Home

IDEAS User Manual 4.0 - Department of Flow and Image Cytometry

1. 2 4 UNDERSTANDING THE IDEAS FEATURES AND MASKS Overview of the IDEAS Features and Masks 000 ADOOC PEIEE eae aaa ee mn ene a PO ea oe ec eC OP a The Base Features at a Glance sorted Alphabetically The Base Features ata Glince Dy Category 24 22 345 25 24285262 e Understanding the Detailed Feature Descriptions Understanding the Size Features 4 suc atte kes Bowe Gee ae Pees epee es Ara A es anu ah ve tse ak to Be ae ee Ss es GE te he ee es E ek eh ek Diameter Teatre eadra a Sst ys ee a po aie ee a Eki h Te CARES ma er arel cial et AN E uats ocd atte my dda doe ect wets Eeng Catule 12 ara 2 4 he Ba ees Se ened Be ERA eae Se eas ee Major Axis and Minor Axis Features 0 00 00000 eee eee Major Axis Intensity and Minor Axis Intensity Features Perimeter Feature nanana eres heh oes donee es acne CG dy tne eh aes oh Sporrea MILT Teu exis oA gee sng wae HG Ale HOGA Gas Thickness Max Peura x co Sara ce be Bete ie ne ew he He ee a a OK ee AK a TMG KNESS Min Feature 2an5 0 Ses date Roos inde eh dae a ee ade ed a Widi Ferre wrae doue eda cer evans cele th cass car nl caso tte e tells AEA Wnderstandms the Locator Features 3 5 scl ol oho ho RGSS ASO Pee Petre ee cade Goo he GA a he See Oe os So ois Seas Angle Intensity Feature cain ees Secs Salles fotlaky aise Centroid X and Centroid Y Features 000 ee a Centroid X Intensity and Centroid
2. i EX B n 5 The coefficient value is automatically recalculated when a new population is selelcted Repeat these steps as required to redefine the coefficients 7 Click Preview Images to view individual objects with corrections applied Double click on an image to add it to the preview window Note the corrections are only applied to on camera channels For example if the object is brightest in channel 3 on the first camera only channels 1 6 are shown corrected for that object When the matrix appears satisfactory click Finish oe o oss area fo ous faos o aoe aoz e a 005 ool O26 nent S o a os oa eee a Cho 2 Positive ChHO8 8 Positive cha 3 Posiive s 0218101101 THF1 merged_comp_control matri All fen oO 2 Positive fee A 3 Positive fen A 4 Positive fen A 5 Positive fen A 7 Positive haee A 6 Positive fen oo 11_ Positive a oO 12_Pasitive Paa Co Gi Enter a name for the compensation matrix file ctm and click Save 47 GETTING STARTED WITH THE IDEAS APPLICATION Save As Compensation Matrix ctm File Save ini E compensation 2 compmatrixJune2O ctm compmatrixJunees ctm My Recent Documents Desktop My Documents My Computer hy Network File name Save Places Save as type compensation matris files ctr r Note The matrix is saved as a compensation matrix file ctm file This file con
3. Quantify capping of cell surface antigens 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 accommodate 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 Womad Frequency 4 Marha io boar Palon Inka Mask Channel 3 Channel 3 APPLICATION EXAMPLE Used in conjunction with the Internalization feature to determine the dis tribution of intensity within a cell UNDERSTANDING THE IDEAS FEATURES AND MASKS 149 RAW CENTROID X AND RAW CENTROID Y FEATURES The centroid X and Y of the original position of the image during acquisition before it was centered IDEAS Data analyzed in IDEAS versions 4 0 or later cut and center objects that were collected as one image in INSPIRE 150 UNDERSTANDING THE IDEAS FEATURES AND MASKS SPOT DISTANCE MIN FEATURE The Spot Distance Min feature provides the shortest distance in microns between two spots connected components in a spot or peak mask This is one of four features that can be used to identify objects with spots that are close together dim bright or small when counting spots in an image To
4. 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 202 UNDERSTANDING THE IDEAS FEATURES AND MASKS SYSTEM MASK The System mask segments objects in an image based on a probability model of how pixels should be grouped together The user sets a weight value that defines a loose or tight grouping A low weight value groups in a more permissive manner Shown is an example of a cell with a apoptotic bleb that is not masked with the System 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 203 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 Intensity Mask on page 196 In the example below cell 10678 1s bright and cell 11992 is dim The 50 Threshold mask is similar for both images whereas the Intensity mask 250 is quite different since only a few pixels in the dim image are greater than 250 counts while most of the m
