new view Click New The new view is automatically added to the list on the left 3 Inthe right section of the window type in a name for the view 4 Click Add Column 5 Define the column by selecting an image and a mask or a composite from the dropdown menu 6 Repeat the previous 2 steps until finished adding columns to the view A column will be added under the column currently selected To insert a column click on the
116. ether too dim to bright or too small to count and can be eliminated from the analysis 192 Understanding the Location Features Valley X and Valley Y Features The Valley X and Y are the exact X Y coordinates of the minimum intensity within the skeletal lines of the input mask The objects condensed shape typically 1 pixel wide skeletal line is determined from the starting mask This is also the origin of the Valley mask See Valley Mask and Skeleton Mask In the figure below the Valley X and Valley Y position of the 7AAD image is shown Inthis example a protein of interest in the PE image localizes to the synapse between two cells 62 ZAAD maae alley _Skeleton M5 7AAD Thin _7 alley Y_Skeleton M5 744D Thin _7 FAAD image PE Image Pixel 43 67 Intensity 38 These features define the origin of the Valley mask 193 Chapter 4 Application Example Measure the exact center of where a synapse between two cells is located 194 Understanding the Shape Features Understanding the Shape Features Shape features define the mask shape and have units that vary with the feature They include the Aspect Ratio Aspect Ratio Intensity Compactness Elon gatedness Lobe Count and Symmetry 2 3 4 195 Chapter 4 Aspect Ratio Feature Aspect Ratio is the Minor Axis divided by the Major Axis and describes how round or oblong an object is See also Major Axis and Mino
117. f a probe Shape Change Wizard Guides you through the process of creating the features and graphs for analyzing the circular shape of a cell using a surface stain or brightfield image Spot Wizard Guides you through the process of creating the mask feature and graphs for analyzing fluorescently labeled spots in images The wizard window is organized so that the instructions for each step are written in the left side of the window the stepwise progress through the wizard is shown in the list on the right side and there may be tips provided at the bottom of the window 24 Guided Analysis Click Next to progress through the wizard or Exit to stop at any time Some steps are optional and a Skip button is provided Follow the instructions in the wizard to complete an analysis in aw a region on the single cells on the Area vs Aspect Ratio scatter plot BET Select nuclear image to create a single cells population 2 Gate single cells 3 Gate cells in best focus 4 Gate fluorescence positives Select an existing single cells population 5 Select subpopulation marker s 6 Gate subpopulation s 7 Select additional subpopulation marker 8 Gate additional subpopulation s 9 Gate apoptotic events Or Single Cells Al A Q Click on the dots in the scatter plot to see the comesponding image in the fage gallery Tip2 f you wish to change the plot properties right click on the plot and sel
118. f the Software Amnis reserves all rights in the Software not expressly granted to you in this Agreement For purposes of this Agreement Execute and Execution means to load install and run the Software in order to benefit from its functionality as designed by Amnis 2 Restrictions Except as expressly specified in this Agreement you may not a copy except in the course of loading or installing or modify the Software including but not lim ited to adding new features or otherwise making adaptations that alter the functioning of the Software b transfer sublicense lease lend rent or otherwise distribute the Software to any third party or c make the functionality of the Software available to multiple users other than the users of the single computer for which it is licensed through any means including but not limited to uploading the Software to a network or file sharing service or through any hosting application services provider service bureau software as a service SaaS or any other type of services You acknowledge and agree that portions of the Soft ware including but not limited to the source code file formats and the specific design and structure of individual modules or programs constitute or contain trade secrets of Amnis and its licensors Accordingly you agree not to disassemble decompile or reverse engineer the Software or data files in whole or in part or permit or authorize a third party to do so ex
119. feature set The default option is used for the default segmentation masks The tight option uses a dif ferent set of features to characterize the background which results in a tighter fit around the cell Examples are shown below Object Object default tight Image Application Examples Used to get a close fit around the cellular area tight option Can be used in lieu of the morph mask for applications where the morph is so tight that it provides incomplete masking sometimes splitting cells into two regions such as a nuclear dye image of cells in anaphase or telo phase Can be used in lieu of the morphology mask with the Similarity feature when measuring nuclear translocation for better separation between untranslocated and translocated cells tight option Used as the default segmentation masks default option 259 Chapter 4 Peak Mask The Peak mask identifies intensity areas from an image that have local maxima bright or minima dark Initially the peak mask will identify all peaks in the image To select peaks which have certain brightness the spot to cell background ratio is used This is the ratio between the spot pixel value to the mean camera background value in the original image Below is an example of the Peak bright option i Spot Mask Peak Mask These peak areas will be masked Application Examples Used with the Spot Count feature to quantify the sp
120. files Note The default template may change between releases of the IDEAS application software In IDEAS versions 3 0 or later a daf file may be used as a template The default template contains over 200 calculated features per object An expanded tem plate is also available that includes over 600 calculated features per object The FlowSight without the Quantitative Imaging upgrade has a limited set of features available Compensation Matrix File ctm The IDEAS application saves the compensation values that are created and saved during the spectral compensation of control files to a compensation matrix file ctm file This file has no associated object data it is created and saved to be applied to experimental files The compensation matrix can be created in IDEAS using single 19 Chapter 2 color control files after acquisition or during acquisition See the INSPIRE or Flow Sight manual for more information Review of Data File Types Table 1 Review of Data File Types Name Created in INSPIRE User creates a cif from the rif and ctm References the cif Created from the daf User creates new ctm when opening a rif or during acqui sition Contains instrument setup data pixel intensity data and uncorrected image data from the INSPIRE appli cation The IDEAS application uses the rif file to create a compensated image file cif file Contains imagery that has been corrected for variations i
121. g Channel 11 12 Click Close 13 To view the resulting morphology masks open the Image Display Properties win dow and if necessary select the new mask s for the channel oOwonNnon ul Icon for Image Display Properties 14 Next you will subtract the nuclear morphology mask from the intracellular mask In the Mask Manager window click New 15 Double click the Morphology Intracellular mask in the list 16 Click the AND button on the toolbar 17 Click the NOT button on the toolbar o 18 Double click Morphology Nuclear mask in the list 19 Enter a new mask name 20 Click OK to add this mask to the list 21 Click Close 22 To view the resulting mask on a Channel 3 image open the Image Display Prop erties window and select the new mask for the channel in the view you are using 93 Chapter 3 Overview of the Analysis Area The Analysis Area provides display space for individual images plots of cellular fea ture values tables of population statistics tables of object feature values and text annotations You can select different layouts for the IDEAS window and placement of the analysis area and expand the Analysis Area by dragging it s boundaries The graphs are created into panels of a default size and can be re sized by dragging acorner or using the size tool The position of the panels is automatically adjusted to fit in the available display space A vertical scroll bar appears when the number of panel
122. ge of intensities from the camera is 0 4095 for the ImageStream or 0 32 767 for EDF mode collection The 1S 100 first generation instrument has a 10 bit camera and therefore the range of pixel intensities is 0 1023 The limits of the graph enable you to use the full dynamic range of the display to map the pixel intensities of the image 255 5 200 0 0 1000 2000 3000 4095 At each intensity on the X Axis of the graph the gray histogram shows the number of pixels in the image This histogram provides you with a general sense of the range of pixel intensities in the image The dotted green line maps the pixel intensities to the display intensities which are in the 0 255 range Manual setting is done by Click dragging the vertical green line on the left side crossing the X Axis at 0 allows you to set the display pixel intensity to 0 for all intensities that appear to the left of that line Doing so removes background noise from the image Click dragging the vertical green line on the right side allows you to set the dis play pixel intensity to 255 for all intensities that appear to the right of that line From the Image Gallery window select the object to use for setting the mapping It appears in the Image Gallery Properties window Tip You might need to select different objects for different channels because an object might not fluoresce in all channels To adjust the pixel mapping for display click drag the vertical
123. gin Analysis 28 Cell Cycle Mitosis 35 Co localization 37 Display Propertes 27 Feature Finder 29 Internalization 39 list 24 Nuclear Localization 41 Open File 26 Shape Change 43 Spot Count 45 Tool 95 Workflow data analysis workflow 16 Zoom Image Gallery 80 289
124. greater the concentration of inten sity inside the cell All pixels are background subtracted The user must create a mask to define the inside of the cell for this feature see About Masks and Overview of the Mask Manager The feature is invariant to cell size and can accommodate concentrated bright regions and small dim spots The ratio is mapped to a log scale to increase the dynamic range to values between inf inf Internalized cells typ ically have positive scores while cells with little internalization have negative scores Cells with scores around 0 have a mix of internalization and membrane intensity Composite Images of brightfield and channel 6 are shown for High Medium and Low Internalization values Mid Internalization Low Internalization High Internalization Application Examples Quantify internalization when supplied with the internal mask Quantify the intensity ratio of a region of interest to the whole cell Used in the Internalization Wizard 238 Understanding the Comparison Features Similarity Feature The Similarity feature is the log transformed Pearson s Correlation Coefficient and is a measure of the degree to which two images are linearly correlated within a masked region The following figure shows two image pairs that are in spatial registry to one another On the left the NF kB green is predominantly located in the cytoplasm of the cell and has a dissimilar distrib
125. green line by click ing near it but not near the yellow cross Tip For fluorescence channels set the vertical green line that appears on the left side to the right of the large peak of background pixel intensities as shown above and set the right vertical green line to the right of the brightest pixel intensities Click Set Linear Curve to make the transformation linear For the brightfield channel set the vertical lines to about 50 counts to the right and left of the his togram to produce an image with crisp brightfield contrast e Tochange the mapping curve to be logarithmic or exponential click drag the yellow cross To restore the mapping to a linear curve Click Set Linear Curve e To see the full scale for the X Axis Click Full Scale e Toset the display mapping of the X Axis to the lowest and highest values for a selected object Click Set Range to Pixel Data 140 Creating Reports and Exporting Data To set the scale of the X Axis to the range of the vertical green lines or of all the pixel intensities for the selected object whichever is larger Click Auto scale e You may enter values manually by selecting the Manual tab Automatic Manual Image Display Mapping x Axis Scale Set Range to Pixel Data Full Scale Set Linear Curve Autoscale 4 Ifyou want to preview the changes in the Image Gallery click Preview Changes in Gallery 5 Continue customizing the Image Gallery display properti
126. h populations either use the tagging tools or gate the cells of interest Next Step Gate spot events A histogram of the Spot Count feature for the last gated population is added to the analysis area Regions have been drawn that include the truth populations Adjust the regions as necessary Note that you may want to adjust your truth populations and repeat the wizard after looking at the images and validating the spot counts o a gt o E E S 2 ra 3 D N E S 0 5 10 15 20 Spot Count_Peak M03 Channel 3 Bright 8 5 The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 46 Building Blocks Building Blocks Building blocks may be used to create a graph for finding single cells focused cells or positive cells based on Intensity The building blocks are shortcuts to creating a graph that provide a limited list of relevant features with set X and Y axis scales set for the graph For more information on creating graphs see Creating Graphs Table 1 Building blocks a ene l one color for all channels two color for all channels for all channels Gradient RMS_MX_ChX Foc for all channels Note Gradient RMS of brightfield is default Area_brightfield default Aspect Ratio_brightfield default Area_sca
127. h translocation Note that for a normally dis tributed population you may want to report the RD of the double positive population in a treated versus untreated sample instead of the percentage gated Nuclear localization of a probe is measured using the Similarity feature in the final graph presented in the wizard The example shown here is of THP 1 cells stimulated with 1 ug LPS for 90 minutes and stained with DRAGS red and NFkB green to measure the nuclear localization of the NFkB Untreated Stimulated For a more thorough explanation of the Similarity feature see Similarity Feature The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 42 Guided Analysis Shape Change Wizard This wizard will create an analysis template for measuring the shape circularity of any population of cells you identify Shape Change To begin double click on Shape Change Follow the instructions to open and analyze your file Step 1 Select the cell morphology image channel From the drop down menu pick the channel for the cell image Step 2 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher
128. hapter 3 Overview of the Data Analysis Tools The IDEAS application provides a powerful tool set that allows you to explore and analyze data The rich feature set lets you create hundreds of your own features to differentiate objects and statistically quantify your results As shown in the following figure the application window is divided into two panels the Image Gallery and Analysis Area which each provide the corresponding tools that you can use for data analysis The layout can be changed to side by side or top and bottom with re sizable panels IDEAS 5o 1OVA D584 A 30 Sodat SE E te FE File Guided Anelyss Analysis Compensation Tools Options Reports Windows Help ax t ARAA KS LEM BAL E gt bie RoR saa Fopusten rangle Ted Sdawle amp Focus boorjugates E Vow ew EF DaS ADAFASE E Das ADAPIAGiT 100 2 0 6 Gradient RMS_EF amo B Pe Image Gallery 9 Population Statistics Ttersty_NC_ChO tensiy_MC C03 btensty_MC_CF03 trtensty_MC Mean Std Dev Nels Mean Populaton 1 555600 138162004 23456004 2 31 te 004 3035 3012529 45195 39 121974 3559 42371 You can create populations of objects by tagging hand selected images drawing regions on graphs and using Boolean logic to combine existing populations After you have created a population you can view it in the Image Gallery or plot it ona graph You can view the statistics for pop
129. he Analysis Area to view pixel positions and intensities as well as generate statistics for an area of the image You can also show the Measurement tool for the image Image panels which are shown in the following figure each contain a toolbar in the upper right corner and a context menu that appears when you right click an image An image in the Analysis Area is three times the size of an image in the Image Gal lery To add an image panel to the Analysis Area e Right click an image in the Image Gallery or Analysis Area and click Add Image to Analysis Areaon the context menu that appears The image panel appears in the Analysis Area 103 Chapter 3 Qa oe To view the individual pixel intensities of a single channel image e Move the mouse pointer across the image The pixel positions and intensities appear under the image The pixel 0 0 is positioned at the upper left of the image Qa ow To display the Measurement tool in an image panel e Right click the image panel and click Show Measurement Tool on the con text menu that appears The 10 micron bar appears 104 Overview of the Analysis Area 0 Microns 10 To examine a line profile or the statistics for an area of an image e While holding the Ctrl key click and drag to create a boxed area on the image The Image Statistics are shown in the image panel The statistics are cal culated for the area that is defined by the box The li
