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IDEAS User Manual 5.0

1. Single focused cells QIQiie a 1e5 Se4 nm a fe Intensity _SSC a Modulation_BF High Modulation z Low Modulation Low Modulation Application Example Quantify image quality and characterize contrast and texture in cells 155 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 and Spot 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 YOYOI Single Parasite Two Parasites Three Parasites Application Examples Counting parasites Counting phagocytosed particles FISH spot counting Counting punctate spots in images Std Dev Feature The Std Dev feature describes the overall distribution of pixel intensities 156 Chapter 5 The Std Dev is the standard deviation of the pixe
2. Single Cols All 7 Select AA subpopulation 8 Gate additional subpopulation s 9 Gate apoptotic events Click on the dots in the scatter plot to see the comesponding image in the age gallery Tip2 f you wish to change the plot properties right click on the plot and select Graph Properties aa 3 S O S n 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 15 Getting Started with the IDEAS Application 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 zi 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
3. D O m gt x eb Q O k gt lt 2 Q9 ped r a9 Major Axis Intensity and Minor Axis Intensity Features o x lt O O r O C F a O O O ed r 4 Max Pixel Feature Q9 red D x lt D TI Q9 eb ma D Median Pixel Feature 5 U x lt D TI O Q p 4 Minor Axis see Major Axis Intensity and Minor Axis Intensity Fea tures ep N D Modulation Feature Perimeter Feature Spot Intensity Min and Spot Intensity Max Features Raw Intensity Feature Strength l Signal Raw Max Pixel Feature Signal Raw Mean Pixel Feature a Signal Raw Min Pixel Feature Raw Median Pixel Feature oe Strength P Signal Saturation Percent Features Strength Shape Ratio Feature Similarity Feature Similarity Feature Spot Area Min Feature ize 120 Chapter 5 Texture Location Signal Strength A o D lO O gjo Ale 015 e T T OR Ro 2 c n 9 a r D OP go O r 5 r gt ps lt gt ped m Q U go O r 5 r gt pi lt 0 x lt a 9 e r J g9 lt TI o fab r p 7 lt 3 3 D r N w iN nm D red r D Pp O a gt Q x lt a O ed
4. Obtain estimate of the mean camera background level Compute background subtracted pixel values in other feature com putations Bked 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 Intensity Feature The Intensity feature is the sum of the background subtracted pixel values within the masked area of the image 158 Chapter 5 Brightfield Scatter CD45 FITC CD14 PE DRAQS 2 oO S E E g Intenstty_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 Max Pixel Feature The Max Pixel feature is the largest value of the background subtracted pixels con tained in the 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 159 single fo a ip m x Intensity_M4_CD 71 T a A gt n mn Tit ial i er Ta ia AT Se a a O
5. _ MO1 GM MC The size of the mask in square microns ARA M01 M12 The ratio of the Minor Axis divided by the Major Axis Bkgd Mean Ch01 Ch12 The average intensity of the camera background Bked StdDev Ch01 Ch12 The standard deviation of the back ground intensities An incremental count of objects Number eaten M01 Ch01 E changes a gp hR radien M12 Ch12 e image to measure the focus qua ity of an image MC ChO1 MC The sum of the pixel intensities in the Intensity ad Ch12 mask background subtracted Minor Axis MO 1 M12 e the narrowest part of the Object Number none The sequence of objects The central tendency of the pixels none along the X Axis and Y axis respec tively Raw Max Pixel teers The largest pixel value Ch12 Raw Min Pixel oe ead oo The lowest pixel value The sum of the pixel intensities in the Uncompensated MC Ch0O1 MC P E mask background subtracted no com Intensity Ch12 pensation applied Understanding the Size Features Size features are in microns and include Area Diameter Length Major Axis Minor Asix Major Axis Intensity Minor Axis Intensity Perimeter Thickness Max and Min Spot Area Min Width and Height 126 Chapter 5 Area Feature The number of microns squared in a mask is equal to the Area In the following fig ure a 1 By MIRAE we viet tie area Is included ip the mask The number of pixels is converted to ym Note that 1 pixel 0 25 um As
6. amp A gt File Guided Analysis Analysis Compensation Tools Options Reports Windows Help o X to A a aAa RLM EBA tTbob ie MBAR AAR Population 2n single T cell amp double amp Focus amp conjugates gt View new X Q Q rey o Q q in r a Q Q iy a gt Drag5 ADAP ActivT Normalized Frequency o bo o 3 0 100 200 300 400 500 600 700 0 20 40 60 8 1e3 0 163 1e4 Area_BF Gradient RMS_BF Intensity_Actin Population Count XGated All 10723 100 A ADAP Actin T cell conjugates 1971 18 4 4 o o D pS j gt O c o E o w o a lt h Intensity_MC_Ch03 Intensity_MC_Ch03 Intensity _MC_Ch03 Intensity_MC Population Mean Std Dev NaNs Mean a Sx Al 1 542e 004 1 555e 004 1 381e 004 a0 conjugates 2 284e 004 1 294e 004 2 345e 004 2 253e 004 1 302e 004 0 2 311e 004 APC T cell annotations can be entered in a text box Current Object 127 a a 4 25017 08 26681 98 30335 10125 29 45195 39 131974 35 69 423 71 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 on a graph You can view the statistics for populations or objects in tables placed in the analysis area Graphs show data plotted with one or two feature values
7. 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 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 _ 30 Chapter 4 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 a light green line and the image next to it in the
8. 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 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 28 Chapter 4 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 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 plo
9. 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 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 area Draw a region to include the cells with high internalization The example below shows the internalization of labeled CpG red 295 x Getting Started with the IDEAS Application T High Internalization 3 For a more thorough explanation of the Internalization feature see Internalization Feature Internalization 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 Nuclear Localization Wizard This wizard will create an analysis te
10. C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 90_30_13 rif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 0ng_2_9 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 15_1_8 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 301 6_ 13 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 45_ 11 _18 cif i C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 60_ 16 23 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 75 PTA cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_26_9 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 15_3_10 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 30_8_15 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 45_13_20 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 60_ 18_ 1 cf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 75 23 6 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 90_ ant 11 cif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 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
11. Maximum Save Corrections to Raw Image File OK e 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 4 Getting Started with the IDEAS Application application 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 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 O analyzed cif and daf files 0 0ng_2_9 ciF 0 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 0 1ng 75_21_4 cif 0 1ng 90_26_9 cif 10ng 15_3_10 cif 10ng 30_8_15 cif 10ng 45_13_20 cif 10n
12. 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 20 Chapter 4 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 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 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
13. ari E m Te ia 5 3 Cc a F r Li 2 Ta LaF MW mT Jeg 4e4 e4 geg Teh 1 2e5 Intensity MC Ch05 To move or resize a region on a graph Click the Move Resize Region toolbar button on the graph panel toolbar a gt 2 Click the region that you would like to move or resize 69 Getting Started with the IDEAS Application 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 1 gt X 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 kl Click and drag to define a rectangular region for rescaling The Zoom Out Scaling toolbar button appears in the graph panel toolbar next to the Scaling toolbar button s Click the Zoom Out Scaling toolbar button to automatically scale the graph The Zoom Out Scaling toolbar button is removed f
14. 5 ao O D a a o N o o 400 600 800 1e3 Max Pixel M4_CD71 then Intensity 0C 30 000 Application Examples Used to estimate the true peak fluorescence activity Is preferred over the Raw Max Pixel for this application Max Pixel to Mean Pixel ratio identifies bright punctate staining vs uni form staining 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 160 Chapter 5 Median Pixel Intensity 230 000 230 000 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 internalization of surface bound antibodies 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 161 FITC Mean Pixel Median Pixel Intensity 230 000 230 000 Application Example Estimate th
15. 6l 47 39 40 12 51 193 Index mask 2 mask color 5 population color 5 population symbol 5 statistics for a graph 5 Diameter 127 Directories changing default 4 Display making composites 56 setting properties 56 setting properties using a wizard 16 views in the Image Gallery 56 E EDF kernels 47 Example data files 4 Exporting data 111 features 111 pixel intensity values 112 F Feature Manager overview 86 tasks 9 tools 87 194 Chapter Appendix A Features angle 134 angle intensity 134 Area 127 aspect ratio 142 aspect ratio intensity 143 Bkdg mean 158 Bked std dev 158 bright detail intensity 150 camera line number 172 camera timer 173 categories 118 centroid delta x and y 136 centroid delta xy 137 centroid x and y 135 circularity 144 compactness 145 contrast 151 create combined 90 create multiple 89 create new 88 89 delete 91 diameter 127 elongatedness 146 find a feature 92 flow speed 173 gradient max 152 195 gradient RMS height intensity internalization lobe count major and minor axis intensity major axis and minor axis max contour position min pixel modulation object number object per mL object per sec overview perimeter raw intensity raw max pixel raw mean pixel raw median pixel raw min pixel saturation count saturation percent shape ratio similarity similarity texture R3 size spot area min 196 Index 153 128 133 158 17
16. 8 Compensated Image File cif 00 2020 c cee cece cece cee cee cee ence eens 8 Data Analysis File GAL cis siscce cee nsan hee arene Sas cleo eoelie ecaeeetin os us dues eaten 8 US 0 01 0 F a 11 nn ne a nL aS See 9 Compensation Matrix File ctm 0000 0 200 c cece cece cece eee ccccceceeeeeees 9 Review of Data File Ty p68 sisi secriicnsaccnecccnsdacauanossednsustebneetensohanageesesteacsiaks 10 Getting Started with the IDEAS Application 0000 0000 000 0 cee 12 General Outline of data analysis 200 0000 00 c cece cece e cece cece cece eee ceeeeeeeeees 12 Guided Analysis aiaeei ne ee ee ee eee oe 12 Applicaton WizZardS crs sc2n 22 cciasiica daca asseesenatelisiieidwdeadernsccanedacticie OERE 14 Open FIE WEZI ee oe facet a Bienes pacjeet inten asec ers plete te eat satiate 15 Display Properties Wi1Zat 2 cod neste se i ceceins oc hee ace cue ade Satie ca icetcteaueekaesses 16 Begin Analysis Wizard ioc 5228 2 ccctscutesssedaheccussencnewusedeechatdestesataetansiaadsases 17 Apoptosis WAZA seeps Series ieas ETE ORE E A EEEE ISEA 18 Cell Cycle Mitosis WZ 0 ste desig sxe testy ce oer naceusinadannnieadaduccaawiecs 20 Co localization Wizard 2 00 220 ccc cece cece cece cece cece cece ee eeeceeeeceeeeeeeees 22 Internalization Wizard 000000000000000000000000000 0000000000000001 001000 000011111 24 Nuclear Localization Wizard 0 2 00 200 2 ccc e ccc e cece cece eee e ee
17. A N EA eee 118 Locau eeren er EE A E E EEES 118 SIAT E e E EE A EA EAEE AAA AA 118 EO UTO a E E E E E E E 118 SOM AIS Olas eeir a seat 118 BS lS NM EE T tare cee eae eee acts E A E S tances 118 Understanding the IDEAS Features and Masks 00 000 220 22002222 20 118 Table of Base Features Alphabetical 0 000000 200 ccc eee eee ec eee cece eee 119 Table of Base Features by Category 0 0 220 c cece cece e cece cece eee eee 122 Table of Basic Features available without QL 02 22 0 0 2222 c eee eee ee 126 Understanding the Size Features 20 0200 000 200 cece cece cece c cece cece ceeceeeeeeees 126 PNT gs CAN csc oie ed econ ae pease laden an oeamen ane EE ene 127 Diameter 1 2 1 6 cee nn AE RS men 127 Heith Sl ak cre 100 2 lt 9 ae ne a tn a ee eee eee ee 128 PS a eS css e sysctl be assis ee acre 129 Major Axis and Minor Axis Features 0 200 2 200 2 c cece cece cece cece ceeeceeees 130 Major Axis Intensity and Minor Axis Intensity Features 0 22 22 130 Perimeter Fedre enea eas heres 2 aaa dnd Bice Sete a EO 131 Spot Area Min Feature 00 0 0 2000 c ccc cece cece ccc cece cee ceeeeeeeceeeeeeeees 132 Thickness Max Feature 2c2csielowsdecicccnseneceateeiededcapacemeneceauedseacacanseeanane 132 e Chapter 1 Thickness Min Feature 2 20 ooo coco L L L bbb LLL LL LoLa 133 Widi Fear tates eens ete Sere eee easa ns E E
18. AM H Batch 4 5 2011 11 28 23 AM H Batch 3 24 2011 11 47 57 AM H E Batch 3 23 2011 2 29 21 PM Batch 3 23 2011 2 00 37 PM Batch 3 2 2011 8 02 21 AM Batch 3 1 2011 1 17 04 PM fH Batch 2 9 2011 11 49 07 AM 2 Click Add Batch The Define a Batch window appears _ 4g Chapter 4 Define a Batch _ pS Input Files Select oof cif or daf files to process Add Files Select a compensation matrix compensated image file or data analysis file ctm cif daf Select a template or data analysis file ast dat Batch Name Batchi 3 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 Select a template file ast or daf Leave blank to use the Default template Set the output files parameters N o1 A 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 8 Click OK The Define a Batch window closes The batch appears in the Batches window _ 49 G
19. 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 0 0ng_2_9 Default daf 1000mg 90_30_13 daf EG 0 0ng_2_9 daf My Recent 0 1ng 15_1_8 daf Documents 0 1mg 30_6_13 daf O 1ng 45_11_18 daf O 1ng 60_16_23 daf Desktop O 1ng 90_26_9 daf 10ng 15_3_10 dar 10ng 30_8_15 daf 0ng 45_13_20 daf 10ng 60_18_1 daf 10ng 75_23_6 daf 0ng 90_28_11 dar My Documents as 1000ng 15 5 12 daf hy Computer 1000ng 30_10_17 daf 1000ng 45_15_ 22 dar e 1000ng 60 20 3 dat a 1000ng 75_25_6 dar My Network Places File name Files of type Data
20. 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 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 with 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 THP1 cells stimulated with 1 ug LPS for 90 minutes and stained with DRAQ85 red and NFkB green to measure the nuclear localization of the NFkB ogi Getting Started with the IDEAS Application Untreated Stimulated mR Loe ee Oe L hg Fi i ig od Se For a more thorough explanation of the Similarity feature see
21. 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 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 itis 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 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 _97 Getting Started with the IDEAS Application The Population Manager provides a central place for maintaining the display pro
22. 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 two 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 e Spot Distance Min is distance 2 in microns e Spot Intensity Max is the Raw Mean Pixel value of spot 2 e Spot Intensity Min is the Raw Mean Pixel value of spot 3 Application Example 167 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 Bright Detail Similarity R3 Feature The Bright Detail Similarity R3 feature is designed to specifically to compare the sm
23. Populations The ast also contains settings for e Image viewing e Image names e 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 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 color control files after acquisition or during acquisition on a FlowSight See the Flow Sight user manual for more information Review of Data File Types Table 1 Review of Data File Types Name Contains i
24. RTX HBA3 ADC Image AF 488 RTX Endosomes image Application Examples Quantify the degree of colocalization between two probes Track internalization and intracellular trafficking of antibody drug con jugates to either the endosomes or the lysosomes Colocalization of Rituxan and compliment C3b 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 169 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 internal compartment and the second is the membrane region 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 fe
25. Select Light Mode Color Mapping E W sue Update All Populations Reset To Standard Cancel 2 Inthe Select Dark Mode Color drop down menu select the color that you want to map 3 To choose a different color click the Select Light Mode Color Mapping color Square and click a new color on the color palette Click Update All Populations 5 If you want to return the settings to the IDEAS defaults click Reset to Standard 6 Click OK to save the changes or Cancel to exit 114 Chapter 4 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 Print Graph t Select options for printing Graph E Legend E Statistics E Cursor E Show Sample Name in Title Size scaling factor Select the checkboxes Graph Statistics Legend Cursor Show Sample Name in Title to include the elements in the report If necessary adjust the size scaling factor Recomended setting is 100 4 Click OK to print the graph 115 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 CC
26. 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 In the 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 154 Chapter 5 Taralick R M K Shanmugan and I Dinstein Textural Features for Image Clas sification EEE Transactions on Systems Man and Cybernetics Vol SMC 3 1973 pp 610 621 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
27. TO LOT I LAG Rte occ crs taciadt R E men aes peeks eel sded oa saacer EENE 180 POCNI E enee eee eee enn ee ade a cae ree nee ee ry Se ee ee ee 181 System MaS oars hertuind aa E EE IR a a AEE OEI DEE EA 182 Threshold MaSK ereere ets r E ET REEE 183 N MEYENII E eene ra TE NR EE ENE EE 184 Table of Contents Troubleshooting 0 0 00 c cece cece ccc ccc cccccccceccccecececeececceeeceeeeeceeees 186 Application Hanging 0 0 0 0 e cece cece cece e eee ee eee eeeceeeceeeesenees 186 COMP CSO 5 eis cheers cranecearnamacraiebtesnediaeonananantanstadasuedadasasusteneeans 186 ie Ui a cas tines cetera teh ae ete eee ae ee eee ines 187 Deleting a Population and Region 0 2 00 222 cece cece eee cece cece cece eeeeeeee 188 Object Number set to Zero 2 20 2 e cece cece ceeeeceeeeeeees 188 Buttons or options in windows are not appearing 20 222 22 e eee eee eee eee 188 GOSS 6 Se ce ee ce en ae ne eee A E 189 DCN aa cy Sesh ead rrscesinesiclecn ede gud atone eames abba neelidcdmeteseeceser 191 XV Welcome to IDEAS 5 0 Welcome to the IDEAS version 5 application documentation for ImageStream and FlowSight data analysis IDEAS 5 0 or later versions are required to open FlowSight data Many new improvements have been added to the application How to use this manual This manual provides instruction for using the Amnis IDEAS application to analyze data files from the Amnis ImageStream