5. Identify single cells vs doublets Cell classification based on shape change Identify recently divided cells in mitosis UNDERSTANDING THE IDEAS FEATURES AND MASKS 155 ASPECT RATIO INTENSITY FEATURE Aspect Ratio M4 Aspect Ratio Intensity is the Minor Axis Intensity divided by the Major Axis Intensity See also Major Axis Intensity and Minor Axis Intensity Features on page 138 The figure below illustrates the difference between Aspect Ratio Intensity and Aspect Ratio See also Aspect Ratio Feature on page 154 Minor A xis Intensity 6 57 Aspect Ratio 0 76 Major 4 oot ir Aspect Fatio Intensity M4 Aspect Ratio 0 57 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 measurement 1s 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 theref
6. 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 six channel masks and a NMC mask that is everything outside of MC The following illustration shows two channel masks UNDERSTANDING THE IDEAS FEATURES AND MASKS 193 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 Object Peak Range Skeleton and Valley Each of the functions masks are defined here Refer to Using the Mask Manager on page 94 for more details about how to create new masks 194 UNDERSTANDING THE IDEAS FEATURES AND MASKS LIST OF FUNCTION MASKS The IDEAS application provides thirteen functions that can be used 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 starting mask Morphology Erode 3 pixels FILL MASK The Fill mask fills in any holes in the starting mask UNDERSTANDING THE IDEAS FEATURES AND MASKS 195 INSPIRE MASK The Inspire mask masks pixels
7. 10 OVERVIEW OF THE IDEAS APPLICATION INSPIRE IDEAS Data Collection spectral Compensation Data Analysis Raw Compensated Data image Apply image Analysis Experimental sampe Filg COMDAROTADT File File TAT n 1 fee E CO pe ce maii PLT Tey he i henna E Batch Processing Apel chen im O Connie further Aik Lerniguete fer Gach paeen wach pareking App Conner tar rsd fie for earch pronening OVERVIEW OF DATA ANALYSIS WORKFLOW 1 Create a compensation matrix using the single color control files Open an experi mental 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 template within the daf file and save the data file and an anlaysis template Note this is usually done on the positive and negative controls to create the appropriate 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 THE IDEAS APPLICATION 11 OVERVIEW OF COMPENSATION ANALYSIS TOOLS AND FILE STRUCTURE Data Acquisition and Compensation on page 12 Data Analysis Tools on page 12 Interface of the IDEAS Application on page 13 Overview of the Data File Types on page 13 DATA A
8. 76 USING THE DATA ANALYSIS TOOLS AB amp AT elas bala fst Baa QUG 6 ESE x R84 RI rb x 1 2 N x 1 pay 2 9 Bg 3 R amp F 2 D w 6 6 E D D 5 4 a 30 fal 5 3 z z 0 I l i O A TT Pagina A AEAN ee TT i D A rimm LIME 1 NAN J3 2e3 le3 O 1e3 2e3 3e3 4e3 1e4 13500 0 5001e3 5e2e3 1e4 Je4 1e3 le2 41 e2 163 5_Intensty 3400 5_Intensty 3400 5_Intensity 3400 Scaling Scaling Scaling Auto C Manual Auto C Manual Auto C Manual X Axis X Axis x Axis Minimum Minimum Minimum Maximum Maximum Maximum Linear C Linear C Linear C Log X gt Log X gt 1000 l Log X gt h10 10 To modify the display characteristics of each population or to change the layering order click Display Properties The Display Properties window opens z Display Properties E DR Modify the display characteristics of populations andor change the layering order Population Line Style m Solid reer Solid jal Solid Populations Histogram Properties Y Axis Units Bin count Frequency Normalized Frequency w 11 Arrange the layering of the populations with the up and down arrows to allow them to be displayed 12 Ifyou want click Populations to open the Population Manager For more information see Using the Population Manager on page 108 13 If you are creating a histogram overlay you can customize it by performing the following steps e To fill or n