130. he File menu choose Open or drag the file into the IDEAS window 2 Select the rif file that you want in the Select File To Load window Tip while browsing for the file to open you can limit the type of file shown in the win dow to rif files 50 Opening data files Select File To Load B tits 4 em 0 0ng_2_9 rif 121906 C16 72 06 DRAQS _ noBF4 rif 4 0 1ng 15_1_8 rif 121906 C16 72 06 DRAQS _ noBF4_m rif My Recent 0 1ng 30_6_13 rif 121906 C16 72 06 FITC_ noBF3 riF Documents 5 0 1ng 45_11_18 rif 0 1ng 60_16_23 riF E 5 0 1ng 75_21_4 rif 0 1ng 90_26_9 rif 5 10ng 15_3_10 rif 10ng 30_8_15 rif it 10ng 45_13_20 rif 10ng 60_18_1 rif 10ng 75_23_6 rif 10ng 90_28_11 iF 1000ng 15_5_12 rif 1 1000ng 30_10_17 rif My Computer 1000mg 45_15_22 rif 1000mg 60_20_3 rif a 1000ng 75_25_8 rif t 1000n9 90_30_13 rif My Network Places File name 0 1ng 1518 1 Raw image files rif v IDEAS files rif cit daf Raw image files ril Compensated image files cif Files of type Data analysis files daf In the next window you will e Choose a compensation matrix e Choose a template e Name the output files e Choose the number of events to process 51 Chapter 3 z Opening C raining Data Files 3 0 NFkB Translocation Dose an A To
131. he control files for compensation To compensate data collected on a single camera instrument the control files must contain all 6 channels To compensate data collected on a two camera instrument the control files must contain all 12 channels Control Files Add Fies Remove Files rer Geet 2 Add compensation control files by clicking Add Files and browsing for the control files for the experiment The files will have the suffix no BF Hold down the con trol key to select multiple files at once 3 When all of the control files for the experiment have been added to the list click Next The control file s are merged and loaded 60 Overview of Compensation 4 Step 2 Verify the channels for each control in the experiment by checking the channel boxes Greats Compare Step 2 Select remove channels for compensation Verify that the selected channels are appropriate for your selected controls Make any appropriate changes and click Next Ch01 v Ch02 V Ch04 v Ch05 V Ch07 V Ch08 Ch10 v Ch11 Previous Cancel The following tables are provided as a guide for each instrument configuration Table 1 First generation ImageStream IS100 C e e T a 470 500nm 400 470nm 500 560nm 560 595nm 595 660nm 660 735nm Table 2 ae 1 camera Ch6 es een DAPI FITC sare OS eo APC Cy7 asRed Table 3 ImageStream 2 camera or FlowSight a a a a
132. he first step is to define your application defaults image gallery settings and statistics report definitions This section covers the fol lowing topics that will help you to create reports Viewing and Changing the Application Defaults Setting the Image Gallery Properties Prepare the image gallery and analysis area for reporting Copy full or partial screens Copy images to the clipboard or save images to files Copy graphs to the clipboard or save graphs to a file Reporting Statistics 131 Chapter 3 Exporting Data into other analysis applications Printing Data Viewing and Changing the Application Defaults To view or change these defaults chooseApplication Defaults from the Options menu Each tab allows you to view or change the default settings 4 ee a Populations Masks Graph Display Graph Export Image Export Colors Default Data Files Directory C Users sfriend Desktop NFKB Translocation FS V Update automatically when file is selected Default Template Files Directory C Users sfriend App Data Roaming Amnis Corporation templates E Update automatically when file is selected V Use default data directory Default Batch Report Files Directory C Users sfriend App Data Roaming Amnis Corporation batches E Update automatically when file is selected Default Compensation Matrix Files Directory C Users sfriend App Data Roaming Amnis Corporation compen
133. he object flow during the run Note Use the statistic Con centration to obtain objects ml of a population OAT Chapter 4 Objects sec Feature The Objects per sec feature returns the local object concentration with respect to time Application Example Monitor the throughput during a run Note Use the statistic Concentration to obtain objects ml of a population 248 Camera Timer Feature Time Feature The Time feature returns the camera timer values that are in ticks converted to secs with a formula Application Example Obtain the time taken to collect a sample 249 Chapter 4 About Masks The set of pixels that contains the region of interest is called the mask In the fol lowing picture the mask consists of the set of pixels on the right image that are col ored cyan The cell is represented in the greyscale image on the left Calculating some feature values such as the Area value requires only amask Calculating others such as Intensity value requires a mask and a channel image There are three types of masks Default masks Combined Masks and Function Masks 1 Default masks named M01 through M12 are either created in INSPIRE during acquisition or created in IDEAS when a rif file is opened The default mask used by INSPIRE during acquisition Inspire is different than the default mask created in IDEAS Default Object when a rif file is opened with QI or from an ImageS tr
134. if file in the 56 Saving Data Files same directory as the rif file The cif file will be larger than the rif file because the cif file contains masking information as well as corrected and or compensated images Saving a Template ast Saving an analysis as a template allows you to apply the structure of the analysis to other cif files Save a template file after saving a daf file A template includes all graph definitions Image Gallery settings feature definitions and statistics settings No data are saved in a template Therefore selected images and populations that are dependent on specific objects such as tagged populations are not saved To save a template 1 Onthe File menu click Save As Template File ast A Save File dialog box appears Enter the name of the file to save 3 Click Save If you select an existing file name a warning appears that asks you to verify the overwriting of the existing file Tip You can change the default template directory in the menu Analysis gt Appli cation Defaults 257 x Chapter 3 Overview of Compensation Spectral compensation corrects imagery for fluorescence that leaks into nearby channels so that you may accurately depict the correct amount of fluorescence in each cell image For example the light from one fluorochrome may appear primarily in channel 3 but some of the light from this fluorochrome may appear in channel 4 as well because the emission
135. image e Overcompensation crosstalk coefficient is too high Plots Intensity mean for the single color positive population is lower than the unlabeled population in the crosstalk channel or the intensity in the crosstalk channel trends diagonally downwards Images the crosstalk channel contains dark spots corresponding to the bright spots in the fluorescent channel of interest 4 Inthe Compensation menu choose View Edit Matrix and manually change the incorrect crosstalk matrix values identified above Start with changes of 1 or 05 and use smaller and smaller increments as you refine the matrix 5 Click Preview and choose the tagged population to view the results of the changed coefficient 6 Repeat steps 4 and 5 until the matrix is corrected 7 Click Save append manual to the matrix name then click OK 8 Open the cif file and use the new matrix to create a new daf file Creating a TIFF If you cannot see the TIFF image that you created trying changing the resolution to 8 bit 271 Chapter Appendix A Deleting a Population and Region Often a user deletes a population but forgets to delete the region Deleting a pop ulation does not delete the region You must delete the region itself Object Number set to Zero When opening a daf file there may be an error if the object number is set to zero This can happen if the data was collected during a crash within INSPIRE This error can be corrected with the fol
136. image above insertion point 7 Columns may be removed by clicking on Remove Column 8 Aview may be edited at any time by selecting the view and following the same procedures 9 Ifyou want to delete a view click the view to select it and then click Delete A confirmation window appears 10 If you want to preview any new changes in the Image Gallery return to the Image Gallery and choose your new view in the View drop down menu Then return to the Image Gallery Properties window and click Preview Changes in Gallery 11 Continue customizing the Image Gallery display properties with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes To create a composite 1 Within the Image Gallery Properties window click the Composites tab The list of existing composites is shown on the left i Image Gallery Properties Display Properties Views Composites NFkB DRAGS E Translocation Name NFkB DRAGS Object 0 NFkB DRAGS NFKB 100 Image DRAGS 100 NFkB al Percent 100 2 Inthe right section of the window type a name for the composite or leave blank to allow the name to be built automatically from the image names added to the composite 142 3 ON OO A Creating Reports and Exporting Data Click Add Image The selected image appears in the Object box Change the Percent if desired The percent specifies the percentage of of the im
137. ine Mask Function window appears with 15 available masks to use SeeAbout Masks for mask definitions e Dilate Erode e Fill e Inspire e Intensity e Interface e Morphology e Object e Peak e Range e Skeleton e Spot e System e Threshold e Valley 89 Chapter 3 G Select an object and image to display Image Ch01 LL er in me REL 10 12 14 16 19 4 Select a function and choose the input mask s channel and scalar parameters as needed The right side of the window adjusts the display and view of the chan nel image e To view a different object in the file select it in the Object list or type it s number e To view a different image for the object select it from the list 5 The Link inputs checkbox is checked by default To modify a mask with different inputs uncheck this box 6 Click OK 7 The new function is added to the mask definition 8 Click OK The new mask name will appear in the list of Masks on the left side To create a new combined mask 1 Select Analysis gt Masks 2 Click New 3 Use the Masks list on the left and the Definition toolbar to build a new mask using the definitions of existing masks with Boolean logic explained in the table below 90 Overview of the Mask Manager Table 1 Mask Tasks and Toolbar Double click the feature in the Masks list Adda mask tothe Or single click the feature in the Masks list and click the
138. ion of this window To change the name or color for each image 1 Select an image in the list of images on the Display Properties tab of the Image Gallery Properties window On the right side of the window you can type a new unique name for the selected image Note that each image is provided with a default name and the image names appear near the top of the Image Gallery Click the colored square for the selected image Click the color that you want in the color palette Click OK to close the palette Tip The grayscale image in each channel is assigned a default color for image display in the gallery Setting the color to white is equivalent to using the original grayscale image The colors are also used to build composite images To fine tune the image display intensity for an image 1 On the Display Properties tab of the Image Gallery Properties window select an image by clicking the image name in the list The graph for the currently selected image is shown in the window and updates as the changes are made Select and image in the image gallery that has intensities for the image channel you are adjusting 139 Chapter 3 Note You will adjust the Display Intensity settings on the graph the Y Axis the value of the display to the X axis the range of pixel intensities The range of pixel intensities will depend on the instrument and the collection mode set during acquisition The display range is 0 255 the ran
139. ion that may be viewed in the Image Gallery or on other graphs To change the attributes of a region or delete a region and the populations dependent on that region see Using the Region Manager To draw a region on a Scatter Plot On the Analysis Area toolbar click either the e Rectangle Region or e Oval Region or La e Polygon Region button on the Analysis Area toolbar b amp b 99 Chapter 3 1 To The Rectangle and Oval tools work by clicking on the graph at the point where you would like to start the region and drag to the region endpoint The region grows as you drag l l 5e3 led 1 5e4 2e4 Click again to complete the region If you are drawing a region on a histogram or scatter plot the Create a Region window appears Name the region Click the colored box to select an alternate color Select Use for statistics only if you do not want to create a population from this region Click OK The region appears on the graph with the name and color that you selected Polygon Tool Option The Polygon tool works by clicking the scatter plot at the point where you would like to start the polygon Click once for each vertex of the polygon Double click to complete the drawing of the region A window appears that allows you to name the population created by the polygon region and to assign the region s display properties Click OK The region appears on the graph with the name and color th
140. is distance Thus the closer the object to a circle the smaller the variation and therefore the feature value will be high Vice versa the more the shape deviates from a circle the higher the variation and therefore the Cir cularity value will be low See also Below is an example using Circularity and Compactness to characterize the shape of peripheral blood mononuclear cells stained with the DNA dye Draq 5 z iD E ji n wE o jz ba E I eS 20 Circu larity_D raq 5 198 Understanding the Shape Features Brighttield Draq 5 Circularity Compactness Application Examples Distinguish singlets and doublets Separate circular and non circular shapes Used in the Shape Change Wizard 199 Chapter 4 Compactness Feature Compactness measures the degree of how well the object is packed together This feature is similar to the Circularity feature but unlike Circularity this feature includes all of the pixels within the mask and is intensity weighted The higher the value the more condensed the object Below is an example using Circularity and Compactness to characterize the shape of peripheral blood mononuclear cells stained with the DNA dye Draq 5 wo Comp actne ss_D raq 5 M Ws 20 Circu larity _D raq 5 Nuclear Brightfield Draq 5 Circularity Application Example Differentiate between rounded objects with smooth boundary to less reg ular objects
141. is required for and RD 7 Select a Feature This is not necessary for the related statistics Count or Objects ml 8 Click Add Statistics The statistic is added to the list 9 Click Close when finished 10 Select a statistic in the list to view the definition or edit any input 11 Change the name of the statistic by unchecking Use default title and typing a new name if desired 12 Delete Columns removes a selected statistic 13 To reorder the list click drag a statistic to it s new location 14 Click Generate Report when complete to generate a report for a current opened daf file A prompt appears to save the text file This text file can be opened from Excel 15 If you do not want to generate a report click OK to save your changes and exit the window 16 The saved template can generate a statistics report for multiple data files by selecting Generate Statistics Report from the Reports menu or during batch proc essing 149 Chapter 3 Generating a Statistics Report using daf Files Once a Statistics Definition has been created the user can generate a statistics report from multiple daf files However these files must use the same template Batch Processing can also generate a statistics report where statistics for each data file will be generated either for rif cif or daf files Generating a statistics report under the Reports menu simply adds the statistics template to the specified daf files To Gene
142. ist on the left A composite can be removed from the list by clicking Delete Continue customizing the Image Gallery display properties with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes Working with Individual Images You can work with individual images in the Image Gallery You can zoom in or out on the images You can add a larger version of an image to the Analysis Area for further analysis show or hide masks for a single image in the Image Gallery and copy one or more images to the Clipboard To manipulate individual images 1 In the Image Gallery right click an image that you are interested in A menu appears 86 Overview of the Image GalleryUsing the Image Gallery Add Image to Analysis Area Show Masks Color On Show Saturation Color Copy Save Image Copy Save Object Images Copy Save Gallery Column Copy Save Gallery e To place the image in the Analysis Area click Add Image to Analysis Area For more information see Analyzing Individual Images To show or hide the masks for the object image click Show Masks or Hide Masks respectively One or the other will appear depending on the current state To turn the colors on or off for the object image click Color On or Color Off respectively One or the other will appear depending on the current state e To show or hide the saturation color for the object image click
143. ity_MC_Ch12 Coefficient value 9 971 Coefficient eror 0 012 Add Graph to Analysis Area 2 If you notice outliers click Add Graph to Analysis Area The plot populates in the Analysis Area 3 Return to the Analysis Area and use the region tools to draw a new region on the plot that defines a new positive control population excluding the outliers Refer to Creating Regions on Graphs for more information e Create a new region to exclude outliers Click the Resize 9 and Zoom 4 buttons on the graph toolbar to more clearly see the population of interest Using one of the region buttons on the tool bar draw a region that contains only the cells you want to use for determining compensation You can click a point on the graph and view the image to help you decide where the region boundaries should be In the example below the Polygon Region tool was selected to draw a border around a selection of cells Clicking within the graph anchors the line and double clicking completes the region 12_Positive ages 2e4 4 Use for statistics only Intensity_MC_ChO7 Intensity_MC_Ch12 For more information refer to Creating Regions on Graphs 4 Assign the new population to the appropriate channel by using the Positive Con trol Populations list for that channel 65 Chapter 3 Best Fit Chol ChO2 ChO3 ChO4 Ch05 Ch06 S Create Compensation Matrix Ch01 Ch02 0