28. The Object Number feature denotes the serial number of a cell in a file Application Example Reference an object in a file Objects ml Feature The Objects per mL feature returns the object concentration with respect to local vol ume Application Example Monitor the object flow during the run Note Use the statistic Con centration to obtain objects ml of a population 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 Time Feature The Time feature returns the camera timer values that are in ticks converted to secs with a formula 173 Application Example Obtain the time taken to collect a sample 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 a mask 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 ID
29. 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 190 Chapter Appendix A Index A Acquisition information 47 Advanced Analysis 34 Analysis Area adding an image panel 12 adding text 78 overview 61 printing 104 113 tools 62 UI 7 Apoptosis wizard 18 Application defaults 4 Area 127 ast about 9 B Batch processing 47 Brightfield information 47 Building Blocks 32 Fluorescence Positives one color 32 Fluorescence Positives two color 32 Focus 32 Single Cell 32 191 Index Single Cell Default 32 Size SSC 32 Tool 63 C Camera settings information 47 Cell Classifiers 47 Cell Cycle using a wizard 20 Channels collected 47 cif merging 44 opening 37 saving 41 cif about 8 Co localization using a wizard 22 Color Show in Image Gallery 54 Compare FlowSight FlowSight QI ImageStream 2 Compensation definition 3 editing the matrix 43 overview 4 view matrix 47 Compensation matrix file about 9 192 Chapter Appendix A Composites Copying images Corrections information ctm about daf about opening saving Data analysis steps Data analysis tools about Data files ast cif ctm daf new from populations opening rif type Defaults application compensation matrix directories 59
30. analysis area m Changes the background of the graphs to black or white Graph Bkgd Tool HH gH gH Tools Graph sizing Changes the size 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 Note that building blocks are available that will help you to create graphs for finding single focused or fluorescent positive events See Building Blocks To create a graph 1 Click the New Histogram or New Scatter Plot toolbar button tia Ee The New Histogram or New Scatterplot window appears respectively _ 63 Getting Started with the IDEAS Application i New Histogram Use the control key to select multiple populations Title and Axes Title X Axis Feature x Axis Labet Y Axis Feature Ch ose Y Axis Feature _ Y Axis Label Frequency Display Properties 2 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 3 Inthe X Axis Feature drop down menu select the feature that you want to graph on the X Axis 4 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 5 If you are creating a scatter plot select a feature and a la
31. analysis files daf k IDEAS files rif cif dat Raw image files rif Compensated image files ci Data analysis files dar _ 39 Getting Started with the IDEAS Application 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 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 if 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 save changes from the File
32. 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 52 Chapter 4 Using 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 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 You can build custom viewing modes as shown in this example For more infor mation see Setting the Image Gallery Properties Z IDEAS OVA_DFSvO4_A 3 0 5 0 File Guided Analy
33. 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 101 Getting Started with the IDEAS Application 1 Select Analysis gt Regions or right click a graph and select Regions The Region Manager window appears Click on the region in the list you want to view ae Region Manager Regions Name Rl Dark Mode Color Use for statistics only Shape Rectangle Vertices X Coordinate Y Coordinate k 99 54751131221 1 041951219512 407 2398190045 0 679024390243 To edit a region 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 CON OOF WD 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 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
34. 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 Cotorcalization 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 aoe Chapter 4 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 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 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 ident
35. in the list 16 Click the AND button on the toolbar i 85 Getting Started with the IDEAS Application 17 Click the NOT button on the toolbar a 18 Double click Morphology Nuclear mask in the list 19 Enter anew 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 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 Overview of the Feature Manager Viewing feature definitions Creating new features with the Feature Manager Ranking features for best 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 feature values or view them as statistics for any population For descrip tions of all the base features see Overview of the IDEAS Features and Masks When the IDEAS application opens a cif or rif file the application calculates the values of fea
36. items Select the Export to option that you want Note that data exported to the Clip board can be pasted directly into a spreadsheet program 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 Click OK 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 A W N o On the Tools menu click Export Image Pixel Values The Export Image Pixel Values window appears cose Export Image Pixel Values Sel Select object toa export Export to z f Clipboard File OF 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 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 On the Tools menu click Export tif Images The Create TIFs From Population window appears 112 Chapter 4 a Create TIFs From Population Select population Select Channels T
37. light 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 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 Compensation Matrix Select a compensation matrix Color Compenszations021810 4101 3L 488 PE PETR PECuS DAFI PO 64 APCCy ctm ChOl Chaz CHOE Cho ChO9 Chia Chit 0 051 0 026 0 002 1 0 036 0 008 0 212 0 015 0 006 0 078 0 012 0 005 0 018 0 005 0 011 0 055 0 005 0 004 0 009 1 0 051 0 045 0 359 0 05 0 008 0 061 0 045 0 004 0 026 0 086 0 002 0 01 1 0 004 0 086 0 267 a o yoloaol oaol oa o es ol oy oy yo a So alo ao ao oa oa ayo o ae 0 0 0 0 0 0 0 0 0 0 0 oo ale aol oa eo aloo Preview a file with this matris applied Select an existing rif file Ovenwnite preview files o o F 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 th
38. 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 Mask Range Mask Application Examples Used with he 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 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 182 Chapter 5 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 Threshold Mask The Threshold mask is used to exclude pixels based on a percentage of the range of intensity values as defined by the starting mask The user chooses the starting mask when creating the Threshold mask See also Intens
39. pixel value within the mask background subtracted M12_Ch12 Min Pixel Feature No No The smallest pixel value within the mask background subtracted Raw Intensity Feature No No The sum of the pixel intensities within the mask Raw Max Pixel Feature Ves Ves MC_Ch01 The largest pixel intensity MC_Ch12 Raw Mean Pixel Feature The average pixel intensity Raw does not have background sub No No tracted Raw Median Pixel Feature eae No No The median pixel intensity Raw Min Pixel Feature Vee Vee MC_Ch01 The lowest pixel value within the mask MC_Ch12 Saturation Count Feature Vee Ves M01_Ch01 The number of pixels in the mask that are saturated M12_Ch12 i Chapter 5 The Percentage of pixels in the mask that are saturated Spot Intensity Min and Spot Intensity Max Features The raw intensity not background subtracted of the dimmest com ponent spot See also Spot Count Feature Spot Distance Min Feature and Spot Area Min Feature Difference of intensity measurements between masks or pix els Bright Detail Similarity R3 Feature No No Measures the correlation of the bright details between image pairs Intensity Concentration Ratio Feature Given two masks the ratio of the intensity in one mask to the total No No intensity in both masks 7 i XCorr Feature The XCorr is a measure of the degree to which two images frequen cies are
40. prior to 4 0 may not be able to be loaded in 5 0 Process FlowSight QI and non QI files b Read and analyze FCS files rm 0 OO New template for finding features Faster processing and loading of data files Ability to use a FlowSight nf file when selecting a compensation matrix Data loading and batching have been optimized to perform instrument cor rection and feature calculation at the same time 3 Non Quantitative Imaging QI a The FlowSight saves the compensation matrix and analysis template used dur ing acquisition as the default matrix and template in the rif file Chapter 1 b Combined feature calculation 1s enabled No new feature or mask calculations can be done without the QI upgrade c The Start Analysis wizard steps you through the analysis of finding the single focused positive cells Morphologically based wizards are not available with out the QI upgrade d Compensation matrices may be applied to compensate Intensity features and Images All other features are not recomputed and are based on uncom pensated imagery e Templates used for loading non QI data files will load only the populations statistics and graphs for the features available and will not load those that require the QI upgrade f Non QI files cannot be merged g Creating data files from populations is not allowed h Instrument segmentation masks are used and are not recomputed in IDEAS 4 Features and Masks a The Gr
41. process of 1000 Advanced 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 e f 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 Name the output files with a new name if necessary 36 Chapter 4 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 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 wi
42. rif 121906 C16 72 06 DRAQS _ noBF4 rif AJ 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 0 1ng 45_11_18 rif 0 1ng 60_16_23 rif 0 1ng 75_21_4 rif 0 1ng 90_26_9 rif 10ng 15_3_10 rif 10ng 30_8_15 rif 10ng 45_13_20 rif 10ng 60_18_1 rif 10ng 75_23_6 rif l 10ng 90_28_11 rif g 1000mg 15_5_12 rif EG 1000ng 30_10_17 rif My Computer 1000ng 45_15_22 rif 1000ng 60_20_3 rif 1000ng 75_25_8 rif 1000ng 90_30_13 rif My Documents File name 0 1ng15_1_8 ri Files of type Raw image files rif Data analysis files da Compensated 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 35 x Getting Started with the IDEAS Application rs Opening C Training Data Files 3 0 NFkB Translocation Dose an Aka To perform fluorescence compensation Select a compensation matis compensated image file or data analysis file ctm cif dat Or Create a compensation matris from control files Mew Maria 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 1000rg 60_20_3 cit Data analysis file dat Enter the number of objects to
43. 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 _4 Getting Started with the IDEAS Application Channel 3 Channel 4 Percent Emission 475 500 525 550 575 600 625 650 6 75 700 725 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 Un compensated Compensated le6 1e5 E E f 5 1e4 bg wT 1000 bof bt bute I ot PT Ett 1000 ot data ota deal 1000 le4 le5 1000 0 1000 le4 1e5 3 Intensity 3_Intensity Uncompensated Compensated SSC Brightfield FITC PE PE Alexa610 Draq 5 ssc Brightfield FITC PE PE Alexa610 Draq 5 Channel 1 Channel 2 Channel 3 Channel S Channel 6 Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Channel 2 Chu Channel 1 _42 Chapter 4 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
44. the customer documents in your training materials or in the knowledge base of your account on the Amnis website 102 Chapter 4 Wagar Dyes Sa ro fare of acne ee N fy ewe ge spre pee eee Ss i s mmm y aaya The following subsections describe how you can print copy or export data directly from the IDEAS application You may create reports with Images Graphs and Statistics in applications such as those in Microsoft Office If you are interested in analyzing data in other analysis applications see the last section Exporting data Reporting Images and Graphs Reporting Statistics Exporting data Feature values pixel intensities TIF files FCS files 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 optimize 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 white background you can change the colors when using th
45. the user to evaluate images with data analysis tools For more information about the QI and non QI FlowSight data see the com parison table 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 There are some differences in the available features and anal ysis that can be done The table below outlines these differences Data comparison o o FlowSight basie FlowSight Q1 ImageStream INSPIRE features Default template and acquisition anal ysis Default compensation IDEAS default fea IDEAS default fea ture set analysis __ ture set no analysis User defined masks INSPIRE mask cal Default Object Default Object Default mask culated during acquijmask computed in mask computed in Chapter 2 ns FlowSight basic FlowSight QI ImageStream iti I I Open File Display Properties and Complete set 10 Complete set 10 Begin Analysis 2 Merging files Intensity and All features and All features and Compensation Images only Images Images Create new files from populations IDEAS Requires version Requires version Can open in 4 0 or 5 0 x 5 0 x 5 0 Populations tab Sample Information populations and counts Populations tab pop Detection tab cell ulations and counts _ classifiers U
46. 0 147 130 130 139 162 155 173 173 173 116 131 163 163 164 165 165 166 166 148 171 168 126 132 Chapter Appendix A spot count spot distance min spot intensity min std dev symmetry 2 table alphabetical list table by category thickness max time valley x and y viewing width and height without QI File name extensions Focus building block graphs copy and paste Graphs apply or remove region creating creating regions legend moving printing resizing regions 156 140 167 156 149 119 122 132 173 141 87 128 133 126 4 32 70 71 63 68 67 67 115 69 197 Index statistics 66 Zoom 70 Guided Analysis 12 H Hardware requirements 3 Histogram tool 62 I IDEAS getting started 12 interface J Image copy 6l individual image 60 Image Display intensity mapping 7 Image Gallery channel view 54 composites 59 overview 53 population 54 printing 104 113 properties 55 56 properties tool 54 resize 55 show hide color 55 show hide masks 54 tools 54 198 Chapter Appendix A UI 7 using 53 views 58 Image panel size change in Image Gallery 56 Individual image display properties 74 manipulating 60 measurement tool 73 pixel intensities 12 show hide mask 76 Internalization using a wizard 24 L Laser information 47 Line Region Tool 63 M Mask Manager tools 83 Masks about 174 combining 83 creating new 80 dilate 175 edit g4 erode 175 199 exa
47. 4 0 rifs 0 1ng 15_1_8 1f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 30_6_13 9f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 45_11_18 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 60_16_23 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 75_ 21 4 Jif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_26_9 rif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 15_3_10 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 30_ E 15 rif i C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 45_13_20 rif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 60_18_1 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 75_23_6 nif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 90_28_11 nif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 15_5_12 rif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 30_10_17 rif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 45_15_22 rif C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 60_20_3 rif C Users sfriend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 75_25_8 rif
48. 79 _ix Chapter 1 Overview of the Mask Managet 0 20 2 200 2 cece cece e cece cece eeeceeeeceeeeeeee 80 Creating New Masks with the Mask Manager 20 220 22022eeeeee eee 80 Viewing and Editing a Mask 2 2 202002 ee cece ee ee 83 Example of Creating a Mask 220 02 000 2000 c cece ccc eee cece c cece ceeeceeeeeees 84 Using the Feature Manager 2 0 0220 2 cece cece eee e cece eee ceeeceeeceeeeeeeees 86 Overview of the Feature Managet 2000 2 200 c cece cece e cece cece eeeeceeeeee 86 Viewing feature definitions 00 02 02 2 cece eee ee eee eee cece eee eee eeeeee 87 Creating New Features with the Feature Manager 20 02 22 02202 88 Ranking features by discriminating powet 22 200 2eeeceeee cece cece eee 92 2 015 a ee ee ene RE et Oe Ee earner ePIC ES 92 3 p 200 0 ome eae eve eee ee ne ee ne ee et ee ee 92 Using the Population Managert 0 0 00 200 2 cece eee e eee ee eee eee cece eee 97 Creating Tagged Populations 00 2 2002 c cece eee e eee e cece cece eee ceeeceeeeeees 100 Creating a tagged population from a file of object numbers 101 Using the Region Managet 20 0 2 00 c cece cece eee c cece cece cceeeceeeeeeeeees 101 Creating Reports and Exporting Data 0 000 00000000220 cece cece eee cece eee eee eee 102 Reporting Images and
49. 88 Chapter 4 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 A O N A single feature uses the definitions of a base feature along with a mask and or an image Click Add Multiple Features in the Feature Manager Sort the feature list alphabetically or categorically Select multiple base features and masks Select one image or check the box to create for all channels using default masks and images ao Add Features Select base features Select feature inputs Texture A C Create for all channels using default masks and images Bright Detail Intensity R3 Bright Detail Intensity AF Contrast MOT Gradient Max W02 Gradient RMS H Contrast Mean H Contrast Std H Correlation Mean H Correlation Std H Energy Mean H Energy Std H Entropy Mean H Entropy Std H Homogeneity Mean H Homogeneity Std Select image SsC lt Ch2 i NFkB Sort Order BF Alphabetical Category DRAGS Select masks 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 _ 89 Getting Started with the IDEAS Application a Confirm Feature Cre
50. Actin 50 Clear Selected Select image Sort Order Alphabetical Category 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 population 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 O5S x Getting Started with the IDEAS Application this case the 2 truth populations picked in step 2 In 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 t 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
51. D 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 ImageStreamx system has a second camera option which enables collection of up to 12 images per object The FlowSight system has 12 channels collection on 1 camera detector Spectral decomposition element cells in flow autofocus velocity Y ry detector g c 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
52. Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 45_ 15 22 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 60L 20_ 3 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 75_25_8 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 90_ 30_ 13 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 0ng_2 9 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 15_ 1_8 daf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 301 6_ 13 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 45_ 11 _18 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 60_ 16 23 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 75 21 4 daf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_ 26 3 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 15_ zE 10 daf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 30 E 15 daf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 45_1 3_20 daf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 60_18_1 daf C Users sfiend Desktop lS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 75 23 6 daf C Users sfriend Desktop
53. E INSTALLATION PROC ESS WILL STOP Disclaimers The screen shots presented in this manual were created using the Microsoft 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 Seattle WA Phone 206 374 7000 Toll free 800 730 7147 Www amnis com _vi Table of Contents Table of Contents Patents and Trademarks 2 2 2 0 00 0 2 e cece ec eceeeeeeeee ii End User License Agreement 20 22 002 2 c ccc cece cece ec cec ccc cecceceeeeeeees lil B Sie FE Gx gc Renee pee eee OO ee eR ne ORE eT ERE ANA Sone tr go Rta a RES vi Technical Assistance 2 22 0 0 0 0 c occ e cece cee cee cee cece cece cece cece cone ceeeceeeseeneees vi Table of Contents 2 02 22 eee eect ees vii Welcome to IDEAS 5 0 2 eee 1 Howto USC this mantab a5 ets gor ea ar een cll tat Sd de elena orale ected easiness l What s New in IDEAS 5 0 00 0000 cee cee cece eee cee eee cence eeeeeeeeeees l Setting Up the IDEAS Application 00 0 0 00 00 ce cece eeeeeeeeees 2 Hardware and Software Requirements 2 2 2 0 220 2220 ccc cece ceecccececceeceeeeee 3 Hardware R CQUICIMCINS sei ooo ors ot elt Sates deeb aU chs Soe Sade coco shoots 3 Software Requirements 22 0 2 00 2220 ccc cece ce cec ccc ccccececceeeecee
54. EAS 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 treamX 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 anda NMC mask that is everything outside of MC The following illustration shows two channel masks ee Chapter 5 that are combined into one mask which is shown in the right most panel 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 Obj
55. Graphs 20 2 00 2222 cece cece cece cece eeeeceeeeeeeeees 103 Prepare the Image Gallery and Analysis Area for reporting 2 103 Copy full or partial screens 00 2 2002 c ccc cece eee eee e cece eee eee eeeeeeeeeees 104 Print directly Analysis Area or Image Gallery 0000 000 2220 2eee eee eee 104 C py As ce sects oss eesti E a E es ee RATEN 104 Copy Graphs and Statistics 2 000 200 c cece cece cece cece cece e cece cece eeeeceeeeeees 105 Reporting Statisties ors 052 2 ctslatieladeicetss sntmace cee suctensuigaeiacesasseenaetseSamaaeosns tees 107 Define a Statistics Report 00 0020 c cece cece cece ccc ccc c ee ceeeeeeeeeeeeeeees 107 Generating a Statistics Report using daf Files 00 0000 020002 22 2e eee ee 110 Reporting Statistics from a Single Graph or Statistics Table 2 2 111 EXODO DAA asarrea eA E TEN 111 Table of Contents Exporting Feature Data 200 0 000 2000 c cece cece ccc ee cece cece eee ceeeceeeeeees 111 Expormng Pixel Data eent a E EAA EIE 112 Creating TIFs From Population for Export 00 2000 2 22 2 cece cece eee eee 112 Prio ae Da e tae ore steele an E EE ease teaheeten 113 Overview of the IDEAS Features and Masks 0 000 2200 222 c eee e eee ee eee 116 About APU Sige ascites ane ance Seana Ge EEEE AERAR 116 Features C atep ONES erona E a AANTEEL 118 E T S E EEE AA S EE A AN E
56. IDEAS Image Data Exploration and Analysis Software User s Manual Version 5 0 September 2011 amnis Amnis Corporation 645 Elliott Ave West Suite 100 Seattle WA Phone 206 374 7000 Toll free 800 730 7147 Chapter 1 Patents and Trademarks Amnis Corporation s 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 are registered or pending U S trademarks of Amnis Corporation All other trademarks are acknowledged af a End User License Agreement 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 wil
57. IF Settings File name prefix Bit Depth bit for display 0 16bit for analysis Pixel Data padded for display 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 A TIF file is created for every selected channel within the selected population oO oO FR ON 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 exporting of data Because the population colors might not show on a white back ground you can change the colors when using the light mode To use light background graphs in the Analysis Area e Click the graph background tool to switch between light or dark mode P4 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 e Select Reports gt Print Image Gallery 113 Getting Started with the IDEAS Application The IDEAS application prints all the images that are visible in the Image Gal lery To map the dark mode colors to light mode colors 1 Select Options gt Manage Color Schemes The Modify Reporting Color Scheme window appears a Modify Reporting Color Scheme Select Dark Mode Color
58. IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 90_28_11 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 15 5 12 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 30_1 0_17 dat C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 45_15_22 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 60_20_3 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 75_25 8 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 90_30_13 daf 2 1 2011 11 53 28 AM 2 1 2011 12 00 41 PM All files were processed successfully C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 0ng_2_9 _4 Default daf_stats b4 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 _5 Z IDEAS 5 0 OVA_DFSv94_A_3 0_5 0 daf Getting Started with the IDEAS Application 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 resizable panels o
59. LAR 133 Understanding the Location Features 000 0 200 2 cece cece cece cece c eee eeeeeees 134 IPN Mey Ut aaah a gs ae rai a beens ees ae as eea aes 134 Angle Intensity Feature 0 00000000 c cece ce cece cece cece cece ce eceeeeseeeees 134 Centoid Features esini ra a hed oun aeacteechalseenancueestaetentieessas 135 Centroid X and Centroid Y Features 0 20 2 200 2 cece eee e cece cece ee 135 Centroid X Intensity and Centroid Y Intensity Features 2 136 Delta Centroid X and Delta Centroid Y Features 2 0 00 020 22222 136 Delta Centroid XY Feature ogo siconse a simetiihiodcasinuwsedesotensheewsancencsceadenss 137 Raw Centroid X and Raw Centroid Y Features 20 20 22 222202 139 Max Contour Position Feature 2 2 2 00 20 2 0 22 cece eee eee eee cece eeee eee 139 Shift X and Shift Y Features 200000 000 2000 c ccc c ec eeeceece eee ee eec cece ceeeeeeeee 140 Spot Distance Min Feature 0 0200 2c ccc c eee e cece cece ccc ecececeeeeeeeeeeeeees 140 Valley X and Valley Y F Caress oc0cc03 cceciwsdscanconusacaeenieededdedaocesacaccaatecss 141 Understanding the Shape Features 0 2 0 00 0 200 220 c cece cece eee e cece cece cece eeeeee 142 Aspect Ratio Feature 0 200 c cece cece cece eee c cee ce eee eceeeeeceeeeceeeeecees 142 Aspect Ratio Intensity Feature 0 00 000220 c cece ce cece cece ce
60. Norm alized Frequency 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 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 beacon teraichamelsy one color for all channels two color for all channels for all channels Gradient RMS_MX_ChxX Focus for all channels Note Gradient RMS of brightfield is default Area_brightfield default Aspect Ratio_brightfield default Area_ scatter Aspect Ratio Intensity _ MX_ChX Intensity MC_ChxX for all fluorescence channels for all channels Intensity_ scatter ingle Cell Default Area_brightfield Aspect Ratio_brightfield Size SSC Area_brightfiel Single Cell To begin choose Building Blocks from the Guided Analysis Menu or click on the Building Blocks icon in the analysis area toolbar 3 Chapter 4 The Building Blocks window opens This window is used to define a graph with a specified set
61. O oF W DY Gate on positive cells for the channels you wish to use for morphological anal ysis 7 Find the feature that separates your populations this may require opening both a and control by using a wizard or the method of finding a feature based on dis criminating tagged populations 8 Refine the analysis and save the template 9 Perform batch analysis on all data files in the experiment using the compensation matrix and analysis template 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 _12 Chapter 4 IDEAS50 Brk DS File Guided Analysis Analysis Compensation Tools Options Reports Windows Help amnis Version 5 0 IDEAS 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 bi at the end of the Start Analysis routine The following wizards are available 13 Getting Started with the IDEAS Application 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 Apoptosis Creates an analysis template for identifying apoptotic events based on brghttield and
62. 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 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 O Komad Frequency Mia onor Palon nemaMark_El channel 3 Channel 3 Application Example Used in conjunction with the Internalization feature to determine the dis tribution of intensity within a cell 139 Shift X and Shift 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 Spot Distance Min Feature The Spot Distance Min feature provides the shortest distan
63. Spot Mask into individual components 179 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 gt selecting small aspect ratio object 3 object 4 selecting small size object 1 object 3 o 2 Selecting small intensity object 1 object 4 FISH IS Masking a cell with one spot using the spot and range masks Range _Area gt 2 Range_AR gt 0 4 Application Examples Use with a 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 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 to the starting mask In the example below the Morphology mask of the image was used as the
64. Starting mask for creating the skeleton 180 Chapter 5 e 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 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 181 Original Image Detail Eroded Image Bright Detail Image The image pairs below show
65. U FOR THE SOFTWARE OR IN THE EVENT THAT AMNIS HAS MADE THE SOFTWARE AVAILABLE TO YOU WITHOUT CHARGE AMNIS 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 a
66. _11 nf O Unprocessed In process Processed 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 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 _44 Chapter 4 To merge cif files 1 Onthe Tools menu click Merge cif Files The Load Multiple cif Files window appears A Load Multiple cif Files x 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 Objects Population Add Files Remove Files Compensated image file cif Select a template or data analysis file ast daf Data analysis file daf r ar cancel 2 To select 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 popula
67. addition with FlowSight QI or ImageStream data users can further define images by creating fea tures a mathematical expression that contains quantitative and positional infor mation about the image The application also contains tools that allow you to view grayscale and pseudocolor images to apply gains and thresholds and to build composite images For individual images tools are available to examine pixel intensities create line profiles of pixel intensities and compute the distribution statistics of the pixels in a region of an image Both morphological measurements and intensity information are available for calculating feature values Histograms and scatter plots display feature data graph ically and the population distribution statistics include a variety of calculations such as the mean standard deviation and coefficient of variation CV Interface of the IDEAS Application The IDEAS Application allows the opening of multiple data files within one instance of the program Each file is divided into two sections the Image Gallery and the Anal ysis Area The placement and size of these areas are adjustable Z IDEAS 5 0 OVA_DFSv94_A_3 0_5 0 daf o x File Guided Analysis Analysis Compensation Tools Options Reports Windows Help gt A 9a aAa k tolk DEALA bla g aeon A a A Population 2n single T cell amp double amp Focus amp conjugates x View new Q a i oS E QQ GS a IM o Q 9 i
68. adient RMS feature has been improved The range is now 0 100 and will require region adjustment for files analyzed prior to IDEAS 5 0 b Improvements have been made to the default segmentation mask and align ment routine 5 Image Gallery 6 User Interface a The statistics are now displayed in tables in the analysis area and the statistics area has been removed b Toolbars have been updated c Analysis is started from the Start Analysis button which goes right into the Open file wizard and other available wizards depending on file type 7 Sample Information a Added population statistics from the acquisition that reflect total counts and col lected counts in the sample 8 Analysis Area a Tables for population statistics and object feature values are now available b Selected panels can be resized cut copied or pasted Selection of panels can be done by clicking on the panel s or drawing a rectangle around the panels Control click to add or remove panels from the selection c Resize a panel by dragging the right or bottom edge or the lower right corner Or choose the size small medium or large in the toolbar d Move a panel by click dragging the title or copy and paste to new location Setting Up the IDEAS Application This chapter describes the hardware and software requirements for the application which includes the procedures for installing remov Welcome to IDEAS 5 0 ing and upgrading the app
69. all bright image detail of two images 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 Colacalized Colocalzed T 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 168 Chapter 5 Original Image DHEIRG EMELE Bright Detail Image The figure below shows the correlation analysis between an image pair H on specifically Localized Cell ADC Image Endosomes image Codocalized Cell th D o PE ant
70. allery Properties window appears with the Display Properties tab displayed Image Gallery roA x Display Properties Views Composites Images Object 0 Ch01 Name Ch02 Color O Ch02 Ch03 Minimum Pixel Intensity 15 Maximum Pixel Intensity 1484 Ch04 Ch05 255 Ch06 Ch07 200 Ch08 Ch09 Ch10 100 Ch11 l Ch12 0 T I l l Ii 0 1000 2000 3000 4100 Automatic Manual Image Display Mapping X Axis Scale Display Width Display Height Set Range to Pixel Data Full Scale Channel Width y l l Set Linear Curve Autoscale Auto Fit Auto Fit 120 32 Preview Changes in Gallery OK Cancel 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 _ 56 Chapter 4 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 displ
71. also bias the outcome during statistical ranking 035 Getting Started with the IDEAS Application The following figure shows the truth populations chosen to find a feature to dis criminate uniform versus polarized actin 2_FITC 2_FITC 2_FITC 2 FITC g 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 MO02 2_Actin 50 Feature Type Area_M01 M02 M03 MOG M07 M09 M11 J Area Area Area Area Area Q a a a o _Morphology M02 2_Actin pa _Object MO02 2_ Actin Tight Area_Threshold MO02 2_Actin 50 Aspect Ratio Intensity_Fill Threshold M02 2_Actin 50 _2 Aspect Ratio Intensity_M01_Ch01 Annans D tin litanna et LNI Sot features by ES New Delete Add Mutiple Features Add Mutiple Features Features ia b In the Add Features window select Category as the Sort Order E 94 Chapter 4 AS Add Features Select base features Select feature inputs m W Size Create for all channels using default masks and images H E Location oe 5 7 Shape Select masks E W Texture Signal Strength H E Comparison H E System Object MO2 2 Actin Tight Threshold MO2 2
72. ample Information 00 00 200 20 cc cece cece eee e eee eee eee eee sees 46 MAC PPO CCS UNS oorsee caine neelactaed arate eters a aE ENEE i 47 Overview of the Data Analysis Tools 0 00 200 e eee e cece cece ec ec cece eeeceeeees 51 Using the Image Gallery 00000000000000000000000000000000 0000000000000000 0 00000001111 53 Overview of the Image Gallery 00 00 0000 c ccc cece cece e cece cece cece eee 53 Image Gallery TOONS 5 eee ce se aticheshane nekencuieacetensiechaddedencsececudendatacamerameacns 54 Setting the Image Gallery Properties 0 0 0 000222 cece cece cece eee e eee eee eee 55 Working with Individual Images 2 0 000022 00 2 e cece eee eee eee eee eee eee 60 Overview of the Analysis Area 0 00 0200 c cece cece cece cece cece ceeceeeeeeeeeeeees 6l Analysis Area Tools 00000000000000000000000000000 0000000000000000 idee leisedaceameaae 62 Creatine Gj 2 0 t oe ee E AET 63 Creating Regions on Graph3s 0 2 2002 2c cece eee e ec ce cece cece eeeeceeeeseeeees 68 Analyzing Individual Images 20 2 200 220 cece cece cece cece cece eeecceeeeeees I2 Viewing the Object Feature Values 0 000 000 0200 e cece eee eee eee eee ee 76 Adding Text to the Analysis Area 00 00 0200 200cc cece eee e cece eee eect eee eeees 78 Population StatiSUCS co cc csc dsccancnzcrakesk uve Secscnscasuseaeeestelededactadacwsescan dete
73. an TR a cell with a mask that includes 2000 pixels is therefore equal to 500 um Brightfield GLa E elec eisai 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 Diameter Feature 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 Areg Ti Diameter 2x The images below depicts beads with a uniform diameter of 9 microns 127 In the next figure note that images with longer shapes that have the same area will have the same diameter value Focus Brighttield Brighttield Brighttield Womad Frequency 7 PiE z z Dlamekr_Ernghiek Brighttield Brighttield Brighttield 1 r Application Example Used to obtain approximate size of the cell 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 128 Chapter 5 Application Example These features can be used to separate rectangular shaped objects For curved objects measurement is more accurate
74. an 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 then a right click in a blank space in the work area allows you to choose paste in the new location k Xolli BBA Hop Ae HSB OB HA a a 2 Aspect Ratio_BF Normalized Frequency o o oy t Ratio Intensity_T o Scatter plots O 7 20 40 60 80 Gradient RMS_BF Channel image with mask 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 cor responding 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 Click Pointer Tool while drawing a regi
75. 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 ina 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 5 0 IDEAS 5 0 is required to analyze data from the FlowSight instrument and offers num berous improvements for analyzing data from any ImageStream instrument Please refer to the web site for the latest improvements and updates to this manual 1 General a b 2 Data IDEAS 5 0 can be operated with a Standard User security profile New user Application Default settings include directory updating default mask color and default statistics Files processed
76. 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 110 Chapter 4 Reporting Statistics from a Single Graph or Statistics Table Statistics can also be reported 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 Exporting Data You can export feature values for a population to the Clipboard a text file ora 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