9. Minor Axis see Major Axis Size Raw Centroid X and Raw Cen Location Intensity and Minor Axis troid Y Features on page 150 Intensity Features on page 138 Modulation Feature on Texture Spot Intensity Min and Spot Signal Strength page 169 Intensity Max Features on page 183 Object Number Feature on System Std Dev Feature on page 171 Texture page 191 Objects ml Feature on System Symmetry 2 3 4 Features on Texture page 191 page 162 Objects sec Feature on System Thickness Max Feature on Size page 192 page 141 Perimeter Feature on Size Thickness Min Feature on Size page 139 page 141 Raw Intensity Feature on Signal Strength Time Feature on page 192 System page 177 Raw Max Pixel Feature on Signal Strength Valley X and Valley Y Features Location page 177 on page 152 Raw Mean Pixel Feature on Signal Strength Width Feature on page 142 Size page 179 Raw Min Pixel Feature on Signal Strength XCorr Feature on page 190 Comparison page 180 UNDERSTANDING THE IDEAS FEATURES AND MASKS 127 THE BASE FEATURES AT A GLANCE BY CATEGORY TABLE 2 LIST OF FEATURES BY CATEGORY FEATURE FEATURE NAME CATEGORY SIZE Size based Features are in microns Area Feature on page 134 The size of the mask 1n square microns Diameter Feature on page 135 Estimates the diameter of the mask based on Area Height Feature on page
10. on page 40 In the Select a template or data analysis file ast daf field select a template 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 Name the output files with a new name if necessary You may change the number of objects to load in the box under Enter the num ber of objects to process The default value is the number of objects in the file GETTING STARTED WITH THE IDEAS APPLICATION 31 Tip you can select a smaller number than the maximum if you have a large number of objects to load This helps save time for creating a template file The IDEAS application randomly loads the specified number of objects within the file 7 Click OK The application then creates the cif and daf files and the daf file is loaded into the Image Analysis area 32 GETTING STARTED WITH THE IDEAS APPLICATION 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 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 z Opening file 1000ng 60_20_3 rif Spectral Compensation Camera Background Auto generate Brightfield compensation values to override matrix Perform correction Select a matrix to perform fluores
11. Gradient Max_MO4_ Ch 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_MO04 Ch4_5 H Energy Ste_MO4_Ch4_5 H Entropy Mean M04 Cht_5 H Entropy Std_MO4 Ch4_5 H Homogeneity Mean M04 Ch4_5 H Homogeneity Std_MO4 Ch4_5 Delete Selected Features Delete any features you do not want to calculate Click OK when finished The new features are added to the list in the feature man ager Close the Add Features window Close the Feature Manager The new features are calculated when the feature man ager closes USING THE DATA ANALYSIS TOOLS 105 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 Hilti XZ C Set Default Name OK Cancel 3 Enter the feature name in the Name box or use Set Default Name after you have created your expression The default name is the name of the definition cre ated 4 Use the toolbar to build a definition mathematical expression of features and op erators TABLE 5 COMBINED FEATURE TASKS AND TOOLBAR TASK Add a feature to the definition Add an operator or a parenthesis to
12. Step Progress ee 1 Select data file to open This wizard will take You through the steps involved in opening ImageStream data files There are 3 types of data files that can be opened in IDEAS Raw Image File rif uncompensated data from the instrument Compensated Image File cif compensated data Data Analysis File daf analyzed data Click the folder button to select the file to open Po Once a data file is open you may begin analysis GETTING STARTED WITH THE IDEAS APPLICATION 19 DISPLAY PROPERTIES Once you have an open data file this wizard is available from the Guided Analysis menu or from the wizard icon This wizard will set the image display mapping for the channel images you select Brightfield and scatter images will be automatically adjusted This wizard is also incorporated into the first steps of the application specific wizards TO BEGIN SELECT WIZARDS FROM THE GUIDED ANALYIS MENU OR CLICK THE WIZARD ICON TO THE LEFT OF THE ANALYSIS AREA oe The Wizards window opens Wizards Select the wizard to use for analysis Description Name Open File Opening ImageStream data files Display Properties Automatically sets image display properties Creates an analysis template for identifying apoptotic evente based Apoptosis on brightfield and nuclear morphology Creates an analysis template that distinguishes mitotic and apoptotic Cell Cycle Mitosis events r Dou