144. j Default Symbol Simple Dot The Masks tab contains the default mask color To change the color of the mask click on the color button Application Defaults EN kx bc Directories Populations Masks Graph Display Graph Export Image Export Colors Default Color The Graph Displaytab contains the default list of statistics shown for a graph Check the box next to the statistic to have it show below the graphs when statistics are shown for a graph The default graph size and font size for the graphs in the anal ysis area may be changed in this tab aie Creating Reports and Exporting Data Graph Display Graph Export Image Export Colors Default Graph Size Small 5 Medium Large Default Graph Font Sizes me 2 hes Labets Tick Mark Labels 10 Regon Nares 0z Update Graphs in Analysis Area Graph Size E Font Sizes Statistics The Graph Export tab contains the default settings for printing and exporting graphs when copying and pasting from IDEAS for reporting into other programs 135 Chapter 3 seen ss Directories Populations Masks Graph Display Graph Export V Graph Size 300 DPI Width 1 027 E Height 0947 in 7 Lock aspect ratio Font Size Titl
145. k This is one of four features that can be used to identify objects with spots that are close together dim bright or small when counting spots in an image To use these features the spots need to be individually masked such as using the Spot or Peak Mask The Spot Area Min Spot Distance Min and Spot Intensity Min or Max fea tures measure properties of different spots in an image and are often used with the Spot Count feature under Texture For more information see Spot Distance Min FeatureSpot Count FeatureSpot Intensity Min and Spot Intensity Max Features e Spot Area Min is the Area of spot 1 Spot Distance Min is distance 2 in microns Spot Intensity Max is the Raw Mean Pixel of spot 2 Spot Intensity Min is the Raw Mean Pixel value of spot 3 Application Example In FISH Spot Counting these features are used to identify objects with ambiguous spots that are located too close together are too dim to count or are too small in order to remove these objects from the analysis 178 Understanding the Size Features Thickness Max Feature Thickness Max measures the largest width of an object This feature is based on an input mask and therefore sensitive to the variation of the input mask shape Select ing an input mask that can accurately capture the object shape is important See also Table of Base Features by Category Length Feature and Shape Ratio Feature for more information Thickness max
146. l Definition Al Note The list of populations is presented as a hierarchy that shows the depend encies of the populations on each other The icon associated with a population indicates how the population is defined eon Defined by The definition of a selected population is shown in the Definition area To edit the display properties of a population OOA ON Within the Population Manager click a population in the Populations list Change the name in the Name box Click a Color square to select a new color on the color palette and click OK Click a display symbol in the Symbol drop down menu Click Close to save the population changes Click Revert to reject the changes To delete a population 1 Within the Population Manager click a population in the Populations list 2104s Using the Population Manager 2 Click Delete A confirmation waming message appears indicating all the dependent pop ulations that will also be deleted 3 Click Yes to confirm To create a new combined population 1 Within the Population Manager Analysis gt Populations click New The right side of the Population Manager window changes to allow you to define a new population Daik Mode Coir C Laht ModeCoior M hy Al F Eao aE Enter a unique population name in the Name box Click a Color square to select a new color on the color palette and click OK Click a display symb
147. l Image Detail Eroded Image Bright Detail Image The image pairs below show objects in grayscale next to their corresponding Spot Masks in cyan Spot masks can be further refined using the Peak and or Range masks See Peak MaskRange Mask Application Examples 263 Chapter 4 Used with the Spot Count feature to enumerate spots in images such as for FISHIS Used with Intensity features to quantify intensity in spots Dark spot finds valleys in images such as the low intensity between 2 stained nuclei and is useful for finding immune synapses Identifies the dark areas in red blood cells or parasitic infections in bright field imagery SIGA About Masks System Mask The System mask segments objects in an image based on a probability model of how pixels should be grouped together The user sets a weight value that defines a loose or tight grouping A low weight value groups in a more permissive manner Shown is an example of a cell with a apoptotic bleb that is not masked with the Sys tem mask weight set at 5 but is masked with the System mask weight set at 2 Application Example Used on brightfield images to capture a low contrast areas such as cells that undergo a blebbing process tails of sperm or other low contrast type of structures 265 Chapter 4 Threshold Mask The Threshold mask is used to exclude pixels based on a percentage of the range of intensity values as defin
148. list you want to view f Region Manager Regions Name R1 Dark Mode Color Use for statistics only Shape Rectangle Vertices X Coordinate Y Coordinate gt 99 54751131221 1 041951219512 407 2398190045 0 679024390243 To edit a region ON oA A ON Within the Region Manager click a region in the Regions list Change the name in the Name box Click a Color square to select a new color on the color palette and click OK Change the X or Y position of the vertices in the Vertices box Select or de select the Use for statistics only box Click Delete to delete a region Click Revert to reject the changes Click Close when finished 129 Chapter 3 Note When a region is deleted all populations that are defined by that region will be deleted A warning dialog box appears listing the populations that will be deleted 130 Creating Reports and Exporting Data Creating Reports and Exporting Data The following six page sample report was created by copying data from IDEAS the ImageStream Data Acquistion forms and excel into a MS word document This template can be found in the customer documents in your training materials or in the knowledge base of your account on the Amnis website me bk ng SaaS Wi Sa 1 2S as Once you have finished analysing an experiment you will want to report the results into third party applications To streamline the proc ess t
149. lls Note that this step is skipped if the cell image channel chosen is brightfield Next Step Gate shape changed events A histogram of Circularity of the last gated population is added to the analysis area Draw a region to include the cells with low circularity scores Shape change is measured in the final graph presented in the wizard Cells with low circularity scores have a highly variable radius In this example monocytes in whole blood were stained with CD14 green Low circularity High circularity For a more thorough explanation of the Circularity feature see Circularity Feature The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 44 Guided Analysis Spot Wizard This wizard will create an analysis template for measuring texture based on spot counting If the low and high spot count data are in separate data files merge the files together before beginning Spot To begin double click on Spot Follow the instructions to open and analyze your file Step 1 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the
150. lowing procedure 1 Select Tools gt Merge rif Files 2 Click Add Files to select the single rif file 3 Click OK Enter a new name if desired The single rif file will merge with itself and rewrite the file with the proper object count Buttons or options in windows are not appearing When the font size setting is set to large some windows will not size properly caus ing buttons or text boxes to not appear To change the font size in Windows go to the Control Panel gt Display gt Appearance and select Font size Normal 272 Glossary Glossary Table 1 Glossary of Terms em The process of collecting data from the ImageStream cell analysis system A type of illumination that uses transmitted light On the ImageS brightfield tream cell analysis system this light is provided by a halogen lamp ee An image that is produced by transmitted light On the ImageStream brightfield image f as cell analysis system this light is provided by a halogen lamp rightfield The camera channel that the brightfield image appears in acquisition The precise adjustment of instrument components based on test results for the purpose of optimizing functionality CD See charge coupled detector CCD One of the six physical partitions on the camera Each camera chan nel collects a different spectral band of imagery which allows for the collection of brightfield darkfield and up to four fluorescence images per object A senso
151. ly if all of the features all channels in the category are selected 122 Using the Feature Manager SAM Size cH Size V Area _Fill Threshold M02 Area_Fill Threshold MO02 2_Actit V Area_M01 Area_M01 V Area M02 Area _M02 First check all then uncheck to deselect all features in a category W Area_M11 Area _M11 V Area _MC Aea MC o j Click Add Statistics k Click Close Repeat until each statistics table contains 1 category of features for ChO2 Actin 6 Launch Excel and then Copy and Paste the statistics into the excel spreadsheet a Select the row of statistics for the 2nd truth population the one not chosen above b Right click in the statistics table and choose Copy Statistics Transposed c Paste into an Excel spreadsheet d Keep all of the features and values selected and sort the data set on the values column heading may be the population name largest to smallest The feature with the largest RD will be at the top Note you may have NaN values for some of the fea tures This means Not a Number and occurs in some cases when there is a division by 0 These can be ignored 7 Validate the features in IDEAS Plot the features with the highest RD for the truth populations and draw regions to discriminate 8 Apply regions to the base population independent controls if available and on mul tiple files and experiments 9 Look for false negative and positive cells 10 Repeat process if
152. masks are recomputed in IDEAS for ImageStream or QI enabled files Combined features can be created using existing features in mathematical expressions in the Feature Manager IDEAS groups the features into eight categories size location shape texture sig nal strength comparison system and combined For more information see Overview of the Mask Manager To calculate the value of a feature the IDEAS application maps the channel image to X and Y coordinates as illustrated by the following diagram Each box in the dia gram represents a pixel The pixel size and field of view per channel is dependent on the magnification used See your INSPIRE Users Manual for more information 160 Understanding the IDEAS Features and Masks Features Categories Size Location Shape Texture Comparison System Size features are in microns and include Area Diameter Length Major Axis Minor Axis Major Axis Intensity Mnor Axis Intensity Perimeter Thickness Max and Min Spot Area Min Width and Height Location features include Angle Angle Intensity Centroid X Centroid Y Centroid X Intensity Centroid Y Intensity Delta Centroid X Delta Centroid Y Delta Centroid XY Max Contour position Spot Distance Min Valley X and Valley Y Shape features define the mask shape and have units that vary with the feature They include the Aspect Ratio Aspect Ratio Intensity Compactness Elon gatedness Lobe Count an
153. may use the wizard Display Properties Wizard to set the pixel intensity mapping to the display range Manual adjustment and other settings are described below Clicking the Image Gallery Properties toolbar button opens the Image Gallery Prop erties window which contains the following tabs e Display Properties Allows you to define the name color and display inten sity mapping for each image Allows adjustment of the image size for the image gallery e Views Allows you to customize the views for the Image Gallery e Composites Allows you to create composites and adjust the amount of color from a channel that is included in a composite image To customize the Image Gallery display properties 1 Click the Image Gallery Properties toolbar button to begin The Image Gallery Properties window appears with the Display Properties tab displayed 138 Creating Reports and Exporting Data Display Properties Views Composites Images Name Ch02 Color i Minimum Pixel Intensity 15 Maximum Pixel Intensity 1484 255 Automatic Manual Image Display Mapping X Axis Scale Digia Mah Dilay Height Set Rangeto Pixel Data Full Scale Channel Width __SetLinearCuve _ Atoscale Auto Fit Arto Fit 120 32 Preview Changes in Gallery To change the size of the panels in the image gallery 1 Display Width and Display Height can be specified or changed to Auto Fit in the lower left sect
154. mple information for any file without loading the file Sample Information SE i Select Data File NFkB Fitc Dg5 No LPS Analyzed _2 daf ER Acquisition Comections Focus Fluidics Detection Camera Settings Illumination EDF Compensation Channels Raw Data File Name f NFKB Fite D5 No LPS_2 nf Acq Date 3 31 2010 Version No Objects Processed Data File Name cif C Users sfriend Desktop 2Cam DEMO NFkB Translocation NFKB Fite Da5 No LPS Analyzed _2 Version 40 4270 No Objects 4721 Sample NFKB Fte Da5 No LPS Analyzed _2 Show Sample Name in Graph Titles Allow Post Processing Acquisition tab File names software version numbers date acquired number of objects sample name Corrections tab Camera background alignment offsets from ASSIST Focus Fluidics tab Core information and sample volume eee Viewing Sample Information Detection tab IS100 and ImageStream lt X only Cell classifier settings during acquisition Population tab FlowSight and lageStreamX MKII only Lists the populations and number acquired Camera Settings tab Bin mode magnification and sensitivity settings Illumination tab Brightfield and laser information EDF tab View kernels used for deconvolution of EDF imagery Compensation tab View the compensation matrix Channels tab Lists channels collected vee Chapter 3 Batch Processing Batch processing allows you to a
155. n Copy graphs to the clipboard or save graphs to a file To copy or save graphs 1 Right click anywhere in the graph and choose Copy Save Graph and or Statistic 2 A preview of the graph is shown Changes from the default settings may be made to the following e Include Graph e Graph Size e Font sizes These first three settings are loaded from the graph you may choose to load the default settings that are set in the applications defaults tab e Include Legend e Include Sample name in title e White background on off e Include Cursor e Include Statistics e Font sizes for statistics 3 If changes are made they can be previewed by clicking Generate Preview Otherwise they will be applied when Save to File or Copy to Clipboard is chosen 4 Option settings may be saved to default settings or loaded from default settings 5 Click Save to File or Copy to Clipboard when done Files may be saved as png bmp or tif formats 6 If Copy to Clipboard was chosen paste into third party application 146 Reporting Images and Graphs A Copy Save Graph and or SEEC 3 A Se Graph R1 050611 FS1QI THP1 ae NFkB Dg LPS Width 3 207 Height 2 960 2 in gt a25 7 Lock aspect ratio 2 ge Font Size N15 Tile 10 Tick mark labels 10 g1 Ais labels 12 Region names 12 2 s 0 F aa or oe A T A Load Graph Settings Load Defaut Settings Similarity_Morphology
156. n Examples Quantify and compare cell shape Identify small medium and large cells 175 Chapter 4 Major Axis Intensity and Minor Axis Intensity Features The Major Axis Intensity is the longest dimension of an ellipse of best fit and is inten sity weighted The Minor Axis Intensity is the narrowest dimension of the ellipse of best fit and is intensity weighted The figure below illustrates the difference between intensity weighted and non inten sity weighted Major or Minor Axis and Aspect Ratio See the Aspect Ratio Intensity Feature for more information Aspect Ratio_M4 l l l l 0 al 6 ike Aspect Ratio Intensity_M4 Application Examples Quantify and compare the image fluorescence shape Identify single cells 176 Understanding the Size Features Perimeter Feature The perimeter feature measures the boundary length of the mask in the number of microns This example uses a 1 pixel wide mask created to illustrate how a perimeter would appear Brightfield Channel 5 PIDNA Application Examples Quantify and compare cell circumference Identify cells with highly irregular surfaces from smooth cells Perimeter of the morphology or threshold masks can identify cells with or without dendrites 177 Chapter 4 Spot Area Min Feature The Spot Area Min feature provides the area of the smallest spot connected com ponent in a spot or peak mas
157. n an object where the object is in contact with a second object Three input parameters are defined First the mask of one of the objects cell of interest Next the mask that covers both objects conjugate A close fitting mask using another function mask such as Object tight can be used for the cell of interest mask A brightfield mask can be used for the conjugate Finally the width of the interface mask from the contact point towards the cell of interest is entered Examples are shown below Callefligemen kine Interface Mask shown on Brightfield Image Conjugate Mask Cell of Composite BF Interest Mask interfaca Mask Image Application Examples Used to quantify synapses in T cell APC antigen presenting cell con jugates 257 Chapter 4 Morphology Mask The Morphology mask includes all pixels within the outermost image contour This mask which is used in fluorescence images is best used for calculating the values of overall shape based features Morphology 258 About Masks Object Mask The Object mask segments images to closely identify the area corresponding to the cell It is based on the assumption that background pixels exhibit high uniformity to each other This helps distinguish the background from the cell pixels The mask characterizes the background pixels using a set of features and then segments the image by determining all the pixels that deviate from the background