77. and is useful for the selection of focused 152 Chapter 5 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 Also used 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 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 153 SN single cells 4 Contrast BF Gradient RMS BF Application Examples Determine overall focus quality of images Used with Contrast to determine focus quality Characterize texture See also Gradient Max Feature and Contrast Feature 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
78. aph that is in the center _ 64 Chapter 4 AB amp RI N Normalized Frequency i a n o 0 7 I 3e3 2e3 le3 0 5 _ Intensity 3400 i 1 le3 2e3 3e3 4e3 Sealing Auto C Manual AEE FoF j RY wo I Normalized Frequency n 3 Fas ae ee le4 5_Intensty 3400 Scaling Auto Manual ACR ELE 13500 0 5001e3 5e2e3 le4 le4 1e3 1e2 10 0 10 le2 1e3 1e4 Frequency 8 0 MNAL TTT E E d Dooa dooa teina 16 z e 5_Intensity 3400 Scaling Auto Manual X Axis X Axis X Axis Minimum Minimum i0000 Minimum Maximum Maximum h s Maximum Linear Linear Linear Log x gt log X gt 1000 Log x gt 10 10 To modify the display characteristics of each population or to change the layering order click Display Properties The Display Properties window opens roo Display Properties Population m reer All Fill CG CG w Line Style Solid Solid Solid Populations Histogram Properties Y Axis Units Bin count default w Frequency O Normalized Frequency 11 Arrange the layering of the populations with the up and down arrows to allow them to be displayed 12 If you want to change the color or sybol of a population click Populations to open the Population Manager For more information see Using the Population Manager 13 If you are creating a histogram overlay you can
79. 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 _ 50 Chapter 4 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 Inthe Batches window click Close C Users sfriend App Data Roaming Amnis Corporation batches Batch 2 1 2011 11 53 28 AM a Batch Report Batch1 C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 analyzed cif and daf files 0 0ng_2_9 _4 Analyzed daf C Users sfriend Desktop S100 NFkB Translocation Dose and Time 4 0 analyzed cif and daf files FITC DRAGQ ctm Alignment Camera Background Brightfield Gain EDF Flow Speed Cell classifiers applied Single objects separated Clipped objects removed Non4ramed objects erased C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 0 0ng_2_9 1if C Users sfriend Desktop IS100 NFkB Translocation Dose and Time
80. at 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 43 Getting Started with the IDEAS Application 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 Merge Raw Image Files gt Select raw image files for merging A of file will be created objects from each file and the a n info iat first nif Note during the lo Edie calc pe gos apply SA deies option needs to be ually Add Files 2 To select the rif files to merge click Add Files The rif file 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 a ie merged rif file O Guests 092011 Demo temp rif ey lt Files to merge O Guests 092011 Demo 092011 X101 Unstimulated __12 rif O Guests 092011 Demo 092011 X101 Stimulated
81. ation The following features will be created if they do not already exist Do vou want to continue Bright Detail Intensity A3_MO4 Ch4 Bright Detail Intensity AY_MO4_ Ch Contrast_MO4_ Ch Gradient Max_MO4_Ch4 Gradient RMS_MO4_Ch4 H Contrast Mean _MO4 Ch4_5 H Contrast Stc_MO4 Ch4_5 H Correlation Mean_MO4 Ch4_5 H Correlation Std_MO4 Ch4_5 H Energy Mean_MO4 Ch4_5 H Energy Std_MO4_Ch4_5 H Entropy Mean_MO4 Ch4_5 H Entropy Std_MO4 Ch4_5 H Homogeneity Mean_MO4 Ch4_5 H Homogeneity Std_MO4 Ch4_5 _Delete selected Features 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 Manager is enabled 2 Select Combined as the Feature Type The editing interface appears Feature Type C Single Combined Name ViSlSlxiZiaiole w Set Default Name OK Cancel 90 Chapter 4 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 express
82. ature is calculated in INSPIRE during acquisition 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 Internalization Feature 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 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 Over view of the Mask Manager The feature is invariant to cell size and can accom modate 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 typically have positive scores while cells with little internalization have neg ative 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 170 Chapter 5 Mid Internalization Single Focused Cells o gt ix 25 Low Internalization High Internalization _ n Normalized Frequency Applicat
83. ay 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 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 7 I 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
84. ay properties with another pro cedure in this section or click OK to finish and save changes or Cancel to finish and discard changes COON OOO A 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 Inthe Image Gallery right click an image that you are interested in A menu appears 60 Chapter 4 To Add Image to Analysis Area Show Masks Color Off Show Saturation Color Copy Image to Clipboard Copy Object Images to Clipboard Copy Gallery Column to Clipboard Copy Gallery to Clipboard 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 To show or hide the saturation color for the object image click Show or Hide Saturation Color respectively One or the other will appear depending on the current state copy images for u
85. 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 Cancel Once a data file is open you may begin analysis Display Properties Wizard This wizard is automatically run when you use the Open File wizard It is also avail able to run in any open data file from the Guided Analysis 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 om The Wizards window opens _16 Chapter 4 Wizards Select the wizard to use for analysis Name Description ifi Open File Opening ImageStream data files Display Properties Automatically sets image display properties tetas Creates an analysis template for identifying apoptotic events based pores on Grightfield and nuclear morphology Creates an analysis template that distinguishes mitotic and apoptotic Cell Cycle Mitosis 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 creat
86. bel for the Y Axis 6 Set the scaling for each axis of the graph The default is Auto which allows the application to automatically scale the graph 7 Toset minimum and maximum values for an axis select Manual 8 Select Linear or Log and enter Maximum and Minimum limits 9 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 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 The 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 The IDEAS application automatically chose 1000 for the scale of the gr
87. box f Sort by Images Used by clicking on the icon D g Check the box for the Ch02 Actin image h Sort the features by Category E Selected Statistics Create New Statistics Statistics Standard Deviation Reference population if required uniform actin Features if required E E E E Sort features by 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 only if all of the features all channels in the category are selected 96 Chapter 4 3E Area_FillThreshold M02 Area_Fill Threshold MO2 2_Actit Area_M01 Area_M01 Area_M02 Area_M02 lAa M11 Area M11 V Area_MC E Prea NC E 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
88. ce cece ceeeceeeeeeee 143 Cire Wlabity BP CANIS oae AAEREN ATENE 144 Compactes Feature aie ass este soli sesame gad ae diet ent E AER 145 Elongatedness Feature sooo ciccdetccwstsecescecnd sachenigceodsaenacscbiesetadessesecetesadeuss 146 RO De C OU UI CAM Sis stieset5 ang tesa cern heasoan cans a REE AEEA 147 Shape Ratio F CANIS soca ees cecciccuetatsdeetasantazamonhudadaetasecoeusanemenbasdedacanss 148 Symmetry 2 3 4 gt lt 1 10 c1 lt an oe ee ree 149 Understanding the Texture Features 00 00 2000 c cece cece eee cece cece ceeeeeeeeee 150 Bright Detail Intensity R3 and Bright detail Intensity R7 Features 150 Contis IE CAT nean iosacedussat haut a EEN NEARNE 151 axils Table of Contents Ensquared Energy Feature 0 0 00 2200 c cece cece cece ecec ce ecceececeeceeeeeceeees 152 Gradient Max TF CAME ec cccdcnstocusegu hat ties coud an ethemannagtatecdeatcn thaasaeacadlewee 152 Gradient RMS Feature cieicocascnscecns ta ttenteoqsecedueseahceasnsiecinasosmsecceereisid dee 153 H SR SEU scepter se eae bate eee teense asa oeaecenes 154 Modulation Feature 0 0 00 2c cece cee cee cee ccc cece cee e ec ee eee e ecco ee ceeeeceees 155 Spot Count Feature iaaccs 26 cts acter ccieer bean Gadtaucuuisasacaaniuciseanaueatewaleaetanwiiees 156 NADEV ev cr 06 1 oe ee en ee ee eee 156 Understanding the Signal Strength Features 000 000 02 00 22 ccc eee eee eee eee 157 Bkgd Mean Feature
89. ce 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 e Spot Intensity Max is the Raw Mean Pixel of spot 2 e 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 140 Chapter 5 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 be
90. ces that are on the original copy of 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 ill Chapter 1 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 wh
91. cross correlated Objects ml Feature A local concentration of all objects per ml Note to get objects per ml of a population use the statistic Con Internalization Feature a System features do not require a mask and tend to deal with system wide metrics centration E O pa O The ratio of the intensity inside the cell to the intensity of the entire cell Similarity Feature Camera Line Number Feature i No Yes An incremental count of objects Camera Timer Feature A DAET No Yes The clock rate in KHz This is relative time Flow Speed Feature Yes Yes The Similarity is a measure of the degree to which two images are Ye The calculated flow speed in mm sec Yes linearly correlated pixel by pixel within a masked region S Object Number Feature Yes The sequence of objects Objects sec Feature A local concentration of number of objects per second Note to get objects per ml of a population use the statistic Concentration Time Feature The camera timer feature converted to seconds ombined _ Any combined feature will be listed under Combined No Yes O lt V Yes lt V O L D a 3 O p O i O i O 125 Table of Basic Features available without QI The default masks used for FlowSight Basic non Ql files is the INSPIRE mask FlowSight Basic Features Mask Channel Brief definition Area
92. ct 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 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 2 3 Go to 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 Select a Tab to see the information for each heading 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 sample information for any file without loading the file _ 46 Chapter 4 a Sample Information x Select Data File NFkB Fite Dq5 No LPS Analyzed _2 daf Acquisition Comections Focus Fluidics Detection Camera Settings Illumination EDF Compensation Channels F Raw Data File Name nif NFkB Fite Dg5 No LPS_2 nf Acq Date 3 31 2010 Version No Objects Processed Data File Name cff C Users sfriend Desk
93. customize it by performing the fol lowing steps e To fill or not fill the line for a population select or clear the Fill checkbox e If you want change the Bin count The default is determined by the X Axis scale of the plots e Decide whether to plot the Y Axis Units as a Frequency or a Normalized frequency percentage 14 Click OK in each window 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 65 Getting Started with the IDEAS Application conjugates 1 8 o ah N Graph Properties Statistics Show Hide Legend Order Plots o D Normalized Frequency o Ww 40 60 Gradient RMS_B Apply Remove Regions Clear Selected Bin 20 Copy Region to Clipboard Paste Region from Clipboard Features Masks Populations Regions Export Statistics To Clipboard Copy Graph Stats To Clipboard Print Graph To show selected statistics for a graph 1 You can show and hide statistics by clicking the Statistics toolbar button in the panel that contains the graph conjugates q m Te 1 6 in _i hm nc d Frequency o 2 Or right click anywhere on the graph and click Statistics on the graph context me
94. d 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 An example is shown below PE Podo Composite 136 PE Podo 2 Composite Centroid Y Chapter 5 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 the lower right Similarly a cell with a large shift in the Y direction and not X are found in the upper left See Centroid Features to measure the X and Y shift together Single Focused cells wo E5 5 ua 2 oO s o O 4 6 Delta Centroid _Ch4Ch6 Application Example Used to identify capped versus not capped cells Use
95. d 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 Drag 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 137 PE Podo Composite gt 2 O Sa C QO PE Podo Composite Delta Centroid Y Delta Centroid X Centroid X Delta Centroid XY v Delta Centroid X 4 Delta Centroid Y Below is an example of using the Delta Centroid XY A bivariate graph of a shape ratio versus Delta Centroid XY can identify cells with caps as shown here Shape Ratio BF 6 9 12 Delta Centroid XY_PODO DRAQ5 Application Examples Quantify the spatial relationship between two fluorescent probes 138 Chapter 5 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
96. dient Max Feature M01 _Ch01 123 The maximum slope of the pixel value changes in the image to fina cna measure focus quality of an image Gradient RMS Feature M01_Ch01 Enumerates changes of pixel values in the image to measure the Yes M12_Ch12 focus quality of an image H Texture Features ie Measures Haralick texture features ie ee Modulation Feature M01 Ch01 Measures the intensity range of an image normalized between 0 Yes M1 2 Ch 12 Spot Count Feature Enumerates the number of spots No No See also Spot Distance Min Feature Spot Area Min Feature and Spot Intensity Min and Spot Intensity Max Features Std Dev Feature No Ves M0O1_Ch01 Describes the overall distribution of pixel intensities M12 Ch12 Signal aces Strength Signal Strength Features are measured in pixel values i Lo Bkgd Mean Feature The average intensity of the camera background LL Bkgd StdDev Feature The standard deviation of the background intensities No ves chot cnt2 Intensity Feature MC Ch01 The sum of the pixel intensities in the mask background sub Yes Yes MC_Ch12 Max Pixel Feature Ven Vien MC_Ch01 The largest pixel value within the mask background subtracted MC_Ch12 Mean Pixel Feature Ves Ves MO01_Ch01 The average pixel value within the mask background subtracted M12 Ch12 Median Pixel Feature Ves Vas M01 _Ch01 The median
97. e average fluorescence activity This feature is preferred over the Raw Median Pixel feature 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 162 Chapter 5 Median Pixel Intensity 230 000 230 000 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 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 Raw Max Pixel Feature The Raw Max Pixel feature is the largest value of the pixels contained in the input mask 163 RTX FITC CD45 PE Brightfield This image is saturated in the FITC channel but not in the PE channel b Q 8 8 b Raw Max Pixel_Ch3 Po 200 A400 600 800 183 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
98. e light mode To use light background graphs in the Analysis Area Click the graph background tool to switch between light or dark mode P4 103 Getting Started with the IDEAS Application To map the dark mode colors to light mode colors 1 Select Options gt Manage Color Schemes The Modify Reporting Color Scheme window appears a Modify Reporting Color Scheme Select Dark Mode Color Select Light Mode Color Mapping C Blue Update All Populations Reset To Standard Cancel 2 Inthe Select Dark Mode Color drop down menu select the color that you want to map 3 To choose a different color click the Select Light Mode Color Mapping color Square and click a new color on the color palette Click Update All Populations 5 If you want to return the settings to the IDEAS defaults click Reset to Standard 6 Click OK to save the changes or Cancel to exit Copy full or partial screens To copy the entire screen to the Clipboard 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 directly Analysis Area or Image Gallery 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 t
99. ect Peak Range Skeleton and Valley Each of the functions masks are defined here Refer to Using the Mask Manager for more details about how to create new masks Dilate Mask The Dilate mask adds the selected number of pixels to all edges of the starting mask Morphology Dilate 2 pixels Erode Mask The Erode mask removes the selected number of pixels from all edges of the start ing mask Morphology Erode 3 pixels 175 Fill Mask The Fill mask fills in any holes in the starting mask 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 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 176 Chapter 5 a Threshold 50 Intensity 250 1023 Interface Mask The interface mask identifies pixels in a
100. ect Ratio Intensity is the Minor Axis Intensity divided by the Major Axis Inten sity See also Major Axis Intensity and Minor Axis Intensity Features 143 The figure below illustrates the difference between Aspect Ratio Intensity and Aspect Ratio See also Aspect Ratio Feature Aspect Ratio 0 99 Major Axis 9 87 Minor Axis 9 76 Aspect Ratio Intensity 0 95 Major Axis Intensity 7 22 Minor Axis Intensity 6 87 Aspect Ratio M4 Aspect Ratio 0 76 Major Axis 1024 Minor Axis J Aspect Ratio Intensity Major Axis Intensity 5 47 Minor Axis Intensity 0 a E q 1 2 Aspect Fatio Intensity M4 Aspect Ratio 0 57 Major Axis 12 76 Minor Axis 7 31 Aspect Ratio Intensity 0 29 Major Axis Intensity 14 22 Minor Axis Intensity 4 05 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 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 this 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
101. ee 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 ess min 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 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 147 Single Focused Cells Application Example Used in cell classification studies Also used to differentiate small round cells from small square cells of similar area 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 148 Chapter 5 Thickness max l l Shape ratio Thickness min Le
102. eeeceeeeeeeeees 26 Shape C hange Wizard esiseina A A E EEEE RE 28 Spot Wizard eener E E ETE EAE ITEE 30 B ldne B01 6 lt lt eresserraser es N E er ae te ater eer ee eee 32 Advanced ANALY G18 ea E EEE E EE EERE 34 Openin CAI ES sses E T aaebotectaeise 34 CSM a TD MICs nies on ores tess R T EES 35 STI ACU TI asa fat ens a ethane aas ansace hn AE 37 QOpenino adaf ae eee nee E ee eer een 39 Vill Table of Contents Sav ng Data Files otros taco scceeeeenalccsdedelecceeecneecenscchactitacdanstactedoeneaaaess 40 Saving a Data Analysis File daf 200 0 0000000022 cece ccc eee ee cece cece eee 40 Saving a Compensated Image File cif 000 000 000220 c eee eee eee eee eee 4 Saving a Template CASU acta siacoecs ete ait Sh asain Buda tones alse heen Gans int heehee 4 Overview of Compensation 200 2220 ccc cece cece c cece ce ceecececeeeceeseeeees 4 Preview and edit a compensation matrix 0 00 220 ccc cceeeeeeeeeceeeeeeees 43 Merging Data Files asict anenensaccivsietestidesancuaeecantedoasdesaasasastensehtassees 44 Merging Raw Image Fess tts et eateries Cerne teed tine Sedat ace acne 44 Merging Compensated Image Files 2 0 0 020022 cee cece cece cece eee eee cece 44 Creating New Data Files coc t0 cece nice daaccnasanedsantesateasheccunsosceeyisaieaseegameasds 45 Creating new data files from populations 2 200 2 22 2 cece eee eee eee 45 Viewing S