13. The Camera Line Number feature returns the camera line number values This feature is obtained from INSPIRE APPLICATION EXAMPLE Used in objects per mL feature CAMERA TIMER FEATURE The Camera Timer feature returns the camera timer values that are in ticks This feature 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 in a file APPLICATION EXAMPLE Determine consistency of flow OBJECT NUMBER FEATURE 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 volume APPLICATION EXAMPLE Monitor the object flow during the run Note Use the statistic Concentra tion to obtain objects ml of a population UNDERSTANDING THE IDEAS FEATURES AND MASKS 191 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 Concentra tion to obtain objects ml of a population TIME FEATURE The Time feature returns the camera timer values that are in ti
14. tains the compensation values and can be opened later for editing To provide the values for fluorescence compensation you select a ctm file when opening a rif file See Opening a rif file on page 29 for more information PREVIEW 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 Open gt Compensation Matrix from the File menu or Select View Edit com pensation matrix from the Compensation menu to view edit or preview the matrix on image data The matrix values are displayed in a table and may be edited 48 GETTING STARTED WITH THE IDEAS APPLICATION Compensation Matrix BEE Select a compensation matris Color Compensation 021810 s101 3L 488 PE PETR PECy5S DAPI PO 64 APCCu ctm Chol ChO2 ChOos Cho4 ChOb Cho ChO3 Chi Chi 0 084 0 08 0 026 0 002 0 121 0 076 0 036 0 008 1 0 235 0 015 0 006 O512 1 0 012 0 005 0 113 O24 0 005 0 011 0 1 0 132 0 005 0 004 0 0193 0 015 1 0 051 0 087 0 02 0 359 0 05 0 174 0 03 0 061 0 045 0 08 0 08 0 026 0 056 0 021 0 026 0 01 1 O02 0 018 0 056 0 267 Z mm m1 l m l So alo AJA Aje Aaaa AAJA m S G ma A E ma S i G a S B m G G aa S GE a B G ma D GE aa G GE ma GA GE m omn S G m A i O m B G a S GE ma G G ma S GE ma B G oa G GGE m B GE mn 0 0 0 0 0 0 0 0 0 0 0 Preview a file with this matris applied Select an existing rif
15. 5 5 20 z5 Damekr_Enghksk 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 i Diam eter 2x The images below depicts beads with a uniform diameter of 9 microns In the next figure note that images with longer shapes that have the same area will have the same diameter value Focus Brighttield Brighttield Brighttield Brighttielc Brighttield Brighttielc r APPLICATION EXAMPLE Used to obtain approximate size of the cell UNDERSTANDING THE IDEAS FEATURES AND MASKS 135 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 on page 159 APPLICATION EXAMPLE These features can be used to separate rectangular shaped objects For curved objects measurement 1s more accurately 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 doughnut shape where 1n 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 1s sensitive to the variation of the inp
16. Cell of Interest heck Conjugate wask Cell of Composite BF Techn teed Interface hask eee APPLICATION EXAMPLES Used to quantify synapses in T cell APC antigen presenting cell conju gates UNDERSTANDING THE IDEAS FEATURES AND MASKS 197 MORPHOLOGY MASK The Morphology mask includes all pixels within the outermost image contour This mask which is used in fluorescence images 1s 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 segmentation masks The tight option uses a different set of features to characterize the background which results in a tighter fit around the cell Examples are shown below Image Object Object default tight 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
17. features see Overview of the IDEAS Features and Masks on page 124 When the IDEAS application opens a cif or rif file the application calculates the values of features as defined by the selected template You can refine your template so that it includes only those features of interest for your experiment You use the Feature Manager to examine existing features and to define new ones To gain access to the Feature Manager select Analysis gt Features or select one of the context menus that are available in the histogram and scatter plot panels 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 You create a combined feature by defining a mathematical expression that includes one or more single features Some features such as Area depend on the boundary of a cell These features require you to select a mask that defines the portion of the image to use for the calculation Other features such as Max Pixel depend on pixel