158. n the camera background camera gains flow speed and vertical and horizontal positioning between chan nels Serves as a database of images used for feature value calculations and imagery display The IDEAS application also performs fluorescence com pensation if necessary when creating a cif file The IDEAS application loads the cif file into a template to create a data analysis file daf file The main working data file that contains the calculated feature values the graphs and the statistics used for analysis The daf file references the cif This file contains no data it contains the structure for the analysis such as definitions for features graphs regions and populations image viewing settings image names and statistics settings Contains compensation values that are created and saved during the spectral compensation of control rif files This file has no associated object data it is created and saved to be applied to experimental rif files Note about Case Sensitivity Even though Windows does not treat file names as case sensitive the IDEAS application depends on the case sensitive rif cif and daf file name extensions to identify the file types Avoid the use of illegal characters for file names such as lt gt ms ie Chapter 3 Getting Started with IDEAS Chapter 3 Getting Started with IDEAS 21 Chapter 3 Getting Started with the IDEAS Application This chapter is di
159. names appear in the list 3 If you want to remove a file from the list select it and then click Remove File 4 When the merge list is complete click OK The Save Merged Raw Image rif File dialog box appears 5 Type a unique file name 6 Click Save The Creating merged rif file window appears Creating merged rif file O Guests 092011 Demo temp rif a Files to merge 5 O Guests 092011 Demo 092011 X101 Unstimulated _12 rf O O Guests 092011 Demo 092011 X101 Stimulated_11 nf Unprocessed In process Processed Cancel When the merge is complete the Merged rif Created message appears 7 Click OK Note The sample information will contain the classifier information for the first file in the merge list however the classifier is turned off when a merged file is loaded To turn the classifier on manually go to the Advanced panel on the open rif window when opening a merged file 69 Chapter 3 Merging Compensated Image Files You can merge cif files together for analysis This option is not available for basic FlowSight files without the Quantitative Imaging QI upgrade To merge cif files 1 Onthe Tools menu click Merge cif Files The Load Multiple cif Files window appears Load Multiple cif Files Select cif files to load Enter the number of objects to load from each file A population will be created for each file Specify the population name File O
160. ne overall focus quality of images Used with Contrast to determine focus quality Characterize texture See also Gradient Max Feature and Contrast Feature 211 Chapter 4 H Texture Features H Texture features include the following H Energy Mean and Std H Entropy Mean and Std H Contrast Mean and Std H Homogeneity Mean and Std H Correlation Mean and Std H Variance Mean and Std Features R M Haralick H defined a set of texture features that characterize the spatial rela tionships amongst the pixel values in an image IDEAS uses a common nor malization method so that images with different intensities can be directly compared For each H texture feature the mean reflects the average value and the standard deviation Std reflects the presence of texture orientation The user defines the texture grain by assigning a granularity value For very fine tex tures this value is small 1 3 pixels while for very coarse textures it is large gt 10 Inthe IDEAS default template the granularity value is 5 While these features have value for distinguishing cellular texture when used individ ually images often contain a mixture of different textures at different grains There fore these features are most powerful when combined Application Example Quantify texture in cells Taralick R M K Shanmugan and I Dinstein Textural Features for Image Clas sification EEE Transactions on Systems Man and
161. ne profile the wavy line in the image panel represents the pixel intensity at each position along the red line of the box 105 Chapter 3 c2 A A Q v 9 Minimum 13 Maximum 1089 Mean 282 95 Std Dev 286 21 Width 49 Height 53 Area 2597 Close Statistics To change the display properties of an image 1 Click the Channel Display Properties button on the image panel toolbar a sga oa The Display Properties window appears e For single channel image you can change the displayed mask and adjust the display intensity mapping For more information see Setting the Image Gal lery Properties 106 Overview of the Analysis Area Select a different mask to display Valley MO5 Drag5 2 X Minimum Pixel Intensity 17 Maximum Pixel Intensity 187 255 200 0 l l 32 50 100 150 209 Automatic Manual Image Display Mapping X Axis Scale Set Range to Pixel Data Full Scale Set Linear Curve Autoscale Foracomposite image you can change the images in the composite and adjust the percent contribution of each image see Setting the Image Gallery Properties 107 Chapter 3 fi a Display Properties Object 42 Com Sex Name TONE Tare ADAP Actin T cell ADAP 100 Actin 100 T cell 100 Add Image Remove Image 2 Click OK To show or hide th
162. ng Data eet aoe ee itt tics tee uit coarsest cat cals 155 Chapter 4 Features and Masks l a anaana aaa occ cc ccc ec ec ec eee eees 157 Understanding the IDEAS Features and Masks 158 Abo tFeat res sasn Shine Gabi n sits Mtn a e aaa e ea tidy st eiadianT teal tc ol 159 Features Categories i i ha ce ee ts poh i Si 161 Table of Base Features Alphabetical 0 0 0 0 0 0 2 e cc ee cece eee 162 Table of Base Features by Category 0 0 002 o eee ce ec cc ec ee cee eeee 164 Table of Basic Features available for FlowSight without QI 169 Understanding the Size Features _ 0 ooo ecco ec eee eee 170 Understanding the Location Features _ 0 0 0 0 c eee ce cee 182 Understanding the Shape Features _ 0 0 00 0 0 o eee ce eee cece cece eee 195 Understanding the Texture Features 2 202 020 0000 0 cc cece cece cee eee eee eee 205 Understanding the Signal Strength Features _ 0 0 0 0 202e cece 216 Understanding the Comparison Features _ 0 0 0 2 2 e eee ee ee 234 Understanding the System Features 0 0 0 0 0 coe e eee ee cece eee 242 Camera Timer Feature 2 2 2 0 0 0 0 0o coco cece cece cee ec ec eee ececcecececceceeteceees 244 ADOUtMOSKS l to hati ele oli sts caso tattle dil tla 250 Appendix Troubleshooting 0 0 0 0 00 ccc cc cece cece ec eccececeececeeeees 269 Gl ssary
163. nical data related thereto nor any direct prod uct thereof are exported or re exported directly or indirectly in violation of or used for any purposes prohibited by such laws and regulations 10 General This Agreement will be governed by and construed in accordance with the laws of the State of Washington without regard to or application of conflict of laws rules or principles The United Nations Convention on Contracts for the International Sale of Goods will not apply You may not assign or transfer this Agreement or any rights granted hereunder by operation of law or otherwise without Amnis prior written consent and any attempt by you to do so without such consent will be void Except as expressly set forth in this Agreement the exercise by either party of any of its remedies under this Agreement will be without prejudice to its other remedies under this Agreement or otherwise All notices or approvals required or permitted under this Agreement will be in writing and deliv ered by confirmed facsimile transmission by overnight delivery service or by certified mail and in each instance will be deemed given upon receipt All notices or approvals will be sent to the addresses set forth in the applicable ordering document or invoice or to such other address as may be specified by either party to the other in accordance with this sec tion The failure by either party to enforce any provision of this Agreement will not con stitute a waive
164. ning files using a wizard 26 Oval Region Tool 95 P Pointer tool 94 Polygon Region Tool 95 Population Manager 124 tools 125 Population Statistics 111 Population Statistics table 95 Populations creating 127 creating a new data file from 71 creating combined 125 deleting 124 display properties 124 during acquisition 73 viewing 124 Printing 145 R Rectangle Region Tool 95 Region Manager 129 Regions editing 129 viewing 129 286 Index Reporting 131 changing color mapping 144 graphs and statistics 146 Images 145 Images and Graphs 144 light mode graphs 144 Statistics 148 statistics from multiple files 150 Reports printing data 145 155 rif about 18 merge 69 opening 50 Sample Information view 72 Saturation view in Image Gallery 80 Saving data files 56 ast 57 cif 56 daf 56 Template 57 Scatter plot tool 95 287 Chapter Appendix A Scatter plots show hide populations 102 Screen resolution 5 Select All Panels Tool 95 Shape Change using a wizard 43 Single Cell Building Block 47 default 47 Size SSC Building Block default 47 Software requirements 4 Spot Count using a wizard 45 Statistics defaults fora graph 8 134 Tagging tool 79 95 Template file about 19 default 15 definition 16 saving 57 Text tool 95 TIFs creating 154 Two color scatter plot building block 47 288 Index Views custom 84 141 Volume information 72 Wizards Apoptosis 33 Be
165. ntemalization of a probe Nuclear Localization Creates an analysis template for measuring the nuclear localization of a probe Shape Change Creates an analysis template for measuring circular morphology Spot Creates an analysis template for measuring texture based on spot counting ee Chapter 3 Application Wizards General Open File Wizard Guides you through the process of opening a data file and setting image display mapping in the image gallery Display Properties Sets image display mapping in the image gallery Begin Analysis Guides you through finding focused single positive cells Feature Finder Guides you through finding focused single positive cells Application specific Apoptosis Wizard Guides you through the process of creating the features and graphs for analyzing apoptosis Cell Cycle Mitosis Wizard Guides you through the process of creating the features and graphs for analyzing the cell cycle and enumerating mitotic events Co localization Wizard Guides you through the process of creating the features and graphs for analyzing the co localization of 2 probes Internalization Wizard Guides you through the process of creating the features and graphs for analyzing the internalization of a probe Nuclear Localization Wizard Guides you through the process of creating the features and graphs for analyzing the nuclear localization o
166. o Intensity_Fill Threshold M02 2_Actin 50 _2 Aspect Ratio Intensity_M01_Ch01 1 Name Aonar Datin Intannths MND CLA l m Sort features by A IB Fal ae New Delete cue Q O Oom b In the Add Features window select Category as the Sort Order E 120 Using the Feature Manager r Z Add Features 2 ee Select base features Select feature inputs Size E Create for all channels using default masks and images E Location fi Shape Select masks W 4 Texture MOS E E Signal Strength M07 H F Comparison Mos aS M11 Hystem MC Morphology M02 2_Actin None Object M02 2_Actin Tight Threshold M02 2_Actin 50 Clear Selected GG Ge elect image 1_BF 2_Actin Ch03 Sort Order 6_SSC 7_DAPI e Alphabetical Category Ch09 o Clear Selected ae Add Features Close c Check Size Shape and Texture base feature boxes d Select the actin masks Morphology Object Thresh old M02 e Select the actin image FITC f Click Add Features to display the list of features to add g In the next window Click OK to add the fea tures Features that already exist will not be recal culated h Click OK and Click Close i Close the Feature Manager by clicking Close and the features will be calculated 5 Add the feature statistics to the populati
167. o the starting mask In the example below the Morphology mask of the image was used as the starting mask for creating the skeleton s s Morphology Skeleton_thin Skeleton_thick Application Examples Thick skeletons can be used with shape based features such as sym metry to accentuate the shape of an object and provide greater sep arations Separate singlets and doublets by computing the area of the thin skeleton mask We have used the object tight for this case Nuclear morphology measurements with lobe count feature for cell clas sification cells 262 About Masks Spot Mask The Spot Mask has two options bright or dark The bright option obtains bright regions from an image regardless of the intensity differences from one spot to another The ability to extract bright objects is achieved using the an image proc essing step that erodes the image and leaves only the bright areas The dark option obtains dark regions The spot to cell background ratio and radius are specified by the user The spot to cell background ratio is the spot pixel value divided by the back ground in the bright detail image A radius value of x implies that the image contains spots with thickness of 2x 1 pixels The figure below illustrates the open residue process The bright areas are eroded from the original image and the detail eroded image is subtracted from the original image resulting in the bright detail image Origina
168. obes of cellular constituents luorescence Light emitted by a fluorescent dye following excitation luorescence com The adjustments made to remove the fluorescence emissions of a calibration 273 Chapter Appendix A pensation fluorochrome into adjacent channels A physical mapping approach that uses fluorescent tags to detect the hybridization of probes with metaphase chromosomes or the less con idensed somatic interphase chromatin fluorescent in situ hybrid ization FISH gain re amplification of a detector signal ravscale The brightness level ranging from black to white of a pixel or group gray of pixels A pixel is equal to a half micron in length with the 40X objective 1 micron with the 20X one ie and 0 33 microns with the 60X objec tive Note that 1 pixel x ym The state of a pixel that hag a value at or above 1023 for the 1S 100 or 4095 for the ImageStream segmentation The process of discriminating an object from its background A custom set of longpass dichroic filters arranged in an angular array The spectral decomposition element directs different spectral bands spectral decomposition to laterally distinct channels on the detector With this technique an element image is optically decomposed into a set of six sub images each cor responding to a different color component and spatially isolated from the remaining sub images The registration error of the six channel images for a single c
169. ol in the Symbol drop down menu Use the toolbar to build the population definition as described in the table and click OK when done oa AON Properties Name R4 And Not Tagged Dark Mode Color L Light Mode Color l Symbol Simple Doty Definition Al Hrga e E 092011 X101 unstimulated _tt cif Si All R1 af R2 3 0 R3 m R4 om OK Cancel Table 1 Population Tasks and Toolbar Task setts popuiatiomte thedet Select the population from the drop down menu 125 Chapter 3 Use the Boolean AND or OR operator m Use the AND operator to include only the objects that Combine two populations are in both of the original populations Use the OR operator to include the objects that are in either one of the original populations Use the Boolean NOT operator o The NOT operator specifies which population will not be used Note you must use AND before NOT Affect the order of operations Use the parentheses toolbar buttons i Click the left arrow button on the toolbar Select objects that are not in the original population Remove an item from the end of the definition See Creating Tagged Populations for information about tagged populations 126 Creating Tagged Populations Creating Tagged Populations You can hand select objects from either the Image Gallery or a graph and group them into a population To create a hand selected population 1 Click the Tagging Mode toolb
170. on Dose and Time 4 0 vifs 0 1ng 75_21_4 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 1ng 90_26_9 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 10ng 15_3_10 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 10ng 30_8_15 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 fs 10ng 45_13_20 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 10ng 60_18_1 daf CA Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 10ng 75_23_6 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 10ng 90_ CA Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 1000ng 15_ C Users sfriend Desktop S100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 30_10_17 dat C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 1000ng 45_15_22 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 60_20_3 daf CA Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 fs 1000ng 75_25_8 daf C Users sfriend Desktop S100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 90_30_13 dat Start time 2 1 2011 11 53 28 AM End time 2 1 2011 12 00 41 PM Result All files were processed successfully Statistics Report C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 0ng_2_9 _4 Default daf_stats bt 27 C
171. on on a graph 1 2 3 4 Right click anywhere on the graph and click Apply Remove Region on the graph context menu that appears The Apply Graph Regions window appears E Apply Graph Regions DER Check the regions to be added to the graph Uncheck the regions to be removed v Note Only regions that have the same scaling types ie linear log as the graph may be added OK Cancel Select the regions that you want to appear on the graph Clear the regions that you want to remove from the graph Click OK To show or hide a population on a scatter plot 1 Click Show Hide Populations on the graph context menu The Show Hide Populations window appears 102 Overview of the Analysis Area 2 Select the populations that you want to appear on the graph 3 Clear the populations that you want to remove from the graph ES Show Hide Populations DER Select the populations to view 4 Click OK Tip On a scatter plot you may show or hide any population on the graph regard less of the features on the axes Each scatter plot has an original or base pop ulation When you show a population on a scatter plot only those objects that are also in the base population will be shown To aid in the identification of the pop ulations shown change the characteristics of the population s in the population manager Analyzing Individual Images To analyze an image in more detail place the image in t