103. eeeeeeeeeeeeees 3 Installing the IDEAS Application 20 0 000 2000 c ccc cece cece cece cece eeeeceeeeeeees 3 Setting Your Computer to Run the IDEAS Application 2 2 22 22 4 Setting the Screen Resolution 0 00 0 2 ccc ccc ce cece cece eee ence eee eeeceeeeeenees 4 Viewing File Name Extensions 2 20 20 20cceec eee ceecceceecceecceceeeceeeeeees 4 Copying the Example Data Files 200 000 0020 c ccc cece cece eee cece eeeeeeeeee 4 Viewing and Changing the Application Defaults 00000 0002002 ccc cece eee eee 4 Overview of the IDEAS Application 00 0 0 02 0 2 Comparing the FlowSight basic Quantitative Imaging and ImageStream data files 2 Understanding the Data Analysis Workflow 2 2 00 200 2 200 220cccccee cece cece eeeeee 3 Overview of compensation analysis tools and file structure 2 22 22 2222 6 Data Acquisition and Compensation 0 202 22 22cccceecceeccececcceeeeeeeeeees 6 Data Analysis Tools 00 0200 20 22 ccc cece cece eee e ccc e eee e eee c eee cece ee eceeeeceeeees 6 Interface of the IDEAS Application 0 2 00 02 0 00 c ccc ce cece cece ceeccceeeees 7 Vil Chapter 1 Overview of the Data File Types 0 00 00 2000 c cece cece cece e cece cece cece eeeceeeeeees 7 Raw Image File rif i ace cet septan ce sttictee actos waemae acetone taccamaaaneeweeadedes
104. eeeees 170 Smary FE COMIN eesin a EEE AE teddanecciaadaas 171 EE CUE a EEE E E ee E E A oeaeece nee none 172 Understanding the System Features 000 0 0002 c ccc cece cece cece cece cece ceeeeeees 172 Camera Line Number Feature j252 lt 0 cc2ccccicescesacccencescevsececavaaecancoebeecseeus ses 172 Camera Timer Feature 250 cus 2cc cs onbaentedetuccussnechcacn aisecnanemenenitaatentieedees 173 Flow Speed F CAM Crs 02 2200 sewcrurinesscccesetcandacieake doctadadastebhac esaulasmavccuanacss 173 Object Number FCatur Gx c cette asin cice cha ntews naan daaceidee sad asinudasheeesaoeeecebet 173 Objects ml ekore 10 ee a eae ee ee eee ee 173 CDISCIS SCC Fea E ie a cca cpa cease dan daeied nan cause ee aRontianoasasimmeaeeteces 173 T 20 eaen E E Sa cinema tees AEE 173 A DOUE MASK sean eee ee ee TO eee ede eee ee eae 174 PINSE IVES Scoop et orice ge etun ee eciee seat eee cence ae eset eee neeee 175 Erodo MaS Gee een ee ee eee ee ee ee eee er ce ane eee 175 PO oa sees seater aay etic ese ante SA alec oma oe eeine EE 176 laspre Maskeen oe feet oy aera see ghee E 176 AAS TUG TEN Mask te ects setae esto seis tats ate ayaa eae ee ee pease AE 176 ECO MIS este heen a cere cera ener arenes seeniscee eet aes 177 Morphology IAG appar dese se enrsec cen ahcttvdg iba neem tatansueesdeckesneasnseteeseteadesctes ag 178 8 2 16 IN AIS ene sere eee ee enema 178 PEAK MaSK eee et nse am erste EAE a AN 179 CT ALEX IAA 2 1c lt a oa a aE E E 180 IC
105. eg soso cases 2 cinelnien aE TEA EE EE AREAS 158 BOG SUDEV TEIE reie E ERES 158 Mitens FeaT agerra ARER TEE OEE 158 Max PIX IF CAV esne O E R 159 Mean Pixel F Cau crcdet aa a E E DESEE 160 Median Pixel F GaC ea O 161 MiP REFE onenn r a A E ENDESAN 162 R w Intensity PCAC sereen A AS AR 163 Raw Max Pixel Feature 2 20 220 002 c cece cece cece ccc eee cccceeccccceeeeceeeeeees 163 Raw Mean Pixel Feature 2 00 02 22000 e cece ec eeccceeeeeceeeeeeees 164 Raw Median Pixel Feature 2 2 20 00000000 c cece cece cece cee eee ec eee eee ece eee 165 Raw Min Pixel F Catt c22 soc ecerdecdsace teuient choadyanscdnliace adseecmanimaieaabensewes 165 Saturation Count Feature 0 2 00 0 0 2 cece cece ccc eee cece cece eeeeeeeeeeeeeeees 166 Saturation Percent Features oc 26 ceccci cows sascnwaccndadebidesedsheccsieesaaxtiaocacnaueeens 166 Spot Intensity Min and Spot Intensity Max Features 0 20 222 202 167 Bright Detail Similarity R3 Feature 00000 000200 e cece eee ee eee cece cece eee 168 Intensity Concentration Ratio Feature 2 0 02202 c eee e cece eee cece cece eee 169 Uncompensated Intensity 2 00 0000 0 occ cece e eee cece eee ceeeeeeeeees 170 Understanding the Comparison Features 20 2 200 22 ccceeeceeeeceeeeeeeeeee 170 Xill Chapter 1 Internalization Feature 2 2 0 00 220 c cece cece eee cece eee eee eee e eee eeeeeee
106. elected and a table is added to the Analysis Area e The selected object is identified in each scatter plot graph with a green cross e The image can be placed in the Analysis area by right click gt Display Single Image 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 e Inthe View drop down menu of the Image Gallery select a specific view The imagery display changes according to the new view To show or hide masks e Click the Show Segmentation Masks toolbar button to toggle between show ing and hiding the selected masks for all images in the Image Gallery _54 Chapter 4 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 Channel amp Tip To hide the mask for a specific channel only set the individual channel mask to None To show or hide color e Click the Show Color toolbar button to toggle between showing and hiding the colors for all images in the Image Gallery See Setting the Image Gallery Properties for more information O To zo
107. elected rows of the table and trans poses the data so that when pasted into other programs such as Excel the rows become columns 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 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 Alternativel
108. end Statistics Cursor C Show Sample Name in Title Size scaling factor 2 3 Select Graph Statistics Legend Cursor and or Show Sample Name and Title depending on what you want to copy Adjust the Size scaling factor as desired It is recommended to set this at 100 5 Click OKto copy the graph and or the statistics to the Clipboard Note The IDEAS application copies the statistics as a metafile If you want to export the data into a table such as that in Microsoft Excel you must instead click Export Statistics to Clipboard on the context menu 106 Chapter 4 aj a eg oo Aspect Ratio BF a k3 0 100 200 300 400 500 600 7o00 Area BF Area BF Aspect Ratio BF 10723 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 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 I
109. es 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 Setting the Image Gallery Properties gJ Display Properties Wizard Step 1 Set image display properties IDEAS will now optimize settings for your image display Choose the image channels used in your experiment Click next to continue Tipt Brightfield and SSC settings are determined automatically Tip2 If you wish to change your image gallery settings click the channel display properties icon in the image gallery toolbar 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 Analaysis 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 Wiz ards from the Guided Analysis menu Begin Analysis 17 Getting Started with the IDEAS Application To begin double click on Begin Analysis Step 1 Gate cells in best focus A Gradient RMS histogram of the All population has been added to the ana
110. etting Started with the IDEAS Application Batches to Run Batch1 Add Batch Edit Batch Remove Batch Submit Batches Processed Batches m Batch 6 29 2011 11 05 33 AM Batch 5 3 2011 10 38 34 AM Batch 5 3 2011 9 51 14 AM 4 Batch 5 3 2011 9 41 07 AM 4 Batch 4 5 2011 11 28 23 AM 4 Batch 3 24 2011 11 47 57 AM Batch 3 23 2011 2 29 21 PM Batch 3 23 2011 2 00 37 PM 4 Batch 3 2 2011 8 02 21 AM Batch 3 1 2011 1 17 04 PM m Batch 2 9 2011 11 49 07 AM 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 e 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 Once you have started processing batches it may use up a fair amount of your computer s processing power Processing Batch Batch1 a Unprocessed Processing Processed nif File 0 0ng_2_9 nf 0 1ng 15_1_8rif 0 1ng 30_6 13 f O 1ng 45_11_18 rf 4 cif File O 0 0ng_2_9 cif O 0 1ng 15_1_8 cif O 0 1ng 30_6 _13 cif O 0 1ng 45_11_18 cif O finn AN 16 23 ci 4 daf File Total elapsed time 0 minutes Tip To cancel the batch processing
111. ew 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 188 Chapter Appendix A Glossary Table 1 Glossary of Terms The process of collecting data from the ImageStream cell analysis acquisition 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 brightfield image ne cell analysis system this light is provided by a halogen lamp calibration Sse a ais results for the purpose of optimizing functionality 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 sensor 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 a
112. 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 Overview of the IDEAS Application INSPIRE IDEAS Data Collection Spectral Compensation Data Analysis l Raw Compensated Data image Apay mage Analysis Experimento Samph File COPERTO File File Template File h Compensation vat rim Batch Processing Overview of Data Analysis Workflow 1 Create a compensation matrix using the 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 Overview of compensation analysis tools and file struc ture Data Acquisition and Compensation Data Analysis Tools Interface of the IDEAS Application Overview of the Data File Types Data Acquisition and Compensation Data are first acquired from the inst
113. g 60_18_1 cif 10ng 75_23_6 cif J 10ng 90_28_11 cif 1000ng 15_5_12 cif 1000ng 30_10_17 ciF 1 1000ng 45_15_22 cif 1000ng 60_20_3 cif 1000ng 75_25_8 cif 1000ng 90_30_13 cif My Network Places File name 0 Ong 2 9 Default daf Files of type Compensated image files cif IDEAS files rif cif daf Raw image files rif Compensated image files cif Data analysis files daf In the next window you will e Choose a template e 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 10ng 60_18_1 dat 3 Chapter 4 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 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 progress bar After the application has successfully opened the cif file the daf file is saved See also Saving Data
114. g the Shape Features Understanding the Texture Features Understanding the Signal Strength Features Understanding the System Features Understanding the Comparison Features About Masks Mask Functions Table of Base Features Alphabetical Delete this text and replace it with your own content eature Name Angle Feature Angle Feature Area Feature Aspect Ratio Feature Aspect Ratio Intensity Feature Signal Bkgd Mean Feature Signal Bkgd StdDev Feature Bright Detail Intensity R3 and Bright detail Intensity R7 Features Bright Detail Similarity R3 Feature Comparison 3 Centroid Features Centroid Features Location Circularity Feature ompactness Feature Contrast Feature Centroid Features Centroid Features Location Diameter Feature Size Elongatedness Feature Elongatedness Feature Flow Speed Feature Gradient Max Feature Texture Gradient RMS Feature Texture o U Q 5 mn A A EN A n n A n Tl OIO DD 3 3 9 Sig 4 i 310 2 o 5 Vs Q 1 D D D 119 H Texture Features Intensity Concentration Ratio Feature Comparison F Signal Intensity Feature Internalization Feature Y N D Length Feature Lobe Count Feature hape ize ize ocation Signal Strength 5 Signal Strength Signal Strength Signal Strength
115. gree to comply fully with all U S export laws and regulations to ensure that neither the Software nor any technical 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 Chapter 1 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 acc
116. he Analysis Area To print the Image Gallery data e Select Reports gt Print Image Gallery The IDEAS application prints all the images that are visible in the Image Gal lery Copy Images To copy the Image Gallery data to the Clipboard 104 Chapter 4 e Right click anywhere in the Image Gallery and then click Copy Gallery to Clipboard The IDEAS application copies all the images that are visible in the Image Gal lery to the Clipboard The images can then be pasted into a third party report ing document ADAF ACHT og aF BDBRecine ol BESO OOE To copy the object s images to the Clipboard e Right click an image in the Image Gallery and then click Copy Object Images to Clipboard Drags ADAP Actin T cell To copy a single image to the Clipboard e Right click an image in the Image Gallery or in the Analysis Area and then click Copy Image to Clipboard Copy Graphs and Statistics You can copy graphs and statistics to other applications Change the graph back ground size axis labels region and population color or symbols as desired before copying To copy a graph and or statistics to the Clipboard 105 Getting Started with the IDEAS Application 1 Select light or dark mode graphs in the analysis area using the tool 2 Right click a graph and then click Copy Graph Stats To Clipboard The Copy Graph window appears Copy Graph Select options for copying Graph C Leg
117. he 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 single cells ce ba i Sy x 120 oOo Feature Valle 300 zi ie i T Saturation Count Channel 3 3 Raw Mas Pixel Channel 3 Saturation Percent Channel 4 2062 Lo T iu T Saturation Count Channel 3 Application Example Measure the validity of the experiment setup Saturated data may not produce useful information Saturation Percent Features The Saturation Percent feature reports the percentage of saturated 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 4 166 Chapter 5 Application Example Measure the validity of the experiment setup Saturated data may not produce useful information 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
118. he 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 100 Chapter 4 6 If you are updating an existing population click the Update button in the Tagged Populations window 7 When you are finished click Close in the Tagged Populations window 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 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 aw Create a Tagged Population From a File Select a comma separated text file that contains the object numbers for the population Create the tagged population Population name Dark Mode Color Light Mode Color Symbol Cancel Browse for the file Name the population select the color symbol and click OK 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
119. ify 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 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 Bright Detail Similarity R3 for the double positive population is added to the analysis area Draw a region to gate on co localized events 299 3 Getting Started with the IDEAS Application EE SS x ooribiaed frega E Bal i J 1 Dr Et el tars may CL ad bd Cpoe Low 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 Internalization Wizard This wizard will create an analysis temp
120. 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 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 truth 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 3 Getting Started with the IDEAS Application Q i oS a
121. in files from an ImageStream or FlowSight with the QI upgrade using the Mask and Feature Manager tools Features are 116 Chapter 5 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 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 Using the Mask Manager and Using the Feature 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 a gt co The pixel size and field of view per channel is dependent on the magnification used See your INSPIRE Users Manual for more information 117 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 Cen
122. ing 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 76 Chapter 4 Current To view and customize the features shown in the Object Data table 1 Click the Object Feature Values tool 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 aTa Getting Started with the IDEAS Application amp Select Object Features Features Aspect Ratio Intensity_MO1_Ch01 i Aspect Ratio iS Cho 4 Select the features to view Multiple features may be chosen by holding down the Cirl 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 For more information see Creating Reports and Exporting Data 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 _78 Chapter 4 Enter title Enter text here 2 Type atitle and text Popu
123. ing views is shown on the left 10 11 vom Image Gallery Properties Display Properties Views Composites Views View Definition H All Channels G BF Name All Channels Ch2 i Column i Cha All Channels i SSC mask M01 Ch2 mask M02 i NFKB mask M03 i Ch4 mask M04 i BF mask M05 i DRAGS mask MOB DRAGS i NFkB SSC Preview Changes in Gallery To create a new view Click New The new view is automatically added to the list on the 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 Then 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 sect
124. ion of features and operators Table 1 Combined Feature Tasks and Toolbar Double click the feature in the Features list Add a feature to Or single click the feature in the Features list and select the definition click the leftmost down arrow button on the toolbar 4 Add an operator or Jick the corresponding button on the toolbar a parenthesis to 107 eal il the definition Enter the number in the box and then click the cor Add anumberto _ responding down arrow button the definition phi If the area is greyed out an operator must be selected first Select the function in the list and then click the cor responding down arrow button The available functions are ABS absolute COS cosine SIN sine SQR square and SQRT square root If the area is greyed out an operator must be selected first Add a function to the definition 5 Click OK 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 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
125. ion 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 59 Getting Started with the IDEAS Application e Image Gallery Properties Display Properties Views Composites NFkB 7 DRAGS E Translocation Name NFkB DRAGS Object 0 3 NFkB DRAQS NFkB 100 Image DRAQS 100 NFkB Percent 100 Preview Changes in Gallery 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 3 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 list on the left A composite can be removed from the list by clicking Delete Continue customizing the Image Gallery displ
126. ion Examples Quantify internalization when supplied with the internal mask Quantify the intensity ratio of a region of interest to the whole cell 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 distribution 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 gt gt 3 5 7 m Cc Le g e ta ge g oF A aa aa a im ST I LL LL Z 100 120 7 AAD Pixel Intensity 7 AAD Pixel Intensity 7 AAD image 7 AAD image 171 Below are examples of cells with varying amounts of similarity between the NF AB 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 u
127. ion 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 that the camera collected for each object that the instrument detected e Instrument settings that were used for data collection e Calibration values from ASSIST e Compensation matrix and template if used while acquiring FlowSight data Compensated Image File cif The IDEAS application 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 e 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 ore