172. on statistics table Do this one category at a time Multiple statistics tables can be added to the analysis area one for each category of features Once the features are calculated you can use the RD Fischer s Discriminant Ratio to a statistics table The RD measures the separation between 2 populations In this case the 2 truth populations picked in step 2 In 121 Chapter 3 order to get the statistic for 1 category at a time select all of the features for the image and then deselect cat egories to leave 1 category for the channel selected a Click on to add a statistics table to the analysis area b Right click in the table and choose Edit Statistics Table c Delete any statistics from the list d Select the statistic RD Mean e Select one of the truth populations in the Reference population box f Sort by Images Used by clicking on the icon SJ g Check the box for the Ch02 Actin image h Sort the features by Category E Selected Statistics Create New Statistics Statistics Standard Deviation MAD RD Mean RD Median cv Reference population if required uniform actin Features if required aE 1_6F 7 2_Actin e Ch03 P 6_SSC 7_DAPI E Ch09 Ch11 i De select all but 1 category by checking and uncheck ing the box for the categories you want to de select Note that the box next to the category will be checked on
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174. ore details about how to create new masks 251 Chapter 4 Dilate Mask The Dilate mask adds the selected number of pixels to all edges of the starting mask Morphology Dilate 2 pixels 252 About Masks Erode Mask The Erode mask removes the selected number of pixels from all edges of the start ing mask Morphology Erode 3 pixels 253 Chapter 4 Fill Mask The Fill mask fills in any holes in the starting mask 254 About Masks Inspire Mask The Inspire mask masks pixels above background and is the mask used during data acquisition in INSPIRE This mask is available to understand what is being masked during collection and is not generally used for feature calculations Note this mask is new in IDEAS versions 4 0 or later Default Inspire 255 Chapter 4 Intensity Mask The Intensity mask masks pixels between the lower and upper raw intensity thresh olds not background subtracted See also Threshold Mask In the example below cell 10678 is bright and cell 11992 is dim The 50 Thresh old mask is similar for both images whereas the Intensity mask 250 is quite dif ferent since only a few pixels in the dim image are greater than 250 counts while most of the metaphase plates in the bright image are masked gt gt 99 a Threshold 50 Intensity 250 1023 256 About Masks Interface Mask The interface mask identifies pixels i
175. orting of data Because the population colors might not show on a white back ground you can change the colors when using the light mode See Application Defaults for more information To use light background graphs in the Analysis Area Click the graph background tool to switch between light or dark mode IP e SeeViewing and Changing the Application Defaults colors tab to set the color mapping desired To print the Analysis Area data e Select Reports gt Print Analysis Area The IDEAS application prints all the graphs statistics text panels and images that are displayed in the Analysis Area To print the Image Gallery data Select Reports gt Print Image Gallery The IDEAS application prints all the images that are visible in the Image Gal lery 155 Chapter 3 To print an individual graph 1 Right click the graph and then select Print Graph on the graph context menu The Print Graph window appears Select options for printing Graph Legend E Statistics Cursor E Show Sample Name in Title Size scaling factor o 50 100 200 300 2 Select the checkboxes Graph Statistics Legend Cursor Show Sample Name in Title to include the elements in the report 3 If necessary adjust the size scaling factor Recomended setting is 100 4 Click OK to print the graph 156 Chapter 4 Features and Masks Chapter 4 Features and Masks 157 Ch
176. osomes image Application Examples Quantify the degree of colocalization between two probes Track intemalization and intracellular trafficking of antibody drug con jugates to either the endosomes or the lysosomes Colocalization of Rituxan and compliment C3b Used in the Co localization Wizard 236 Understanding the Comparison Features Intensity Concentration Ratio Feature The intensity concentration ratio is defined as the ratio of the intensity inside the first input mask to the intensity of the union of the two masks the higher the score the greater the concentration of intensity inside the first mask All pixels are background subtracted The ratio is invariant to cell size and can accommodate concentrated bright regions and small dim spots The ratio is mapped to a log scale to increase the dynamic range to values between inf inf This feature is a generalization of the Internalization feature See Internalization Feature for more information Application Example Quantify relative intensity concentrations between different cellular com partments Internalization is a special case of this where the first mask is the intemal compartment and the second is the membrane region 237 Chapter 4 Internalization Feature Single Focused Cels 25 The Internalization feature is defined as the ratio of the intensity inside the cell to the intensity of the entire cell The higher the score the
177. ov ing and upgrading the application The following subsections cover this information Setting Up the IDEAS Application Installing the IDEAS Application Setting Your Computer to Run the IDEAS Application Viewing and Changing the Application Defaults Hardware and Software Requirements This section states the minimum and the recommended hardware and software requirements for running the IDEAS application Hardware Requirements The minimum hardware requirements are 4 GB of RAM and a dual core Intel proc essor However due to the large size of the image files that the ImageStream cell analysis system creates a larger amount of RAM will prevent paging and thus increase performance Software Requirements IDEAS 6 0 64 bit version requires that the Windows 7 operating system be installed on your computer IDEAS 32 bit version requires Windows XP Windows 2000 or later version of the operating system The latest service pack and any critical updates for the operating system must be included You must also install the Micro soft NET Framework 2 0 which requires Microsoft Internet Explorer 5 01 or later Important If the software requirements are not met Setup will not block installation nor provide any warnings Note that service packs and critical updates are available from the Microsoft Secu rity Web Site Installing the IDEAS Application If the IDEAS application has previously been installed the previous version will
178. perform fluorescence compensation Select a compensation matrix compensated image file or data analysis file ctm cif daf Or Create a compensation matrix from control files New Matrix To use a custom template for analysis Select a template or data analysis file ast daf Name the output files to be created Compensated image file cif 1000ng 60_20_3 cif Data analysis file daf 1000ng 60_20_3 dat Enter the number of objects to process 1000 of 1000 3 Click the folder next to Select a compensation matrix compensated image file or data analysis file ctm cif daf field to choose the matrix that was gen erated from the controls used for the experiment If the rif file contains a com pensation matrix used during acquisition it will be entered into this box If you leave it blank the default compensation matrix will be used but this is not rec ommended unless you do not want to compensate your data If a compensation matrix for the experiment has not been made click New Matrix For more information on creating a compensation matrix see Creating a New Compensation Matrix File 4 Inthe Select a template or data analysis file ast daf field select a tem plate file to load by clicking the folder and browsing for the file If left blank the Default template with the basic features masks and settings will be used Flow Sight files use the acquisition template as the default 5
179. r Axis Features Aspect Ratio Minor Axis Major Axis inor Axis See also Elongatedness Feature and Shape Ratio Feature for other shape ratios Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightWidth Thickness min Length ss max ess min inor Axis ajor Axis Application Examples Quantify the roundness of the mask Identify single cells vs doublets Cell classification based on shape change Identify recently divided cells in mitosis 196 Understanding the Shape Features Aspect Ratio Intensity Feature Aspect Ratio Intensity is the Minor Axis Intensity divided by the Major Axis Inten sity See also Major Axis Intensity and Minor Axis Intensity Features The figure below illustrates the difference between Aspect Ratio Intensity and Aspect Ratio See also Aspect Ratio Feature N io Aspect Ratio_M4 l l I i 6 A 1 2 Aspect Ratio Intensity_M4 Application Examples Quantify the roundness of the fluorescent image Better resolution for identifying single cells vs doublets in experiments using a DNA dye Cell classification based on fluorescent morphology 197 Chapter 4 Circularity Feature This feature measures the degree of the mask s deviation from a circle Its meas urement is based on the average distance of the object boundary from its center divided by the variation of th
180. r any breach of this limited warranty Amnis will at its option and expense promptly correct or replace the Software so that it conforms to this limited warranty Amnis does not warrant that the Software will meet your requirements that the Software will operate in the combinations that you may select for Execution that the operation of the Software will be error free or uninterrupted or that all Software errors will be corrected The warranty set forth in this Section 5 does not apply to the extent that Amnis provides you with the Software or portions of the Soft ware for beta evaluation testing or demonstration purposes 6 DISCLAIMER THE LIMITED WARRANTY SET FORTH IN SECTION 5 IS IN LIEU OF AND AMNIS EXPRESSLY DISCLAIMS ALL OTHER WARRANTIES AND CONDITIONS EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO ANY IMPLIED WARRANTIES AND CONDITIONS OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT AND ANY WARRANTIES AND CONDITIONS ARISING OUT OF COURSE OF DEAL ING OR USAGE OF TRADE NO ADVICE OR INFORMATION WHETHER ORAL OR WRITTEN OBTAINED FROM AMNIS OR ELSEWHERE WILL CREATE ANY WARRANTY OR CONDITION NOT EXPRESSLY STATED IN THIS AGREEMENT 7 Limitation of Liability AMNIS TOTAL LIABILITY TO YOU FROM ALL CAUSES OF ACTION AND UNDER ALL THEORIES OF LIABILITY WILL BE LIMITED TO THE AMOUNTS PAID TO AMNIS BY YOU FOR THE SOFTWARE OR IN THE EVENT THAT AMNIS HAS MADE THE SOFTWARE AVAILABLE TO YOU WITHOUT CHARGE AMNIS
181. r for recording images that consists of a particular type of inte charge coupled detec grated circuit one that contains an array of linked or coupled capac tor CCD itors Under the control of an external circuit each capacitor can transfer its electric charge to either of its neighbors The mean normalized standard deviation expressed as a per coefficient of variation centage The CV measures the variation of a feature value inde CV pendent of the population mean value The formula is CV 100 x standard deviation mean The process of removing intensity specifically intensity that was derived from fluorescence crosstalk that originated from dyes cen tered in other channels The IDEAS application performs com pensation on a pixel by pixel basis The set of values that report the relative amount of fluorescence of compensation matrix each probe in each channel The compensation matrix is used to sub tract intensity originating from dyes centered in other channels Leakage of fluorescence signal from a fluorochrome into adjacent crosstalk charnels A type of illumination in which the sample is illuminated at angles that darkfield do not directly enter the objective On the ImageStream cell analysis system 90 degree angle side scatter from the 488 nm laser provides the darkfield imagery FISH S fluorescent in situ hybridization FISH A fluorescent dye used to label cellular constituents or specific fluorochrome 5 pr
182. r is enabled 2 Select Combined as the Feature Type The editing interface appears Feature Type C Single Combined Name saxe wt ay Set Default Name Ok Cancel 3 Enter the feature name in the Name box or use Set Default Name after you have created your expression The default name is the name of the definition created 4 Use the toolbar to build a definition mathematical expression of features and operators Table 1 Combined Feature Tasks and Toolbar Double click the feature in the Features list Add afeatureto Or single click the feature in the Features list and select the definition click the leftmost down arrow button on the toolbar Add an operator or Click the corresponding button on the toolbar a parenthesis to PPRA the definition Enter the number in the box and then click the cor Add a numberto responding down arrow button the definition rm EXE If the area is greyed out an operator must be selected first Select the function in the list and then click the cor Add afunctionto responding down arrow button the definition The available functions are ABS absolute COS cosine SIN sine SQR Square and 117 Chapter 3 SQRT square root If the area is greyed out an operator must be selected first Remove anitem Click the left arrow button on the toolbar 5 Click OK 6 Click Close Note When you close the Feature Manager the IDEAS applic
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184. rate a Statistics Report 1 Select Reports gt Generate Statistics Report The current daf file appears in the window with the specified statistics columns e Select a daf or template file that contains a statistics report definition 092011 X101 unstimulated _1t Report title 092011 X101 unstimulated_1t daf_stats Similarity Files Count All CountR1 Count R2 Count R3 Count R4 Gated R4 Median R3 2 Pick a Report Definition The definition may be obtained from a daf or ast file 3 Change the Report title if desired 4 Additional daf files can be added or removed with the Add Files or Remove Files buttons 5 Reorder the files as desired by selecting files and then right click the new loca tion in the list and choose move here You can Ctrl select multiple files in the desired order and then move all at once by right clicking in the desired location and choosing move here 6 Click OK A prompt will confirm that the daf file will be saved The report title name will be used as the default file name for the report In the above example the file gen erated will be named Report 1 txt If the report title contains illegal characters such as gt lt the default filename will change to Statistics Report txt Tab delimited text format is used for the report 150 Reporting Statistics Reporting Statistics from a Single Graph or Statistics Table Statistics can also be r
185. re Channel mask Enumerates the number of spots Std Dev Feature Morphology Describes the overall distribution of pixel intensities Object Signal no Strength Signal Strength Features are measured in pixel values aa Intensity Feature Morphology The sum of the pixel intensities in the mask background sub Object Thresh tracted old 30 50 70 31 Chapter 3 The statistics Count and Percent Gated are added to the Population Statistics table in the analysis area and a statistics definition is added to the template To view the definition choose Define Statistics Report from the Reports menu 3322 Guided Analysis Apoptosis Wizard The apoptosis wizard will guide you through the process of creating the features and graphs to measure apoptosis using the images of the nuclear dye and brightfield Begin by opening a data file and then choosing the Apoptosis wizard Apoptosis To begin double click on Apoptosis Follow the instructions to analyze your file Step 1 Select the nuclear image channel From the drop down menu pick the nuclear channel image Step 2 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in
186. rea M10 Mask M01 Name Aspect Ratio Intensity_M01_Ch01 Area_M11 Area_M12 Image Ch01 Area_MC Aspect Ratio Intensity_M02_Ch02 Lonant Dastin intansi MNI ChN2 Sort features by A IN 2 l a El New Delete Edit J f Add Multiple Features Close 2 Choose an icon to sort the features Table 1 Sorting Features eature Icon Definition Sorts features alphabetically BI fos features based on the images used Sorts features based on the masks used Sorts features by category such as size loca tion shape texture signal strength and system B Sorts by base features such as area aspect ratio intensity and object number 3 Click a feature in the Features list to view its definition in the right side of the win dow Creating New Features with the Feature Manager To create a new single feature A single feature uses the definitions of a base feature along with a mask and or an image sfide Using the Feature Manager 1 Click New in the Feature Manager The right hand area of the Feature Manager is enabled Feature Type Single Angle C Combined 4 Name Mask Combined Mask Set Default Name OK Cancel 2 Select Single as the Feature Type The Mask and Image lists become visible depending on the single feature selected Feature Type Single Similarity E C Combined Name Mask Combined Mask z
187. rogress bar After the application has successfully opened the cif file the daf file is saved See also Saving Data Files Opening a daf file A daf file contains the calculated feature values so that they will not need to be recal culated as described in Overview of the Data File Types To open a daf file the IDEAS application requires the cif file to reside in the same directory The daf file does not contain any image data so you can think of the cif file as the database that contains the imagery Because all of the feature values have been saved in it the daf file should open quickly To open a daf file To use a wizard to do this see Open File Wizard otherwise 1 From the File menu choose Open or drag the file into the IDEAS window 2 Select the daf file that you want in the Select File To Load window Tip while browsing for the file to open you can limit the type of file shown in the win dow to daf Select File To Load Look in analyzed cif and daf files 0 0ng_2_9 Default daf 1000ng 90_30_13 daf Q 0 0ng_2_9 daf My Recent 0 1ng 15_1_8 daF Documents 0 1ng 30_6_13 daF 0 1ng 45_11_18 daF 0 1ng 60_16_23 daF 0 1ng 75_21_4 daF 0 1ng 90_26_9 daF 10ng 15_3_10 daF 10ng 30_8_15 daf 10ng 45_13_20 daF My Documents sd 10ng 60_18_1 daf 10ng 75_23_6 daf 3 10ng 90_28_11 daf 1000ng 15_5_12 daF My Computer 1000mg 30_10_17 daf