128. 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 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 164 Chapter 5 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 Raw 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 ie NO _ my LW AN WAT i en 90 120 150 180 Raw Min Pixel a O c D 5 D LL D D N w Po Application Example Quantify spectral absorbance using the brightfield image Identify over compensated images Measure the level of malaria infection in RBCs 165 Saturation Count Feature T
129. ity 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 2 Threshold 50 Intensity 250 1023 Application Example Used with the Area feature to define apoptotic cells 183 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 rectangle 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 184 Troubleshooting Troubleshooting This chapter covers common issues and provides solutions 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 qui
130. k Yes to confirm To create a new combined population 1 Within the Population Manager Analysis gt Populations click New _ 98 Chapter 4 The right side of the Population Manager window changes to allow you to define a new population Properties Name Dark Mode Color Light Mode Color t Symbol Simple Dot inition M aGBocoe 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 symbol in the Symbol drop down menu Use the toolbar to build the population definition as described in the table and click OK when done oF W N Properties Name R4 And Not Tagged Dark Mode Color Light Mode Color Symbol Simple Dot x Definition All Hq ramie 3 092011 X101 unstimulated_1t cif 3 All RI y R2 3 00 R3 mei R fo Tagged OK Cancel Table 1 Population Tasks and Toolbar nee poputo te Meca Select the population from the drop down menu Use the Boolean AND or OR operator 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 Select objects
131. l 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 Apoptotic Cells with high Scatter Std Dev and BF_Modulation Live Cells with low Scatter Std Dev and BF_Modulation Application Example Quantify intensity variation within a mask Distinguish apoptotic and necrotic cells Understanding the Signal Strength Features Signal Strength features include the following Bkgd Mean and Bkgd StdDev features describe the background of the image e Intensity and Raw Intensity features quantify the intensities in the region of interest 157 e Raw Max Raw Min Raw Mean and Raw Median Pixel report single pixel values in an image e Max Min Mean and Median Pixel report background subtracted single pixel values in an image e Saturation Count and Saturation Percent quantify the saturated pixels e Spot Intensity Min is used when counting spots Note that when the name includes Raw this means that there is no background subtraction Bked 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
132. late 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 24 Chapter 4 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 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 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
133. lation 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 column header in the table and the menu opens BR OND Edit Statistics Table Insert Column Edit Column Delete Column Delete All Colurnns Order Columns Copy Statistics Copy Statistics Transposed 5 Edit Statistic Table opens a Statistics Properties window to enable changes to multiple column statistics 6 Toaddasingle statistic column select Insert Column 7 Select Edit Column to make a change 8 To delete a single column right click on that column and select Delete Column 79 Getting Started with the IDEAS Application 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 s
134. lication 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 512 MB of RAM and a 1 GHz processor However due to the large size of the image files that the lmageStream cell analysis system creates a larger amount of RAM will prevent paging and thus increase per formance Software Requirements IDEAS 5 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 be removed duri
135. ling to license the Software to you only upon the condition that you accept all the terms contained in this Agreement By clicking on the I accept button below or by downloading installing or using the Software you have indicated that you understand this Agreement and accept all of its terms If you are accepting the terms of this Agreement on behalf of a Company or other legal entity you represent and warrant that you have the authority to bind that Company or other legal entity to the terms of this Agreement and in such event you and your will refer to that Company or other legal entity If you do not accept all the terms of this Agreement then Amnis is unwilling to license the Software to you and you must return the Software to Amnis for a full refund if you have paid for the license to the Software or if Amnis has made the Software available to you without charge you must destroy all copies of the Software Your right to return the Software for a refund expires 30 days after the date of purchase 1 Grant of License Conditioned upon your compliance with the terms and conditions of this Agreement Amnis grants you a non exclusive and non transferable license to Execute as defined herein the executable form of the Software on a single computer solely for your internal business purposes You may make a single copy of the Software for backup purposes provided that you reproduce on it all copyright and other proprietary noti
136. 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 _9 Getting Started with the IDEAS Application Ranking features by discriminating power General Example 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 process to find features that separate two populations of cells from each other with minimal knowledge of the feature set A general description of the steps is followed by a specific example 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 atime 4 Create any additional masks and features you think may help differentiate the truth populations 5 Calculate the statistical disc
137. low the Valley X and Valley Y position of the 7AAD image is shown In this example a protein of interest in the PE image localizes to the synapse between two cells Gz 440 Value alley Skeleton M5 7440 Thn 43 alley Skeleton M5 7440 Think 6r Pee ee Ate Ly P re i m P t a i a oO ng amo a I ee m r m t E re l H E l H PAA iage PALA page Comeo site Fisel 43 67 Intensity 38 These features define the origin of the Valley mask 141 Application Example Measure the exact center of where a synapse between two cells is located 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 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 Minor Axis Features 142 Chapter 5 Aspect Ratio Minor Axis Major Axis Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightVidth Thickness min Length ss max ess min _ 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 Aspect Ratio Intensity Feature Asp
138. lustrates 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 130 Chapter 5 Aspect Ratio 0 99 Major Axis 9 87 Minor 4 Aspect Ratio Inten Major Axis Intensity Minor Axis Intensity ci mT Oo Po to 41 mw t i 2 Gs Cc a D mi Ll Aspect Ratio 0 765 Major Axi Minor Major Axis Minor Aspect Ratio 0 57 Minor Axis 7 31 Aspect Ratio Intensity 0 29 Major Minor Axis Intensity 4 05 Application Examples Quantify and compare the image fluorescence shape Identify single cells 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 PI DNA 131 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 Spot Area Min Feature The Spot Area Min feature provides the area of the smallest spot connected com ponent 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 spot
139. ly obtained using the thick ness features 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 Shape ratio Thickness min Length Thickness min 129 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 Minor Axis Major Axis Application Examples Quantify and compare cell shape Identify small medium and large cells 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 il
140. lysis 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 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 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 presented in the order of acquistion 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 Apoptosis Wizard The apoptosis wizard will guide you through the process of creating the features and g
141. make sure that the Hide extensions for known file types check box is not selected 3 Click OK Copying the Example Data Files lf the CD or DVD includes data files copy them to a single directory on your hard drive Sample data files are also available on your workstation or at www amnis com login for customers with an Amnis 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 BR OND Viewing and Changing the Application Defaults To view or change these defaults clickApplication Defaults on the Options menu e The Directories tab allows you to change the default Data Template Batch or Compensation Matrix file directories and choose the option for updating the location as you select files Welcome to IDEAS 5 0 The Populations tab allows you to view or change the default color or symbol for populations The Masks tab allows you to view or change the default mask color The Statistics tab allows you to view or change the default list of statistics shown for a graph Defaut Data Files Directory f Customer Training and Application Support_ 120309515100 Training 04 Update automatically when file is selected Defaut Template Files Directory 0 2011 Data v081811 X177 UVa Eg E Update automatically when file is selected Use default data directory Defaut Batch Report Files Directo
142. mentation 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 Image default tight 178 Chapter 5 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 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 b f y Spot Mask Peak Mask These peak areas will be masked Application Examples Used with the Spot Count feature to quantify the speckleness of cells Separate connected spots in a
143. 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 _ 40 Chapter 4 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
144. mplate 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 N R Chapter 4 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 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 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
145. mples fill intensity list of morphology object peak range show in Image Gallery skeleton spot system threshold valley viewing definitions Merging cif files Merging raw images files Mitosis using a wizard Name and Color Image Gallery Nuclear Localization using a wizard Nuclear translocation using a wizard 200 Index 84 176 176 81 178 178 179 180 54 180 181 182 183 184 83 44 44 20 56 26 26 Chapter Appendix A O Object Data 76 Object Feature Values Table 63 One color histogram building block a2 Opening files using a wizard 15 Oval Region Tool 63 P Pointer tool 62 Polygon Region Tool 63 Population Manager 97 tools 99 Population Statistics 79 Population Statistics table 63 Populations creating 100 creating a new data file from 45 creating combined 98 deleting 98 display properties 98 during acquisition 47 viewing 98 Printing 104 R Rectangle Region Tool 63 201 Index Region Manager 101 Regions editing 102 viewing 101 Reporting 102 changing color mapping 104 graphs and statistics 105 Images 104 Images and Graphs 103 light mode graphs 103 statistics 107 statistics from multiple files 110 Reports printing data 104 113 rif about 8 merge 44 opening 35 S Sample Information view 46 Saturation view in Image Gallery 54 Saving data files 40 ast 41 cif 41 202 Chapter Appendix A daf 40 Template 41 Sca
146. n e Minimum standard statistical definition e Mode standard statistical definition e RD Median the ratio discriminant Fisher s discriminant using the Median and MAD 108 Chapter 4 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 e 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 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
147. n 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 Cell of Interest hisk Mt ac Mask shown on Brighttield Image 177 Conjugate hask Cell of Composite BF nera ea M ieta me Image Application Examples Used to quantify synapses in T cell APC antigen presenting cell con jugates 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 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 feature set The default option is used for the default seg
148. n of probes with metaphase chromosomes or the less con densed somatic interphase chromatin fluorescent in situ hybrid ization FISH gain The amplification of a detector signal etal 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 ela fesel Missin 0 33 microns with the 60X objec tive Note that 1 pixel x ums The state of a pixel that has a value at or above 1023 for the 1S 100 or 4095 for the IlmageStream 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 cell 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 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
149. n the file select it in the Object list or type it s number e Toview a different image for the object select it from the list The Link inputs checkbox is checked by default To modify a mask with different inputs uncheck this box Click OK The new function is added to the mask definition Click OK The new mask name will appear in the list of Masks on the left side _ 8 Chapter 4 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 Table 1 Mask Tasks and Toolbar Double click the feature in the Masks list Adda mask tothe lOr single click the feature in the Masks list and click the definition leftmost down arrow button on the toolbar 4 Use the Boolean AND or OR operator 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 o The NOT operator specifies which mask will not be mask used Affect the order of Use the parentheses toolbar buttons operations AE Remove an item Click the left arrow button on the toolbar from the end of the z definition Combine two masks 4 Addmasks and Boolean logic
150. nderstanding 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 fluorescence 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
151. ng installation To install IDEAS software 1 Download the application Setup file from your account at www amnis com or insert the CD or DVD that is labeled IDEAS application View the contents in My Computer or Windows Explorer Double click Setup exe Follow the instructions until the installation process has completed MadCap help viewer is installed and opened during installation or upgrade Select the language and check box to not show this dialog again DO oO FR ON Chapter 1 7 An icon 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
152. ngth Thickness min See also Aspect Ratio Feature and Elongatedness Feature for other shape ratios Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightAWVidth Thickness min Length ss max ess min Application Example Measure object s elongatedness to provide shape classification 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 149 2 lobe symmetry 3 lobe symmetry 4 lobe symmetry Single Focused Cells ll E X Single Focused Cell eT x Single Focused Cells fet 18 Frequency ta om r Frequency E ma oo 20 Symmeby 4_Ch Application Example Classify different white blood cells based on the morphology of the nuclear image 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 Ent
153. nsation Matrix File 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 V lt gt 10 Getting Started with the IDEAS Application Getting Started with the IDEAS Application This chapter is divided 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 Open one data file the or control Create and save a compensation matrix for the experiment Select focused cells Select single cells or conjugates Select channels for subpopulation markers and gate to define subpopulations
154. nstrument setup data pixel intensity data rif and uncorrected image data from the INSPIRE appli Raw Image File Se SPN cation The IDEAS application uses the rif file to create a compensated image file cif file Contains imagery that has been corrected for variations in the camera background camera gains flow speed and vertical and horizontal positioning between chan nels User creates a cif Serves as a database of images used for feature value from the rif and ctm 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 References the cif 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 daf Data Analysis File Created from the daf User creates new ctm when opening a rif or during acqui sition on a FlowSight Compe
155. nu that appears The Statistics window appears 66 Chapter 4 3 4 5 Edit Statistics Table Insert Colurnn Edit Column Delete Column Delete All Columns Order Columns Copy Statistics Copy Statistics Transposed To display the statistics for the graph select Show statistics To close the Sta tistics Area for the graph select Hide statistics Select the statistics that you want to display The selected statistics will be dis played for each population on the graph The statistics that are supported are the Count Percent Total Percent Gated Percent Concentration count sample vol ume Mean Median Standard Deviation MAD Median Average Deviation RD Mean RD Median CV Mininum Maximum Geometiric Mean Mode var lance and NaN not a number 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 Note The legend contains an entry for each population on the graph If the graph is a scatter plot the 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 e Tomove the legend click and drag it You cannot drag the legend past the b
156. nuclear morphology Cell Cycle Mitosis Creates an analysis template that distinguishes mitotic and apoptotic events Codocalization 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 intemalization 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 Creates an analysis template for measuring texture based on spot counting Spat Cancel Application Wizards General e Open File Wizard Guides you through the process of opening a data file and setting image display mapping in the image gallery e Display Properties Sets image display mapping in the image gallery e Begin Analysis Guides you through finding single focused positive cells Application specific e Apoptosis Wizard Guides you through the process of creating the features and graphs for analyzing apoptosis e Cell Cycle Mitosis Wizard Guides you through the process of creating the features and graphs for analyzing the cell cycle and enumerating mitotic events e Co localization Wizard _14 Chapter 4 Guides you through the process of creating the features and graphs for analyzing the co localization of 2 probes e Internaliza
157. of features available depending on the purpose of the graph 1 Choose the specific Building Block from the drop down menu Building Blocks Select Predetined Building Block Fluorescence Posittyes One Color Fluorescence Posittyes Two Color Focus Single Cell Single Cell Default 2 Choose the population s to graph Gr me Building Blocks Select Predefined Building Black Fluorescence Positives One Color wt Use the control key to select multiple populations E All B 03 Single celle Sra Ae apoptotic 3 Choose the X Axis Feature and the Y Axis feature if applicable 33 Getting Started with the IDEAS Application Title and Axes Ses Feature Intensity MIC COT Intensity MEC CAO Intensity ML Cho Intensity ML ChOS 1 Axis Label Intensity MC Cho Y Ayie Feature LUntensity MEC ChOB 4 ClickOK 5 The graph is added to the analysis area Advanced Analysis Opening data files saving Data Files Overview of Compensation Creating a New Compensation Matrix File Viewing Sample Information Merging Data Files Creating New Data Files Batch Processing 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 in