188. ry The Directories tab contains the default Data Template Batch or Compensation Matrix file directories Setting Up the IDEAS Application Populations Masks Graph Display Graph Export image Export Colors Default Data Files Directory C Users sfriend Desktop NFKB Translocation FS v Update automatically when file is selected Default Template Files Directory C Users sfriend App Data Roaming Amnis Corporation templates G Update automatically when file is selected V Use default data directory Default Batch Report Files Directory C Users sfriend App Data Roaming Amnis Corporation batches Gaa Update automatically when file is selected Default Compensation Matrix Files Directory C Users sfriend App Data Roaming Amnis Corporation compensation Update automatically when file is selected V Use default data directory The Populations tab contains the default color or symbol for populations To change the default settings click on the color to or choose a default symbol from the list Chapter 1 Application Defautts eh eS Directories Populations Masks Graph Display Graph Expot Image Export Colors The Masks tab contains the default mask color To change the color of the mask Default Color Default Symbol Simple Dot click on the
189. s Delta Centroid XY can identify cells with caps as shown here Application Examples Quantify the spatial relationship between two fluorescent probes 188 Understanding the Location Features Identify false apoptotic positive cells in the TUNEL and Annexin V assays Quantify shape change Quantify capping of cell surface antigens Raw Centroid X and Raw Centroid Y Features The centroid X and Y of the original position of the image during acquisition before it was centered IDEAS Data analyzed in IDEAS versions 4 0 or later cut and center objects that were collected as one image in INSPIRE 189 Chapter 4 Max Contour Position Feature The Max Contour Position is defined as the location of the contour in the cell that has the highest intensity concentration It is invariant to object size and can accom modate localized intensity concentrations The actual location in the object is mapped to a number between 0 and 1 with 0 being the object center and 1 being the object perimeter which allows one to compare the results across cells of different sizes An example is shown below All Channel 3 Nommadzed Frequency Maicon Pcellon inkimeaMark_ cr Channel 3 Channel 3 Application Example Used in conjunction with the Internalization feature to determine the dis tribution of intensity within a cell 190 Understanding the Location Features Shift X and Shift
190. s exceeds the space available on the window The panels can be re tiled using the arrange analysis area tool As illustrated by the following figure the Analysis Area can contain several types of panels histogram histogram overlay scatter plot tables of population statistics or object feature values channel image composite image and text Each panel will contain its own toolbar and context menu To move a panel click on the name at the top of the graph and drag it to a new location A graph may be selected and thena right click in a blank space in the work area allows you to choose paste in the new location ROLE BSA Loeb ke Hene saa doublet A toolbar is visible at the top of the Analysis Area The following table describes the function for each tool Analysis Area Tools Table 1 Analysis Area Tools Provides the normal mode of interaction with the graphs Clicking a point on a scatter plot graph causes the IDEAS application to display the corresponding image in the Image Gallery if the population that is currently displayed in the Image Gallery contains that point Click the bin in a histogram to select the bin In the Image Gallery you can view images of cells in the bin by choosing the Selected Bin pop ulation Le Pointer Tool 94 Overview of the Analysis Area Click Pointer Tool while drawing a region on a graph to cancel the cre ation of a region Allows you to create a
191. s for a graph 1 You can show and hide statistics by clicking the Statistics toolbar button in the panel that contains the graph 97 Chapter 3 conjugates A Q k a 1 8 on gt MN o two d Frequency 2 Or right click anywhere on the graph and click Statistics on the graph context menu that appears The Statistics window appears Graph Statistics IZ Count View statistics E Total V Gated Plotted Objects mL Mean E Median Std Dev MAD CV Minimum Maximum Geo Mean Mode Variance NaN Select the statistics that you want to display Check the View statistics box and the box next to the statistic to be displayed for each population on the graph The statistics that are supported are the Count Percent Total Percent Gated Per cent Concentration count sample volume Mean Median Standard Deviation MAD Median Average Deviation RD Mean RD Median CV Minimum Max imum Geometiric Mean Mode variance and NaN not a number 4 When finished click Close To show the legend for a graph 1 Right click anywhere on the graph and click Show Hide Legend on the graph context menu that appears If the legend was hidden it appears on the graph If the legend was shown it dis appears from the display 98 Overview of the Analysis Area Note The legend contains an entry for each population on the graph If the graph is ascatter plot the
192. sation a E Update automatically when file is selected v Use default data directory 132 Creating Reports and Exporting Data The Directories tab contains the default Data Template Batch or Compensation Matrix file directories _ e a Populations Masks Graph Display Graph Export image Export Colors Default Data Files Directory C Users sfriend Desktop NFKB Translocation FS 6 3 V Update automatically when file is selected Default Template Files Directory C Users sfriend App Data Roaming Amnis Corporation templates Update automatically when file is selected v Use default data directory Default Batch Report Files Directory C Users sfriend App Data Roaming Amnis Corporation batches Update automatically when file is selected Default Compensation Matrix Files Directory C Users sfriend App Data Roaming Amnis Corporation compensation Update automatically when file is selected V Use default data directory 133 Chapter 3 The Populations tab contains the default color or symbol for populations To change the default settings click on the color to or choose a default symbol from the list Z Application Defaults M b ams Directories Populations Masks Graph Display Graph Export image Export Colors Default Color i
193. sets 029 005 014 048 147 008 Horizontai 008 032 013 07 0 028 Ofsets 001 008 019 038 002 0 38 ie BEB 8G 7 Comected during acquisition 7 Perform normalization 4866 of 4866 V Apply cell classifiers V Erase nonframed objects Minimum Maximum _ Save Comections to Raw Image File Cook Lancet Make any changes to the corrections that you need and then click OK Opening a cif file A cif file is generated when corrections are applied to a rif file as described in Over view of the Data File Types When opening a cif file the IDEAS application cal culates feature values and creates a daf file to display images and graphs When opening a cif file an analysis template is selected The template provides the initial characteristics of the analysis Opening the cif file causes the IDEAS appli cation to calculate feature values and to use populations graphs and image viewing settings to display the cells as defined by the template To open a cif file To use a wizard to do this see Open File Wizard otherwise 53 Chapter 3 1 From the File menu choose Open or drag the file into the IDEAS window 2 Select the cif file that you want in the Select File To Load window Tip while browsing for the file to open you can limit the type of file shown in the win dow to cif Select File To Load Look in analyzed cif and daf files N 0 0ng_2_9 ciF G 0
194. t control files are collected without brightfield illumination or scatter The IDEAS application performs brightfield compensation when it loads a rif file The process of creating the compensation matrix is described in the next section Creating a New Compensation Matrix File The compensation matrix is a table of coefficients The IDEAS application uses this table to place the detected light that is displayed in each image into the proper chan nels on a pixel by pixel basis The coefficients are normalized to 1 Each coefficient represents the normalized amount of the leakage of the fluorochrome into the other channels The default matrix which is used if no compensation matrix is chosen is the identity matrix shown below 59 Chapter 3 Ch01 Ch02 Ch03 Q R Ch05 Ch06 Ch07 Ch08 Ch093 Ch10 Ch11 Chi2 1 0 0 0 0 0 0 0 0 9 090 0 9 090 0 090 oj o 9 9 9 9 9 9 9 9 9 o o o ojo o jo o o o o o o o ojo o jo o o o o o S Se eee ee se eee So o o o o o o o o o o S O 9O H 90 90 90 90 90 0 0 o SO O O O0 0 90 0 0 0 0 o 0 9 9 9 9 9 9 9 9 o o To generate a new compensation Matrix file 1 Start the Compensation Wizard in one of two ways Click the New Matrix button when opening a rif file OR select Compensation gt Create New Matrix The compensation wizard opens to Step 1 Create Compensation Matrix Z z Step 1 Select t
195. t saturated preferably single color If single color cells are not available choose cells with a distinct staining pattern in the peak channel 2 Create Intensity scatter plots of adjacent channels in order to observe the over or under compensation 3 Identify the matrix values that need adjusting by inspecting the scatter plots and images Each column contains the coefficients for the peak channel into the cor responding crosstalk channels rows For example the crosstalk of channel 2 green into channel 3 is highlighted in the matrix below 270 Appendix Troubleshooting Compensation Matrix Select a compensation matrix 081109 G2A1 shape change MCP1_2 cif ChO1 ChO2 ChO3 ChO4 ChO5 ChO6 ChO ChO8 ChO9 ChiO Chii Chi2 1 0 048 0 0 0 0 0 0 003 1 0 0 02 PARE 1 0 085 0 0 017 0 044 0 001 0 002 0 001 o ao oa o jojojojojojoj ojojo o ojojojojoj ojojojo o jojojojoj ojojojojo olololojlojojojolojo Preview a file with this matrix applied Select an existing rif file Overwrite preview files Select a population from the current file m Iz Preview e Undercompensation crosstalk coefficient is too low Plots Intensity mean for the single color positive population is higher than the unlabeled population in the crosstalk channel or the intensity in the crosstalk channel trends diagonally upwards Images the crosstalk channel contains an apparent fluorescent mirror
196. ter 3 Begin Analysis Wizard This wizard is available once a data file is open and will guide you through choosing the focused cells then single cells then choosing subsets of fluorescent positive cells for phenotypic analysis before progressing on to a morphological analysis Open a data file using the Start Analysis button or by choosing Wizards from the Guided Analysis menu The wizards selection screen will appear once the data file is open If you have an open data file and want to access this wizard choose Wizards from the Guided Analysis menu Begin Analysis To begin double click on Begin Analysis Step 1 Gate cells in best focus A histogram of the brightfield channel Gradient RMS values for the All population has been added to the analysis area Click on the bins in the histogram to view the images in each bin The cells with better focus have higher Gradient RMS values Begin your region at the bin after the Gradient RMS value you wish to exclude and continue the region to the maximum in the plot You may choose an already existing population Step 2 Gate single cells A scatter plot of the brightfield Area versus Aspect Ratio for the population chosen in step one has been added to the analysis area Single cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by a light green line and the image next to it
197. tfield channel set the vertical lines to about 50 counts to the right and left of the his togram to produce an image with crisp brightfield contrast 83 Chapter 3 e Tochange the mapping curve to be logarithmic or exponential click drag the yellow cross To restore the mapping to a linear curve Click Set Linear Curve e To see the full scale for the X Axis Click Full Scale e Toset the display mapping of the X Axis to the lowest and highest values for a selected object Click Set Range to Pixel Data To set the scale of the X Axis to the range of the vertical green lines or of all the pixel intensities for the selected object whichever is larger Click Auto scale e You may enter values manually by selecting the Manual tab Automatic Manual Image Display Mapping x Axis Scale Set Range to Pixel Data Full Scale Set Linear Curve 4 Ifyou want to preview the changes in the Image Gallery click Preview Changes in Gallery 5 Continue customizing the Image Gallery display properties with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes To customize the Image Gallery views images and masks 1 Within the Image Gallery Properties window click the Views tab Note The Image Gallery view can be customized to view any combination of channel images or composites The default view All Channels is a view that displays all image channels that were in
198. the color that you want in the color palette Click OK to close the palette Tip The grayscale image in each channel is assigned a default color for image display in the gallery Setting the color to white is equivalent to using the original grayscale image The colors are also used to build composite images 82 Overview of the Image GalleryUsing the Image Gallery To fine tune the image display intensity for an image 1 On the Display Properties tab of the Image Gallery Properties window select an image by clicking the image name in the list The graph for the currently selected image is shown in the window and updates as the changes are made Select and image in the image gallery that has intensities for the image channel you are adjusting Note You will adjust the Display Intensity settings on the graph the Y Axis the value of the display to the X axis the range of pixel intensities The range of pixel intensities will depend on the instrument and the collection mode set during acquisition The display range is 0 255 the range of intensities from the camera is 0 4095 for the ImageStream or 0 32 767 for EDF mode collection The 1S100 first generation instrument has a 10 bit camera and therefore the range of pixel intensities is 0 1023 The limits of the graph enable you to use the full dynamic range of the display to map the pixel intensities of the image 255 5 200 100 0 E I I Ii 0 1000 2000 300
199. the image in the cif file The masks labeled M01 through M12 contain pixels that are detected as brighter than the background In addition the application generates a Combined Mask named MC and a Not Combined Mask Not MC for each object A combined mask consists of the union of the masks of all the channels of the object A Not Com bined Mask is all of the pixels with no intensities above background You might need to adjust the masks or create new ones that include only a specific area of a cell such as the nucleus You can combine masks by using Boolean logic or you can adjust them by applying functions Creating New Masks with the Mask Manager There are two ways to work with new masks in the Mask Manager First masks can be created by using functions which allows you to choose an input mask and if needed adjust the channel and scalar input Alternatively masks can be created by combining masks through Boolean logic This option is not available for basic Flow Sight files without the Quantitative Imaging QI upgrade To create anew mask using Functions 1 Select Analysis gt Masks The Mask Manager opens with a list of existing masks on the left 2 Click New The right side of the window is enabled to define a new mask 88 Overview of the Mask Manager Mask Manager xs reece e E S A a D T T N Masks Name MOI M02 M03 M04 M05 Definition MOG m01 Click Function The Def
200. the lower right Similarly a cell with a large shift in the Y direction and not X are found in the upper left See Delta Centroid XY Feature to measure the X and Y shift together 186 Understanding the Location Features Single Focused cells a oO Fe 6 1 gt v 2 2 a a 4 6 Delta Centroid_Ch4Ch6 Application Example Used to identify capped versus not capped cells Used to measure shifts in X or Y direction between two images Delta Centroid XY Feature The Delta Centroid XY feature measures the distance between the Centroid feature of two images using the user provided masks for each image Either one or both the centroids of the images may be intensity weighted X and Y pixel coordinates are cal culated from an origin in the upper left corner to obtain the centroid positions and the distance between the centroids is converted to microns In the example below an image pair is shown stained with the nuclear dye Draq 5 and a PE labeled antibody that is differentially expressed two cells either uniformly or in the pseudopod The two cells are identified by their different Delta Centroid XY values 187 Chapter 4 Composite gt 2 O Cc Q O Composite Delta Centroid Y Delta Centroid X Centroid X Delta Centroid XY v Delta Centroid X Delta Centroid Y Below is an example of using the Delta Centroid XY A bivariate graph of a shape ratio versu
201. the scatter and brightfield channels If necessary the column can be set to the identity values by double clicking on the heading Inspect the coefficients in the matrix by double clicking on the coefficient Coefficients highlighted by red have errors greater than 1 A graph representing the coefficient appears The population potted in the graph is the positive control population of the column of the coefficient The X Axis represents the intensity in the assigned channel of the fluorochrome The Y Axis represents the intensity in the channel of leakage The coefficent value and error are also displayed Matrix Coefficient Inte 0 3_Positive me I 1 40000 60000 80000 Intensity_MC_ChO3 I 20000 Coefficient value 9 49 Coefficient error 0 00036 Add Graph to Analysis Area You can use the automatically generated control populations as they are or you can refine them and create different populations by using the region tools See the option below to remove objects from the population By default the populations are named 3_ Positive 5 Positive and so on You can view the populations in the Image Gallery Some populations may be empty To choose a different population use the arrow and select the population from the list The hierarchical relationship is shown in the population list Assign 63 Chapter 3 populations only to the channels that correspond to the fluorochromes used
202. tinue the region to the maximum in the plot You may choose an already existing population Step 3 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 4 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken SATE Chapter 3 Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip Next Step Gate double positives A scatter plot of the last gated or selected population of the Intensity values for the co localization channels is added to the analysis area Draw a region around the dou ble positive cells for the co localizing probes Next Step Gate co localized events A histogram of