158. ogarithmic or exponential click drag the yellow cross e Torestore the mapping to a linear curve Click Set Linear Curve e Tosee 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 e 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 i arual Image Display Mapping Asis Scale Set Range to Piel 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 included during acquisition of the file _ 58 Chapter 4 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 exist
159. ole or in part or permit or authorize a third party to do so except 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 1s 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 A
160. om 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 p zoom out po reset zoom To show saturation e Click the Show Saturation Color toolbar button Saturated pixels in images if any appear in red a 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 set tings are described below ee Getting Started with the IDEAS Application 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 G
161. on on a graph to cancel the cre Ls Pointer Tool ation of a region Allows you to create a population of hand picked objects For more infor Tagging Tool mation see Creating Tagged Populations le New Histogram Tool Creates a new histogram New Scatter Plot Too Creates a new scatter plot Refer to 62 Chapter 4 Tool Description 2 i gt Populations Statistics Greaes a iable tecisilay rondan stai able On l 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 Text Tool to the Analysis Area Draws a horizontal line on a histogram to define a region z Line Region Tool Rectangle Region Tool Draws a rectangular region on a scatter plot b 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 com plete the region Wizards Tool Short cut to using Wizards for guided analysis Building Blocks Tool Short cut to using Building Blocks for guided analysis Select All Tool Selects all panels in the analysis area Tiles graphs in the analysis area after changing the size of the analysis Tile Graphs Tool area to fit all graphs to the new space E Polygon Region Tool L Layout Tools Switches the layoutof the image gallery and
162. or histogram to identify as many populations as you want This step will be repeated until you choose Skip 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 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 oh es Getting Started with the IDEAS Application QQ BsSiSislx Fa aago Normalized Frequency m N el 6 50 Channel 6 Asea_Threshold M06 Chann tes 1 Ses 24 Bright Detail Intensity R3_MC_Channel 6 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 Co localization Wizard The co localization wizard will
163. ordance with this sec tion The failure by either party to enforce any provision of this Agreement will not con stitute a waiver of future enforcement of that or any other provision Any waiver modification or amendment of any provision of this Agreement will be effective only if in writing and signed by authorized representatives of both parties If any provision of this Agreement is held to be unenforceable or invalid that provision will be enforced to the maximum extent possible and the other provisions will remain in full force and effect This Agreement is the complete and exclusive understanding and agreement between the parties regarding its subject matter and supersedes all proposals understandings or com munications between the parties oral or written regarding its subject matter unless you and Amnis have executed a separate agreement Any terms or conditions contained in your purchase order or other ordering document that are inconsistent with or in addition to the terms and conditions of this Agreement are hereby rejected by Amnis and will be deemed null 11 Contact Information If you have any questions regarding this Agreement you may con tact Amnis at 2505 Third Avenue Suite 210 Seattle WA 98121 DURING INSTALLATION IF YOU AGREE TO THE FOREGOING TERMS AND CONDITIONS AND DESIRE TO COMPLETE INSTALLATION OF THE SOFT WARE PLEASE CLICK THE I ACCEPT BUTTON OTHERWISE PLEASE CLICK THE I DO NOT ACCEPT BUTTON AND TH
164. 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 E Export Feature Data Select daf files to process Select features to export 081103 G2A1 shape change MCP1 2defauit dof Area_MO02 Area_MO06 Area_MC Aspect Ratio Intensity_M01_Ch01 Aspect Ratio Intensity_M02_Ch02 Aspect Ratio Intensity_M06_ChO06 Aspect Ratio_M01 Aspect Ratio M02 Aspect Ratio_MO06 Bkod Mean_Ch02 Bkod Mean_Ch06 Bkgd StdDev_Ch01 Select 4 population m Tl sorouety Export to Order by Clipboard Object C Export all used features Text File O Feature 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 111 Getting Started with the IDEAS Application 7 If you haven t defined any populations All is the default To make a new pop ulation refer to Creating Tagged Populations In the Select feature values to export area select features by clicking items in the list or hold down the Ctrl while clicking to select multiple
165. oundary of the graph panel Moving a Graph e With any graph in the Analysis Area you can move it to another location by clicking on the name 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 Focus EK A A ii E E _67 Getting Started with the IDEAS Application 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 on a his togram All other types of regions may be drawn only on a 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 region 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 kJ e Polygon Region button on the Analysis Area toolbar Refer to C
166. p 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 Population Manager zs Populations Properties Sark 092011 X101 unstimulated _1t cif i Name All All RI Dark Mode Color Light Mode Color I Symbol Simple Dot Definition All L_New Delete i 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 icon Defined by e feson The definition of a selected population is shown in the Definition area To edit the display properties of a population O O A W DY 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 3 Within the Population Manager click a population in the Populations list Click Delete A confirmation warning message appears indicating all the dependent pop ulations that will also be deleted Clic
167. pness 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 151 intensities are computed using the 3x3 block around the pixel This is similar to the Gradient RMS calculation with different weighted assignments to the pixel arrays with no background subtraction Example images are shown in the figure below single cells 30 Le co Ta E z a uy 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 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 intensity 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 Gradient Max Feature The Gradient Max feature measures the sharpness quality of an image by detecting largest change of pixel values in the image
168. pper right gt Oo c v e a w pa wu l 0 3 Similarity _NFkB 7 AAD Application Examples Quantify translocation Identify copolarization of two probes 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 MC It is computed using the normalized cross correlation between the two input images Application Examples Used as a mask independent measure of similarity between two images Understanding the System Features The system features do not require a mask Camera Line Number Feature The Camera Line Number feature returns the camera line number values This fea ture is obtained from INSPIRE Application Example 172 Chapter 5 Used in objects per mL feature 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 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 Object Number Feature
169. r 4 Table of Base Features by Category 3 9 O Q r 4 Valley X and Valley Y Features XCorr Feature mn A Q r DF TI o 0 r z p 121 Table of Base Features by Category Table 1 List of Features by category Feature name Size based Features are in microns S Yes Yes _ MO1 M12 MC The size of the mask in square microns Diameter Feature Estimates the diameter of the mask based on Area M01 M12 Height Feature Based on a bounding rectangle the Width is the smaller side and Yes Yes M01 M12 the Height is the longer side of the rectangle ve fe Length Feature Major Axis and Minor Axis Features Describes the widest part of the mask and the narrowest part of the No Yes M01 M12 mask respectively Major Axis Intensity and Minor Axis Intensity Features aae M01_Ch01 Based on a bounding ellipse the Minor Axis is the narrow part and No Yes _ M12 Ch12 the Major Axis is the widest part Minor Axis Major Axis and Minor Axis Features No Yes M01 M12 Perimeter Feature Describes circumference of the mask M01 M12 Spot Area Min Feature The Area of the smallest spot in the mask See also Spot Dis No No tance Min Feature Spot Intensity Min and Spot Intensity Max Features and Spot Count Feature Thickness Max Feature N Yes M01 M12 Describes the longest width of the mask Thicknes
170. r C User stiend App Data Roaming Amnis Cormoration batches E Update automatically when file is selected Defaut Compensation Matrix Files Director C Wsers stiend App Data Roaming Amnis Corporation compensation co E Update automatically when file is selected E Use default data directory Overview of the IDEAS Application 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 directory 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
171. r 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 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 the analysis area Gate on the apoptotic cells with low nuclear area and high brightfield contrast _19 Getting Started with the IDEAS Application 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 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
172. raphs 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 18 Chapter 4 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 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 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 scatte
173. reating Regions on Graphs for more details ce 1 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 Fest led 1 5e4 Zed 2 Click again to complete the region If you are drawing a region on a histogram or scatter plot the Create a Region window appears 3 Name the region 4 Click the colored box to select an alternate color _ 68 Chapter 4 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 that 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 Ly To Draw a region on a Histogram On the Analysis Area toolbar click the Line Region tool 2 Drag the line across the histogram double
174. rimination 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 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 _92 Chapter 4 actin distribution Bias introduced during the selection of truth populations will likely
175. rom the graph panel toolbar To resize a graph e Select the graph s to be resized and then click the sizing button tool small medium or large oy gy ge e 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 _70 Chapter 4 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 region on a graph 1 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
176. ropy H Homogeneity and H Variance Modulation Spot Count and Std Dev Contrast Gradient Max and Gradient RMS are generally used to determine best focus Bright Detail Intensity R3 and Bright detail Intensity R7 Features 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 150 Chapter 5 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 apoptotic cells i mE Ji D LL Low Bright Detail Intensity RS values identity non apoptotic cells Pal High Bright Detail Intensity R3 values identify apoptotic cells 1 l l 0 deJ 1 2e4 1 5e4 bes Jez Bright Detail Intensity AS Threshold M5 ChS 3041 _Ch5 Application Example Identify cells that have bright specks such as Apoptotic cells Contrast Feature The Contrast feature measures the shar
177. rtion 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 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 r Feature Manager 092011 X101 unstimulated_1 daf 8 ee eee x Feature Type Aspect Ratio Intensity_M01_Ch01 Aspect Ratio Intensity_M02_Ch02 vr Benart Batin Intancihs MN CLN Sort features by a 3 ts 3 a Delete Edt Add Multiple Features 2 Choose an icon to sort the features Table 1 Sorting Features A sors features alphabetically BI fos features based on the images used e Son
178. rument using the Amnis INSPIRE application Next the IDEAS application processes and analyzes the image data The IDEAS application contains the algorithms and tools that are needed to analyze the imagery Preprocessing algorithms and tools correct for instrument biases including flow speed variations spatial alignments illumination irregularities and camera back ground Compensation for spectral crosstalk is calculated from control files and applied to experimental files A compensation matrix may be created on the instru ment during acquisition see your INSPIRE or FlowSight user manual for details After the preprocessing completes the IDEAS application allows for the inter rogation of the image data segmenting out cells and other objects of interest Using a default template the application calculates the values for standard features to be used in Subsequent analyses Guided analysis for many common applications is available through the use of wizards Finally the application displays imagery and feature calculation results and it defines cell populations in a host of plots and his tograms Data Analysis Tools Data in the IDEAS application can be further explored by using the data analysis tools For example populations of cells can be identified by drawing regions on his tograms or scatter plots or by tagging individual objects The IDEAS application pro vides standard distribution statistics for all defined populations In
179. s your sole and exclusive remedy and Amnis entire liability for 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 _ jy End User License Agreement 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 YO
180. s Min Feature N Describes the shortest width of the mask Width Feature Based on a bounding rectangle the Width is the smaller side and the Height is the longer side of the rectangle Location Features are in X Y pixel coordinates from an origin in the upper left corner pixels or contour Angle Feature No N The angle of the major axis from a horizontal plane in radians O Angle Feature The angle of the major axis intensity from a horizontal plane in radi No No Centroid Features The central tendency of the pixels along the X Axis and Y Axis No Yes M01 M12 respectively The central tendency of the pixels along the X Axis and Y Axis M12_Ch12 122 O O Yes M01 M12 Yes Yes M01 M12 Chapter 5 In Expanded Mask_Image In Default Feature name The distance between the X or Y Centroids of two images The distance between the Centroids of two images Max Contour Position Feature The location of the contour in the cell that has the highest intensity concentration E a a a a oe a we a Spot Distance Min Feature The shortest distance between two components spots See also Ves No Spot Area Min Feature Spot Intensity Min and Spot Intensity Max Features and Spot Count Feature Valley X and Valley Y Features No No E The X Y coordinates of the minimum intensity within the skeletal lines that are used when creating
181. s 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 cv s See coefficient of variation CV 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 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 channels A type of illumination in which the sample is illuminated at angles that 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 See fluorescent in situ hybridization FISH A fluorescent dye used to label cellular constituents or specific fluorochrome probes of cellular constituents Light emitted by a fluorescent dye following excitation fluorescence com The adjustments made to remove the fluorescence emissions of a darkfield 189 Glossary A physical mapping approach that uses fluorescent tags to detect the hybridizatio
182. s button on the image panel toolbar T cell a The Display Properties window appears e Forsingle channel image you can change the displayed mask and adjust the display intensity mapping For more information see Setting the Image Gal lery Properties _74 Chapter 4 Select a different mask to display Valley MO5 Drag 2 Minimum Pixel Intensity 17 Maximum Pixel Intensity 187 Automatic Manual Image Display Mapping A Axis Scale Set Range to Pixel Data Full Scale Set Linear Curve Autoscale Cancel e Foracomposite image you can change the images in the composite and adjust the percent contribution of each image see Setting the Image Gallery Properties a Getting Started with the IDEAS Application ow Display Properties Object 42 Composite ADAP A Name PRR aes Object 0 ADAP Actin T cell ADAP 100 pent Actin 100 ADAP i T cell 100 Add Image Remove Image 2 Click OK To show or hide the 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 e Toe 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 CUa e View
183. s features based on the masks used Sorts features by category such as size loca tion shape texture signal strength and system E Sorts by base features such as area aspect Getting Started with the IDEAS Application 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 Delete this text and replace it with your own content 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 1 Click New in the Feature Manager The right hand area of the Feature Manager is enabled Feature Type Single Angle v Combined Name _ _ _ Mask Combined Mask z 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 v C Combined Name Mask Combined Mask 7 Image 1 Channel 1 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 _
184. s 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 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 e Spot Intensity Max is the Raw Mean Pixel of spot 2 e Spot Intensity Min is the Raw Mean Pixel value of spot 3 Application Example InFISH 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 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 132 Chapter 5 Selecting 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 Le n gth a pikitia o Shape ratio Thickness min Length Thickness min Thickness Min Feature Thickness Min measures the smalles
185. s 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 187 Troubleshooting 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 following procedure 1 Select Tools gt Merge rif Files 2 Click Add Files to select the single rif file 3 Click OK Enter an
186. scence 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 and must reside in the same directory as the corresponding cif file The daf file contains e Feature definitions e Population definitions e Calculated feature values e Image display settings Chapter 3 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
187. se in reports In the Image Gallery right click an image that you are interested in A menu appears Add Image to Analysis Area Show Masks Color Off Show Saturation Color Copy Image to Clipboard Copy Object Images to Clipboard Copy Gallery Column to Clipboard Copy Gallery to Clipboard To copy the single channel image to the Clipboard click Copy Image to Clip board To copy all of the channel images of 1 object to the Clipboard click Copy Object Images to Clipboard To copy the single channel image for all of the displayed images to the Clip board click Copy Gallery Column to Clipboard To copy all the visible images in the Image Gallery to the Clipboard click Copy Gallery to Clipboard 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 61 Getting Started with the IDEAS Application The graphs are created into panels of a default size and can be re sized by dragging a corner 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 panels exceeds the space available on the window The panels c
188. selecting Generate Statistics Report from the Reports menu or during batch proc essing 109 Getting Started with the IDEAS Application 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 Generate a Statistics Report 1 Select Reports gt Generate Statistics Report The current daf file appears in the window with the specified statistics columns Generate Statistics Report Select a daf or template file that contains a statistics report definition 092011 X101 unstimulated_t Report title 092011 X101 unstimulated_1t dat_stats 7 golo Similarity Similarity Files Count All Count R1 Count A2 Count R3 Count A4 Gated R4 Median R3 MAD R3 Add Files Remowe Files 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
189. sis Analysis Compensation Tools Options Reports Windows Help Population 2n single T cell amp double amp Focus amp conjugates View Custom ADAP ActinT in I ADAP ActinT 54 x Getting Started with the IDEAS Application Image Gallery Tools Table 1 Image Gallery Tools Allows you to create a population of hand Ko Tagging Mode Tool picked objects See Creating Tagged Pop ulations a Provides custom display features See Set Image Gallery Properties Tool Iting the Image Gallery Properties FI show segmentation Mask Too fiche masks on the imagery See Show Segmentation Mask Tool Using the Image Gallery g Sets the Image Gallery color See Using Show Color Tool the Image Gallery Click on the tool and it will show any sat a Show Saturation Color Tool urated pixels will turn red See Using the Image Gallery Zoom in or out and reset zoom on the image gallery See Using the Image Gallery To view the imagery for a population 1 Inthe Population drop down menu of the Image Gallery click the population that you want The list includes all the populations as well as the currently selected bin from a histogram To create a population refer to Creating Tagged Pop ulations 2 To select 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 s
190. stance of the IDEAS application File Open Save Data Analysis File daf Save as Data Analysis File Used Features Only Save as Template ast Save All Close Exit 3A Chapter 4 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 the 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 Select File To Load Look in gt rifs v O 2 egm N 0 0ng_2_9
191. t 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 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 cells 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 CD 14 green 29 Getting Started with the IDEAS Application Eg Tag PATEE Fierro F eq gy bi z Cmui hpa L
192. t A 8 BF Draa5 ADAP Actin T BF Dreas Intensity_ADAP 16 O 100 200 300 400 500 600 700 40 60 1e3 0 1e3 1e4 Area _BF Gradient RMS_BF Intensity_Actin Population Count XGated All 10723 100 nikies Qheh ADAP Actin T cell conjugates 1971 18 4 o D Qa GA cB o o h Fi a c o E 2 T pe D a Q lt 1 Image Gallery Intensity _MC_Ch03 Intensity _MC_Ch03 Intensity_MC_Ch03 Intensity_MC Mean Std Dev NaNs Mean 1 542e 004 1 555e 004 0 1 381e 004 2 284e 004 1 294e 004 0 2 345e 004 ocus 2 253e 004 1 302e 004 0 2 3116 004 annotations can be entered in a text box 2 253e 004 6839 24 0 3 167e 004 as aaea e The Image Gallery displays the images of populations of cells segmentation masks and composite images For more information refer to Using the Image Gallery e The Analysis Areadisplays plots and distributions of cellular feature values Individual images text panels and feature values for objects and populations in tabular form For more information refer to Overview of the Analysis Area 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 applicat
193. t 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 107 Getting Started with the IDEAS Application The Statistics Report Definition appears Statistics Report Definition pS Statistic Columns Add Columns Delete Columns Report tile 092011 X101 unstimulated_1t dat_stats Enter a Report title Under the Statistic Columns clickAdd Columns The Add Statistic Report Col umn window opens Select the statistic s in the Statistics list e Count the absolute count of the populations e Y Total percentage of a population as a percentage of All e Gated the percent of one population as a percentage of another but not used for tagged populations e the percentage of one population as a percentage of another also is used for tagged populations e QObjects mL the concentration of the population in the sample run e CV the coefficient variable e Geometric Mean standard statistical definition e Maximum standard statistical definition e Mean standard statistical definition e Median standard statistical definition e MAD standard statistical definitio
194. t 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 amp 7 fe oc fk 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 D x Chapter 4 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 a c To examine a line profile or the statistics for an area of an image e 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 line profile the wavy line in the image panel represents the pixel intensity at each position along the red line of the box s JA Getting Started with the IDEAS Application Minimum 32 Maximum 275 Mean 103 65 Std Dev 65 13 Area 433 Close Statistics To change the display properties of an image 1 Click the Channel Display Propertie
195. t the application oF O N 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 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 not saturated preferably single color If single color cells are not available choose cells with a distinct staining pattern in the peak channel Create Intensity scatter plots of adjacent channels in order to observe the over or under compensation Identify the matrix values that need adjusting by inspecting the scatter plots and images Each col
196. t 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 information Thickness max Le n gth oe AA Si Shape ratio Thickness min Length Thickness min 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 133 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 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 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 the image frame Angle Intensity Feature Angle Intensity is the angle of the major axis intensity from a horizontal plane in radi ans 134 Chapter 5 Brightfield 7AAD Composite Horizontal Plan amp Major Axis _ Major A
197. that are not in the original population _ 99 Getting Started with the IDEAS Application Affect the order of operations Use the parentheses toolbar buttons i Click the left arrow button on the toolbar _ Remove an item from the end of the definition see Creating Tagged Populations for information about 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 Within the Image Gallery click the Tagging Mode toolbar button to begin Ro The Tagged Population window appears a Tagged Populations Tagged Populations Image viewing mode Update existing P00 All Channels x Create new 2 Select either Update existing or Create New e ToCreate New double click images within the Image Gallery and select Save Create a new population name and display properties in the Create a New Population window 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 t
198. the cif file in the 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 2 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 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 spectrum of the probe extends beyond the channel 3
199. the Valley Mask Shape Features define the mask shape and have units that vary with the feature Aspect Ratio Feature The ratio of the Minor Axis divided by the Major Axis M01 M12 Aspect Ratio Intensity Feature M01 Ch01 The ratio of the Minor Axis Intensity divided by the Major Axis Inten Yes Yes M1 2 Ch 12 Circularity Feature No The degree of the mask s deviation from a circle Compactness Feature No No Describes the density of intensities within the object Elongatedness Feature The ratio of the Height Width which use the bounding box ile Lobe Count Feature No No The number of lobes in a cell Also see Symmetry Shape Ratio Feature ratio of Thickness Min Length features ee 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 four fold variation of the Shapes See also Lobe Count Feature fatthegraniartyarconpientyottiemage Texture cate the granularity or complexity of the image Bright Detail Intensity R3 and Bright detail Intensity R7 Features The Intensity of the pixels in the bright detail image using a 3 or 7 Ves MC_Ch1 MC __ pixel structuring element Also see Spot Mask for a description Ch6 of the bright detail image Contrast Feature Enumerates changes of pixel values in the image to measure the Yes Yes focus quality of an image Gra
200. 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 _57 Getting Started with the IDEAS Application 2 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 3 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 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 l
201. therefore the Cir cularity value will be low See also Compactness Feature Below is an example using Circularity and Compactness to characterize the shape of peripheral blood mononuclear cells stained with the DNA dye Drag 5 144 Chapter 5 Comp actne ss_Draqs 10 1 5 ei 25 Circu larity Drag 5 Nuclear Application Examples Brighttield Drag 5 Circularity compactness aa Distinguish singlets and dou b 227 0 942 blets Separate circular and non circular shapes Compactness Feature Compactness measures the degree of how well the object is packed together This feature is similar to the Cir cularity 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 See also Circularity Feature Below is an example using Circularity and Compactness to characterize the shape of peripheral blood mononuclear cells stained with the DNA dye Draq 5 145 mo Comp actne ss_Draqs 10 18 Circu larity Drag 5 el 25 Muclear Brighttield Drag 5 Circularity compactness Pa othe arte Fi C 227 0 942 Application Example Differentiate between rounded objects with smooth boundary to less reg ular objects Elongatedness Feature Elongatedness is the ratio of the Height over Width of the object s bounding box See also Width Feature 146 Chapter 5 S
202. thin 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 Rif File Option Setting Advanced Corrections 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 application sup port 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 Soo 7 Apply Bright Field Crosstalk Compensation v Perform correction Corrected during acquisition Fluorescence compensation matrix if cif daf or ctm h01 h02 h06 4 C C c 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 Sslelslslola c C 1 0 0 1 0 0 0 0 0 0 Minimum 20 99 Maximum 23 33 Change Correction Offsets Select Change Compensation Matrix Choose EDF Kemels Reference Camera 1 1 V Perform correction Excitation Kemels Reference Camera 2 9 1 ope 8 BEEBE SsE amp s Offsets 0 29 0 05 0 14 0 48 1 47 0 08 Horizontal 0 08 0 32 0 13 0 1 0 0 28 Offsets 0 01 0 08 0 19 0 38 0 02 0 38 Change Alignment Offsets V Corrected during acquisition V Perform nomaiization V Apply cell classifiers v Separate single objects v Erase non4ramed objects v Remove clipped objects Minimu m
203. tion created from each file N of W 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 Onthe Tools menu click Create Data File from Populations The Create cif and or rif From Populations window appears _45 Getting Started with the IDEAS Application in Create cif and or rif From Populations o 8 x Select populations Sari NFkB Fite Dq5 No LPS Analyzed _2 cif All f R1 R2 3 0 R3 New Raw Image File nf 3 New Compensated Image File cif J _ Cancel 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 sele
204. tion Wizard Guides you through the process of creating the features and graphs for analyzing the internalization of a probe e Nuclear Localization Wizard Guides you through the process of creating the features and graphs for analyzing the nuclear localization of a probe e 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 e 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 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 i LE C Instructions Draw a region around the single cells on the Area vs Aspect Ratio scatter plot 1 Select nuclear image to create a single cells population 2 Gate single cells Or 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
205. 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 _ 83 Getting Started with the IDEAS Application P Mask Manager x a a ee Masks C Name 01 M02 M03 M04 i mos Definition i M06 M01 2 Click a mask in the Masks list to view the definition in the Definition area 3 Click Close To edit a mask function 1 Inthe Mask Manager window select the mask that contains the function you want to edit Click Edit 3 Remove the definition for the combined mask using the back arrow tool as needed Refer to Creating New Masks with the Mask Manager for more infor mation on the definition tools NO 4 Orclick the Function button on the toolbar for a function mask The Define Mask Function window appears Refer to Creating New Masks with the Mask Man ager for more information Function 5 Click OK when finished Example 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 _ 84 Chapter 4 Gre
206. top 2Cam DEMO NFkB Translocation NFKB Fite Dq5 No LPS Analyzed _2 Version 40 4270 No Objects 4721 Sample NFkB Fite Dq5 No LPS Analyzed _2 Show Sample Name in Graph Titles v 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 Detection tab lmageStreamX only Cell classifier settings during acquisition Population tab FlowSight 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 Batch Processing Batch processing allows you to automatically 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 _47 Getting Started with the IDEAS Application The Batches window appears It lists a record of all batches you have proc essed Batches Batches to Run Batch1 Processed Batches a Batch 6 29 2011 11 05 33 AM Batch 5 3 2011 10 38 34 AM Batch 5 3 2011 9 51 14 AM Batch 5 3 2011 9 41 07
207. troid 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 and 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 Understanding the IDEAS Features and Masks This section contains the following subsections which describe the features that the IDEAS application uses for data analysis Table of Base Features by Category Table of Base Features by Category Table of Base Features by Category 118 Chapter 5 Understanding the Location Features Understandin
208. tter plot tool 62 Scatter plots show hide populations 71 Screen resolution 4 Select All Panels Tool 63 Shape Change using a wizard 28 Single Cell Building Block 32 default 32 Size SSC Building Block default 32 Software requirements 3 Spot Count using a wizard 30 T Tagging tool 54 62 Template file about 9 default 2 definition 3 saving 41 Text tool 63 TIFs creating 112 203 Two color scatter plot building block Views custom Volume information Wizards Apoptosis Begin Analysis Cell Cycle Mitosis Co localization Display Propertes Internalization list Nuclear Localization Open File Shape Change Spot Count Tool Workflow data analysis workflow Zoom Image Gallery 204 Index 32 58 47 18 17 20 22 16 24 14 26 15 28 30 63 54
209. tures 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 6 Chapter 4 expression that includes one or more single features that exist in the 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 po
210. umn 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 186 Chapter Appendix A Compensation Matrix Select a compensation matrix 081109 G241 shape change MCP1_2 cif ChO1 ChO2 ChO3 ChO4 ChOS ChOB ChOF ChOS ChO9 ChIO Chi Ch12 1 0 048 0 0 0o03 1 0 ou DHI 0 085 0 017 0 044 0 001 0 002 0 001 mm mm mm So oo So oo co o oyloaol ol o o oa oy es oyoa o yoloaol ol o o o s joy oay oa o yoyloaloaol o o esjoy o y oay oa oy yoloal oa o ye Joy o oy oay a o yoyloaloa e o o o oy oay a o ol oal ye o o o y o o oay a oy yoy eTo olo oy o oy oay ya oo e Aa aloo oo alo ia jo oalo o o o o o oay a 0 0 0 0 0 0 0 0 Preview a file with this matris applied Select an existing rif file Overwite preview tiles Select a population from the current file aooo 7 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 image e Overcompensation crosstalk coefficient is too high Plots Intensity mean for the single color positive population i
211. xis 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 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 135 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 Centroid X Y A Centroid X Y PE A sa os Yinensiy shin ___sa ___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 Centroi
212. y Note Only regions that have the same scaling types ie linear log as the graph may be added OK Cancel 2 Select the regions that you want to appear on the graph 3 Clear the regions that you want to remove from the graph 4 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 2 Select the populations that you want to appear on the graph 3 Clear the populations that you want to remove from the graph fee 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 Th Getting Started with the IDEAS Application population 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 populations shown change the characteristics of the population s in the pop ulation manager Analyzing Individual Images To analyze an image in more detail place the image in the 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 contex
213. y 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 a new 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 _ 80 Chapter 4 Mask Manager Definition M01 3 Click Function The Define Mask Function window appears with 15 available masks to use e Dilate See Dilate Mask e Erode See Erode Mask e Fill See Fill Mask e Inspire See Inspire Mask e Intensity See Intensity Mask Intensity Mask e Interface See Interface Mask e Morphology See Morphology Mask e Object See Object Mask e Peak See Peak Mask e Range See Range Mask e Skeleton See Skeleton Mask e Spot See Spot Mask e System See System Mask e Threshold See Threshold Mask e Valley See Valley Mask 81 N Getting Started with the IDEAS Application SA l Select an object and image to display Link inputs Object Image 17 ChM Number of Pixels w Pp py 0 2 Ae ele eet 4 6 8 10 12 14 16 19 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 Toview a different object i
214. yscale Images WEE Ee A AND ROT B 1 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 2 Select Analysis gt Masks Click New 4 Click on the Function toolbar button to adjust the mask that will define the whole cell The Define Mask Function window appears Q 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 using 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 O ON OA a 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

IDEAS User Manual 5.0

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