203. tion 64 creating 59 definition 16 editing the matrix 67 overview 58 validating matrix 63 276 view matrix 73 Compensation matrix file about 19 creating 59 Composites 85 142 Copying images 87 Corrections information 72 ctm about 19 creating 59 daf about 18 opening 55 saving 56 Data analysis steps 22 Data analysis tools about 78 Data files ast 19 20 cif 18 20 ctm 19 20 daf 18 20 new from populations 71 opening 50 rif 18 20 277 Index Chapter Appendix A type 20 Defaults application 5 color mapping dark light mode 10 137 compensation matrix 15 directories 6 133 graphs 9 135 image export 9 136 mask 15 mask color 8 134 population color 7 134 population symbol 7 134 statistics fora graph 8 134 Diameter 172 Directories changing default 6 133 Display making composites 81 138 setting properties 81 138 setting properties using a wizard 27 views in the Image Gallery 81 138 EDF kernels 73 Example data files 5 278 Exporting data 152 features 152 pixel intensity values 153 Feature Finder wizard 29 Feature Manager overview 113 tasks 117 tools 114 Features angle 183 angle intensity 183 Area 171 aspect ratio 196 aspect ratio intensity 197 Bkdg mean 217 Bkgd std dev 218 bright detail intensity 206 camera line number 243 camera timer 244 categories 161 centroid delta x and y 185 centroid delta xy 187 centroid x andy 184 circularity 198
204. tion creates a cif file when the user opens a rif file and applies a compensation matrix The segmentation algorithm automatically defines the bound aries of each object creating a mask that is used for calculating feature values The applied compensation matrix performs pixel by pixel fluorescence compensation prior to segmentation During the creation of the cif file the application makes cor rections to the imagery These corrections include e Removal of artifacts from variability in the flow speed camera background and brightfield gains Alignment of the objects to subpixel accuracy which allows the viewing of compensated imagery composite imagery and the calculation of multi image feature values such as the Internalization value e Coincident objects are cut apart to place into individual image frames Note that this will increase the number of objects in the file Multiple cif files can be created from a single rif file by applying a different flu orescence compensation matrix or correction each time a rif file is opened and choosing a unique name for the cif file Similarly you can create a new daf file from a single cif file by creating a new name and applying a different analysis template Data Analysis File daf The IDEAS application creates a daf file while it is loading a cif file into a template file ast The daf file is the interface to visualize and analyze the imagery that the cif file contains an
205. tion of up to 12 images per object The FlowSight system has 12 channels collection on 1 camera detector Spectral decomposition element A A cells in flow O autofocus velocity ry detector brightfield illuminator About Features The IDEAS application provides a large selection of criteria or features for analyz ing images A feature is described by a mathematical expression that contains quan titative and positional information about the image A feature is applied to specific locations of an image by the use of a mask that identifies pixels within the region of interest of the image A few system features such as Object Number Camera Back ground and Flow Speed do not require calculations masks or image intensity infor mation There is a slight difference in features created during data acquisition and those in IDEAS During acquisition features are created with the INSPIRE mask Features and masks are calculated in IDEAS for files collected with the ImageStream or a FlowSight with the Quantitative Imaging QI upgrade New masks and features can be created in files from an ImageStream or FlowSight with the QI upgrade using the Mask and Feature Manager tools Features are 159 Chapter 4 created in IDEAS using base feature algorithms such as Area or Intensity along with a mask and or a channel image for files created with an ImageStream or with the QI upgraded FlowSight machine The default
206. tions based on Inten sity and include the channel s you are going to use for morphological analysis Click Next to add the scatter plot to the analysis area Click Skip if you do not wish to use this step Draw regions in the scatter plot to identify as many populations as you want This step will be repeated until you choose Skip or Finish 29 Chapter 3 Step 4 Assign truth populations Choose two truth populations of cells that represent one morphological phenotype dif ference you wish to separate Use the tagging tool icon to hand tag images or select pre existing gated populations Note If there are more than one phenotypic dif ferences repeat the process for each with new truth populations Step 5 Select channels and feature categories Choose the channel and feature category you wish to explore Multiple rows may be entered The features in the table below will be created and calculated All of the default features newly created features and user defined features in the chosen cat egories will be evaluated for their ability to separate the truth populations The three highest ranking features for each category will be saved and available for evaluation Step 7 Results The top ranking features are listed in the table with their category and channel A Sta tistics table is added to the analysis area that lists the features with the RD Mean for the truth populations RD is the Fischer s discriminant ratio which is the
207. tiple features may be chosen by holding down the Ctrl key 5 Click OK The features appear in the Object Data table 6 Toadd selected objects to the table right click and choose Add Current Object To export or copy feature values e Right click in the table and choose Copy feature values to clipboard Adding Text to the Analysis Area To add text to the Analysis Area 1 Click the Text button on the Analysis Area toolbar A A text panel is added to the analysis area 110 Overview of the Analysis Area Enter title Enter text here 2 Typeatitle and text Population Statistics The Population Statistics table displays selected statistics for chosen populations The statistics that are supported are the Count Percent Total Percent Gated Per cent Concentration count sample volume Mean Median Standard Deviation MAD Median Average Deviation RD Mean RD Median CV Minimum Max imum Geometric Mean Mode variance and NaN not a number To view and customize the population statistics Click the Populations Statistics tool Click the next to the population to expand the list of populations Columns can be moved by click dragging Right click in the grey area in the table or on a column heading and the menu opens A wWhND Edit Statistics Table Insert Column Edit Column Delete Column Delete All Columns Order Columns Copy Statistics Copy Statistics Transposed 5
208. tle 092011 X101 unstimulated _1t daf_stats 2 Entera Report title 3 Under the Statistic Columns clickAdd Columns The Add Statistic Report Col umn window opens 4 Select the statistic s in the Statistics list e Count the absolute count of the populations e Total percentage of a population as a percentage of All 148 Reporting Statistics e Gated the percent of one population as a percentage of another but not used for tagged populations the percentage of one population as a percentage of another also is used for tagged populations e Objects mL the concentration of the population in the sample run e CV the coefficient variable Geometric Mean standard statistical definition e Maximum standard statistical definition e Mean standard statistical definition e Median standard statistical definition e MAD standard statistical definition e Minimum standard statistical definition Mode standard statistical definition e RD Median the ratio discriminant Fisher s discriminant using the Median and MAD e RD Mean the ratio discriminant Fisher s discriminant using the Mean and StdDev e Standard Deviation standard statistical definition e Variance standard statistical definition NaN stands for not a number the number of objects whose values are not valid numbers 5 Select a population to base on the selected statistic s 6 Select a reference population if necessary This
209. tter Aspect Ratio Intensity __MX_ChX Intensity_MC_ChX for all fluorescence channels for all channels Intensity_scatter ingle Cell Default Area_brightfield Aspect Ratio_brightfield Size SSC Area_brightfield To begin choose Building Blocks from the Guided Analysis Menu or click on the Building Blocks icon in the analysis area toolbar Single Cell v The Building Blocks window opens This window is used to define a graph with a specified set of features available depending on the purpose of the graph 1 Choose the specific Building Block from the drop down menu Building Blocks Select Predefined Building Block C 1D Fluorescence Positives One Color Fluorescence Positives Two Color Focus Single Cell Single Cell Default 2 Choose the population s to graph ts Chapter 3 Building Blocks Select Predefined Building Block Fluorescence Positives One Color v Use the control key to select multiple populations So All B N Single cells B m A2 N apoptotic 7 none d mei A4 v 3 Choose the X Axis Feature and the Y Axis feature if applicable Title and Axes Title 3 R3 i Intensity_MC_Ch02 ee intensity MC Ch03 Intensity_MC_Ch04 Y Axis Feature Llntensity MC_Ch06 Y Axis Label 4 Click OK 5 The graph is added to the analysis area 48 Advanced Analysis Advanced Analysis Opening data files Saving Data Files Ov
210. ty The closer this ratio is to 1 0 the better focused the image This feature is mainly used for single uniform particles such as beads The figure below shows the image quality test using the Ensquared Energy feature 209 Chapter 4 Gradient Max Feature The Gradient Max feature measures the sharpness quality of an image by detecting largest change of pixel values in the image and is useful for the selection of focused objects This figure shows the change in intensity across the red line The top image has a larger slope change than the lower image Application Example Determine peak focus quality of images Alsoused to characterize texture However the Gradient RMS and Con trast feature are more robust for these applications See also Gradient RMS Feature and Contrast Feature 210 Understanding the Texture Features Gradient RMS Feature The Gradient RMS feature measures the sharpness quality of an image by detecting large changes of pixel values in the image and is useful for the selection of focused objects The Gradient RMS feature is computed using the average gradient of a pixel normalized for variations in intensity levels This is similar to the Contrast cal culation with different weighted assignments to the pixel arrays and with background subtracted Example images are shown in the figure below D i Contrast_BF Gradient RMS_BF Application Examples Determi
211. ulations or objects in tables placed in the analysis area Graphs show data plotted with one or two feature values and tools are provided that allow you to draw regions for the purpose of generating new populations You can show any population on a plot Every image is linked to the feature data Selecting an individual data point in a graph allows you to view it in the Image Gallery or look at its feature values in the Statistics Area Any object that is selected in the Image Gallery is also shown on the plots in the Analysis Area 78 Overview of the Image GalleryUsing the Image Gallery Overview of the Image GalleryUsing the Image Gallery This section contains the following subsections which describe how to view pop ulations of objects in various ways view masks customize the Image Gallery dis play and hand select objects for a population Overview of the Image Gallery Using the Image Gallery Setting the Image Gallery Properties Working with Individual Images Creating Tagged Populations Overview of the Image Gallery The Image Gallery displays the imagery and masks of any population of objects A toolbar is provided in the upper left corner of the panel as shown in the following figure The Image Gallery also makes different viewing modes available for the imagery The default template contains the viewing modes which allows you to view all channel images in grayscale or color or each channel image individually Tip
212. uorescence in each cell image Compensation is defined as the correction of the fluorescence crosstalk When creating the cif the IDEAS appli cation also automatically performs corrections to the raw imagery using values saved from the instrument at the time of data collection These corrections include flowspeed normalization brightfield gains and spatial registry A template is used to define the features graphs image display properties and anal ysis for the daf Within the daf file the user can perform many analyses using the tools and wizards within the application and save the results as a template file ast The IDEAS application then calculates feature values and shows the data as spec ified by the selected template Once a data analysis file daf file or compensated image file cif file is saved it can be opened directly for data analysis You would only open a cif if you wanted to change the template or a rif file to change the compensation The diagram on the next page displays this workflow 16 Understanding the Data Analysis Workflow INSPIRE IDEAS Data Collection Spectral Compensation Data Analysis Raw Compensated Data Image Apply Image Analysis File pensat File File wif ee mper Template e0 e0e matrix File o eeeesees _ i Compensation Seessees 3 Ctm Matrix lt Q im s HHHH Batch Processing Overview of Data Analysis Workflow 1 Create a compensation matrix using the
213. ures that are not included in the feature finder template See theFeature Finder Wizard for more details General 1 Set image display and draw preliminary regions to include cells of interest i e sin gle focused positive cells 2 Visually inspect overall quality of images and experiment to determine whether to proceed or redo the experiment 3 Create two tagged truth populations of cells that represent the phenotypes you wish to discriminate Perform the discrimination on one characteristic difference at a time 4 Create any additional masks and features you think may help differentiate the truth populations 118 Using the Feature Manager Example 5 Calculate the statistical discrimination RD between the two populations afforded by features in 1 category at a time Pick the top feature for each cat egory 6 Plot the features with the highest RD for the truth populations for each category 7 Validate by applying the feature to the base population independent controls if available and on multiple files and experiments Treatment induced actin polarization The data file is available for practice Log in to your account on the Amnis website and look in the folder Training data files e Cells were incubated with inducing compound for 1 hour e The nucleus was probed with DAPI and actin stained with FITC e Large event image files were collected on the ImageStream e Compensation and analysis was done
214. ustment of the image size for the image gallery e Views Allows you to customize the views for the Image Gallery e Composites Allows you to create composites and adjust the amount of color from a channel that is included in a composite image 81 Chapter 3 To customize the Image Gallery display properties 1 Click the Image Gallery Properties toolbar button to begin The Image Gallery Properties window appears with the Display Properties tab displayed Display Properties Views Composites Images Name Ch02 Color Minimum Pixel Intensity 15 Maximum Pixel Intensity 1484 255 Automatic Manual Image Display Mapping X Axis Scale Pap aa Display Height Set Range to Pixel Data Full Scale Channel Width Set Linear Curve Attoscale Auto Fit Auto Fit 120 325 Preview Changes in Gallery To change the size of the panels in the image gallery 1 Display Width and Display Height can be specified or changed to Auto Fit in the lower left section of this window To change the name or color for each image 1 Select an image in the list of images on the Display Properties tab of the Image Gallery Properties window On the right side of the window you can type a new unique name for the selected image Note that each image is provided with a default name and the image names appear near the top of the Image Gallery Click the colored square for the selected image Click
215. ution compared to the 7 AAD image red When the inten sity of the green is high the intensity of the red is low and vice versa The Similarity value for this cell is 2 067 indicating that the image pair has a high degree of dis similarity Analysis of the image pair on the right shows that when the intensity of the green is high the intensity of the red is high and the Similarity value is a high positive number Untranslocated Translocated NF kB image 7 7 Cc Le 2 Es e E ae g a A m E m ug o x a 1 LL LL Z Z 100 120 7 AAD Pixel Intensity 7 AAD Pixel Intensity 7 AAD image 7 AAD image Below are examples of cells with varying amounts of similarity between the NF kB image in green and 7 AAD image in red shown here as a composite image The most dissimilar image pairs in the upper left to the most similar image pairs in the upper right Frequency I L 0 3 Similarity NFkB 7 AAD 0 1 0 5 0 9 14 18 Application Examples 239 Chapter 4 Quantify translocation Identify copolarization of two probes Used in the Nuclear Localization Wizard 240 Understanding the Comparison Features XCorr Feature The XCorr feature is a measure of similarity or sameness between two images the higher the value the more similar the images It is robust to intensity variations and relative shifts between the images and is typically used with the combined mask
216. utomatically analyze a group of files with one tem plate when a compensation matrix has already been generated for the experiment To perform batch processing 1 Onthe Tools menu select Batch Data Files The Batches window appears It lists a record of all batches you have proc essed Batches to Run Edit Batch Remove Batch Submit Batches Processed Batches 9 Batch 6 29 2011 11 05 33 AM H Batch 5 3 2011 10 38 34 AM H E Batch 5 3 2011 9 51 14 AM H E Batch 5 3 2011 9 41 07 AM H E Batch 4 5 2011 11 28 23 AM H E Batch 3 24 2011 11 47 57 AM H Batch 3 23 2011 2 29 21 PM H E Batch 3 23 2011 2 00 37 PM H Batch 3 2 2011 8 02 21 AM H Batch 3 1 2011 1 17 04 PM M E Batch 2 9 2011 11 49 07 AM 2 Click Add Batch The Define a Batch window appears oe N oo f Batch Processing deine a bach Input Files Select nif cif or daf files to process Select a compensation matrix compensated image file or data analysis file ctm cif daf Select a template or data analysis file ast daf V Overwrite existing files File suffix To select the files for the batch click Add Files Navigate to the files and select by clicking on the file Select multiple files to add by holding down the Ctrl key while selecting the files e Toremove files from the Files to Process list click Remove Files Select a compensation matrix from a file ctm cif or daf
217. ve changes from the File menu Remember that the daf file does not contain image information so opening the daf file requires the related cif file to reside in the same directory To save a daf file 1 Onthe File menu click Save as Data Analysis File daf 2 Entera file name Note that the default directory is the one where the cif file was saved If you select an existing file name a warning appears that asks you to verify the overwriting of the existing file 3 Click Save The data is now ready for analysis You can create graphs view imagery and dis play feature values and statistics After you have defined an analytical procedure in the daf file you can save the file as a template which allows you to use the procedure for analyzing other files Refer to Overview of the Data Analysis Tools for more information option Saving a data analysis file with only the feature values used When you want to reduce the size of a data analysis file you may save the daf with only the features that are used for analysis of statitics or graphs On the File menu click Save as Data Analysis File Used Features Only and follow the instructions 2 3 above Saving a Compensated Image File cif The IDEAS application creates and saves a cif file when a rif file is opened By default the application names the cif file with the same name that the rif file has replacing the rif extension with cif The application also places the c
218. vers common issues and provides solutions Always make sure you are using the most up to date version of IDEAS by logging in to your account at www amnis com and down loading the software Application Hanging If the IDEAS application is hanging there may be a memory issues especially with large file processing You must use the Task Manager to force quit the application Press and hold Ctrl Alt Delete The Window Task Manager appears Under the Applications tab select IDEAS Application If the status is Not Responding select End Task The manager will force quit the application after a confirmation oR WD Compensation Sometimes an applied matrix produces poorly compensated data This can happen for a number of reasons 1 miscalculation of the compensation matrix by inclusion of inappropriate events such as doublets saturated pixel events or artifacts 2 controls used for matrix calculation differ significantly from the experimental sam ples different cell type different probe or 3 cells exhibit substantial auto fluorescence This protocol describes a method for manually adjusting and validating a compensation matrix for difficult samples To troubleshoot and repair a compensation matrix 1 Create a population of cells that are miscompensated using the tagging tool See Creating Tagged Populations Choose single cells that are exhibiting crosstalk Choose a range of intensities from negative to bright but no
219. vided into two sections First guided analysis is described using the analysis wizards and second advanced anal ysis with more detailed instructions that describe how to open com pensate merge save and create data files without using the wizards Building blocks are also discussed which provide a short cut method to building commonly used graphs Guided analysis makes it easy to start analyzing your data Once you are familiar with the basic analysis available you may want to perform more advanced analysis Note that data files from FlowSight without the Quantitative Imaging upgrade have a limited feature set and limited wizard analysis General Outline of data analysis Note that these steps apply to any type of data analysis whether you use a wizard or not 1 Open one data file the or control 2 Create and save a compensation matrix for the experiment 3 Using an application wizard or the begin analysis wizard e Select focused cells e Select single cells or conjugates e Select channels for subpopulation markers and gate to define subpopulations e Gate on positive cells for the channels you wish to use for morphological anal ysis 4 By using an application wizard evaluate the feature for your analysis and refine as needed Or follow the feature finder wizard to find the feature that separates your populations Note that if the morphological differences are in separate files this may require merging both a and
220. w Min Pixel Feature The Raw Min Pixel feature is the smallest value of the pixels contained in the input mask The example below illustrates quantifying the level of malarial infected cells by using Min Pixel values of brightfield imagery Brightfield YoYol Brightfield YoYol Malarla Infected RBC Infected RBC p Wu NAM Mt Wa 120 150 180 210 Raw Min Pixel N wo Normalized Frequency Application Example Quantify spectral absorbance using the brightfield image Identify over compensated images Measure the level of malaria infection in RBCs 229 Chapter 4 Saturation Count Feature The Saturation Count feature reports the number of saturated pixels in an object See also Saturation Percent Features In the figure below objects with saturated pixels are lined up at the Raw Max Pixel value of 1023 and a selected image is shown with saturated pixels in red pp single cells Finkei pA x 120 w T oo T Saturation Count_Channel 3 ap o Feature Value l l l 0 300 600 900 1 2e3 Saturation Count_Channel 3 31 Raw Max Pixel_Channel 3 Saturation Percent_Channel3 4 2062 Application Example Measure the validity of the experiment setup Saturated data may not produce useful information 230 Understanding the Signal Strength Features Saturation Percent Features The Saturation Percent feature reports the percentage of saturate
221. w in IDEAS 6 0 IDEAS 6 0 offers numerous improvements for analyzing data from any ImageS tream or FlowSight instrument Please refer to the web site for the latest improve ments and updates to this manual 1 General e New user application default settings include setting graph and statistics exporting formats e Color mapping tool has been improved in the application defaults settings e Files processed prior to IDEAS version 4 0 may not load in 6 0 Data e New Feature Finder wizard for choosing a feature e Tagging tool updates the number tagged e Tagging tool defaults the population symbol to a solid diamond and a new color for each population for easier viewing in graphs Features and Masks e The Valley mask has been improved Image Gallery e The measurement tool is now available in the gallery Analysis Area e Moving panels in the analysis area is now easier Click and drag on the panel anywhere except the toolbar e Panels are aligned by their tops by lines e Cursor changed to indicated current state Reporting e Copy Save graphs and images has been improved Images may show scale bar adjust font sizes and save as tif png or bmp files at 300 DPI Graph default settings may be used for fonts graph sizes and resolution Chapter 1 Setting Up the IDEAS Application This chapter describes the hardware and software requirements for the application which includes the procedures for installing rem
222. wing and Changing the Application Defaults 0 000000002 eee ce cece ee eee 5 Chapter 2 Overview of IDEAS 0o0 0 o oo cc cece ccc ec ec eee scenes 13 Comparing the FlowSight basic Quantitative Imaging and ImageS tram Gata MNOS jee ocean ceca teas siete a E A ES 15 Understanding the Data Analysis Workflow 0 0 02 00202 16 Overview of the Data File Types 0 0 0 0 oo occ cece ec ee ec eee ees 18 Chapter 3 Getting Started with IDEAS 0 21 Guided Analysis fr 05525205 55 cco ae eee aa ieee eee ad haa heen na ys ees 23 Application Wizards t ctl Le Ask E A EAA 24 Open File Wizard cnsecc2t20352 2uvet suey a000 aaao aoaaa aeeoa ar ao raorao aran 26 Display Properties Wizard cares b ou haa tasi atlas ued hea celia aaah gil arrian 27 Begin Analysis Wizard 0 0 0 0 0c cece cece cee ec cece cececececeeeeeececececeseeeeneeeees 28 Feature Finder Wizard _ 2 00 020 ooo coon ncn 29 Apoptosis Wizard ae se niente etd donne cal canis naatde debi enn slien auin mus tte Rendon dade 8 ance 33 Cell Cycles Mitosis Wizard fect sane tense tect cu deles e i lee eeta 35 Co localiz tion Wizard s onina a ea a ae aa 37 Internalization Wizard ci c i4e ee hie oils let Ee ils titel eda dali uesciiite eal 39 Nuclear Localization Wizard s co5 22 Ssicantck dg enacrieeslnshind de ssi aback tltsaudcaucensegaenee 41 Shape Cha ge Wizard oesseveniiidieiiii sa e tu ele tenet E EEEE ea oe ats 43 SpOt
223. wo spots See also Spot Area Min Feature Spot Distance Min Feature and Spot Count Feature The following diagram illustrates these features e Spot Area Min is the Area of spot 1 Spot Distance Min is distance 2 in microns Spot Intensity Max is the Raw Mean Pixel value of spot 2 Spot Intensity Min is the Raw Mean Pixel value of spot 3 Application Example In FISH Spot Counting these features are used to identify ambiguous spots that are located too close together too dim to bright or too small to count and can be eliminated from the analysis 232 Understanding the Signal Strength Features Uncompensated Intensity The Uncompensated Intensity feature is the sum of the background subtracted pixel values within the masked area of the image with no compensation applied This is the Intensity of the uncompensated image This feature is calculated in INSPIRE during acquisition 233 Chapter 4 Understanding the Comparison Features The Comparison features describe the difference of intensity measurements between masks or pixels in different images or the same image with different masks These include Bright Detail Similarity R3 Intensity Concentration Ratio Internalization and Similarity agge Understanding the Comparison Features Bright Detail Similarity R3 Feature The Bright Detail Similarity R3 feature is designed to specifically to compare the small bright image detail of two imag
224. xample of Creating a Mask Here is an example of creating a mask of the cytoplasm In this example cells were stained with a green intracellular marker in Channel 2 and a red nuclear dye in Channel 11 You can generate a cytoplasm specific mask by first refining the intracellular and nuclear masks and then removing the nuclear mask pixels from the intracellular mask 1 2 3 Greyscale Images Observe the default masks in the Image Gallery Since the default masks are designed to capture all the light in an image they tend to include light that exists beyond the perceived boundaries of the images In this case both the intra cellular and nuclear masks need to be refined Start by creating morphology masks for both channel images because the Morphology mask is designed to conform to the shape of the image Note that the Object mask function may also be used in place of the Morphology mask function Select Analysis gt Masks Click New 92 Overview of the Mask Manager 4 Click on the Function toolbar button to adjust the mask that will define the whole cell The Define Mask Function window appears Function Select Morphology in the Function list Select a starting Mask Select Channel 2 intracellular marker on the left side of the window Click OK Click Set Default Name or enter a new mask name 10 Click OK to add this mask to the list 11 To make the Morphology Nuclear mask repeat steps 3 10 usin
225. y existing population Step 3 Gate single cells A scatter plot of Area versus Aspect Ratio has been added to the analysis area Sin gle cells will have an intermediate Area value and a high Aspect Ratio Click on the dots to view the image associated with that dot Note that the image is surrounded by alight green line and the image next to it in the image gallery is not it s neighbor in the plot The images are presented in the order of acquistion Step 4 Optional Answer Yes if you want to define subpopulations in your experiment Select subpopulation marker s Choose one or two channels you wish to use to identify populations based on Inten sity Click Next to add the scatter plot or histogram to the analysis area Click Skip if you do not wish to use this step Next Step Gate subpopulations step number sequence is dependent on the number of times the subpopulation marker step is taken Draw regions in the scatter plot or histogram to identify as many populations as you want This step will be repeated until you choose Skip 39 Chapter 3 Next Step Optional Select additional subpopulation marker s OR Gate internalization positives A scatter plot of Max Pixel versus Intensity for the internalizing probe is added to the analysis area Draw a region to include the positive cells Next Step Gate internalization events A histogram of the Internalization feature for the positive cells is added to the anal ysis
226. y organization and an overview of the work flow Understanding the Data Analysis Workflow Overview of compensation analysis tools and file structure The Amnis cell analysis systems possess unique capabilities that neither flow cytometry nor microscopy alone can deliver The IDEAS application provides an image gallery to view every cell in the data file along with linked graphical data for confident gating and image confirmation The application contains powerful algo rithms that facilitate and quantify the image analysis of ImageStream and FlowSight QI data Examples include the analysis of molecule co localization and trans location the analysis of cell to cell contact regions and signaling interactions the detection of rare molecules and cells and a comprehensive classification of large numbers of cells The IDEAS application acquires data from INSPIRE com pensates the images and allows the user to evaluate images with data analysis tools For more information about the QI and non QI FlowSight data see Comparing the FlowSight basic Quantitative Imaging and ImageStream data files 14 Comparing the FlowSight basic Quantitative Imaging and ImageStream data files Comparing the FlowSight basic Quantitative Imaging and ImageStream data files There are three types of instruments that collect data for Image Analysis in IDEAS The FlowSight without Quantitative Imaging QI The FlowSight with the QI upgrade and the ImageStream
227. ymmetry 2_Ch Application Example Classify different white blood cells based on the morphology of the nuclear image 204 Understanding the Texture Features Understanding the Texture Features The Texture features determine local intensity variations in images and include Bright Detail Intensity R3 and Bright Detail Intensity R7 Contrast Gradient Max Gradient RMS H Texture H Contrast H Correlation H Energy H Entropy H Homogeneity and H Variance Modulation Spot Count and Std Dev Contrast Gradient Max and Gradient RMS are generally used to determine best focus 205 Chapter 4 Bright Detail Intensity R3 and Bright detail Intensity R7 Fea tures The Bright Detail Intensity R3 and Bright Detail Intensity R7 features compute the intensity of localized bright spots within the masked area in the image Bright Detail Intensity R3 computes the intensity of bright spots that are 3 pixels in radius or less while Bright Detail Intensity R7 computes the intensity of bright spots in the image that are 7 pixels in radius or less In each case the local background around the spots is removed before the intensity computation The figure below shows the process of obtaining the localized bright spots in the image Original Image Detail Eroded Image Bright Detail Image The graph below illustrates the use of the Bright Detail Intensity R3 feature on a nuclear image to separate apoptotic cells from non
228. ze Location Signal Strength Signal Strength Signal Strength Signal Strength Signal Strength Signal Strength Signal Strength hape Comparison Comparison ize exture ocation Signal Strength exture exture ize ize ystem ocation ize Comparison Chapter 4 Table of Base Features by Category Table 1 List of Features by category Feature name Size based Features are in microns Area Feature M12 The size of the mask in square microns MC Diameter Feature E the diameter of the mask based on Area No Yes orm 1 Height Feature Based on a bounding rectangle the Width is the smaller Yes Yes M01 M12 side and the Height is the longer side of the rectangle Length Feature Ficasuvetts ingest parto themask Major Axis and Minor Axis Features Describes the widest part of the mask and the narrowest No Yes M01 M12 part of the mask respectively Major Axis Intensity and Minor Axis Intensity Features Based on a bounding ellipse the Minor Axis is the nar No Yes row part and the Major Axis is the widest part Minor Axis Major Axis and Minor Axis Features Major Axis and Minor Axis Features ma M12 Perimeter Feature EE circumference of the mask eee _ Spot Area Min Feature The Area of the smallest spot in the mask See also Spot Distance Min Feature Spot Intensity Min and Spot N Intensity Max Features and Spot Count Feature Thickness Max Feature a Thickness Min Feature D ain
229. ze of selected graphs to small medium or large Creating Graphs You can add two types of graphs to the Analysis Area e Histogram Graphs a single feature e Scatter Plot Graphs two features 95 Chapter 3 Note that building blocks are available that will help you to create graphs for finding single focused fluorescent positive events or a size versus scatter plot See Build ing Blocks To create a graph without using a building block 1 10 11 Click the New Histogram or New Scatter Plot toolbar button N lel The New Histogram or New Scatterplot window appears respectively Use the control key to select multiple populations S J SpeedBeads_100R2_500AI_R2s3percent_1 cif Title and Axes Title Al X As Feature Choose X Axis Feature X Adis Label 7 Nommalize Y Axis Frequency Bin count default Select the one or more populations to graph by clicking them To select more than one population use the Ctrl key The title defaults to the selected population You can edit the title In the X Axis Feature drop down menu select the feature that you want to graph on the X Axis If you want to change the label for the X axis edit the text in the X Axis Label field The label defaults to the name of the selected feature If you are creating a scatter plot select a feature and a label for the Y Axis If you are creating a histogram you can choose to normalize the Y axis fr

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