18. population of cells you identify TO BEGIN DOUBLE CLICK ON SHAPE CHANGE Follow the instructions to open and analyze your file The analysis includes e Opening the data file e Setting the display properties e Gating on single cells e Gating on focused cells e Gating on fluorescent positive cells e Creating and graphing the feature Circularity of a surface stain or brightfield image e Creating a statistics report Shape change is measured in the final graph presented in the wizard Cells with low circularity scores have a highly variable radius In this example monocytes in whole blood were stained with CD14 green Eg 14g ESETE E Aioreured forga La r 1m H Cenimy Mophatog a CNL For a more thorough explanation of the Circularity feature see Circularity Feature on page 156 26 GETTING STARTED WITH THE IDEAS APPLICATION 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 eraph For more information on creating graphs see Creating Graphs on page 75 TO BEGIN CHOOSE BUILDING BLOCKS FROM THE GUIDED ANALYSIS MENU OR CLICK ON THE BUILDING BLOCKS ICON TO THE LEFT OF THE ANALYSIS AREA The Building Blocks window opens This window is used to define a graph w
19. the definition Add a number to the definition 106 USING THE DATA ANALYSIS TOOLS TOOLBAR Double click the feature in the Features list Or single click the feature in the Features list and select click the leftmost down arrow button on the toolbar Click the corresponding button on the toolbar x 7 gt Enter the number in the box and then click the correspond ing down arrow button y 314 If the area is greyed out an operator must be selected first TABLE 5 COMBINED FEATURE TASKS AND TOOLBAR TASK TOOLBAR Adda function to Select the function in the list and then click the correspond the definition ing 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 Remove an item Click the left arrow button on the toolbar from the end of the definition el 5 Click OK 6 Click Close Note When you close the Feature Manager the IDEAS application calculates val ues 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
20. 1 Intercellular Muclear Intercellular Muclear g g 1 1 Observe the system masks in the Image Gallery Since the system 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 intracellular and nuclear masks need to be refined Start by creating morphology contour masks for both channel images because the Morphology mask is designed to conform to the shape of the image Select Analysis gt Masks Click New Click on the Function toolbar button to adjust the mask that will define the whole cell The Define Mask Function window appears Function Select Morphology in the Function list Select a starting Mask Select Channel 3 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 5 12 Click Close 13 To view the resulting morphology masks open the Image Display Properties win dow and if necessary select the new mask for the channel Oo ON DW WI Icon for Image Display Properties 98 USING THE DATA ANALYSIS TOOLS 14 15 16 17 18 19 20 Al 22 23 24 25 26 27 28 29 30 31 1 Intercellular Muclear g 1 Next you will subtract the nuclear morphology mask from the intracellular mas
21. 4 199 UNDERSTANDING THE IDEAS FEATURES AND MASKS FISH IS Masking a cell with one spot using the spot and range masks E 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 cir cular 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 starting mask for creating the skeleton 200 UNDERSTANDING THE IDEAS FEATURES AND MASKS alela elake tere kY Skeleton_thin Skeleton_thick APPLICATION EXAMPLES Thick skeletons can be used with shape based features such as symmetry to accentuate the shape of an object and provide greater separations Separate singlets and doublets by computing the area of the thin skeleton mask We have used the object tight for
22. 4 Ch APPLICATION EXAMPLE Classify different white blood cells based on the morphology of the nuclear image 162 UNDERSTANDING THE IDEAS FEATURES AND MASKS UNDERSTANDING THE TEXTURE FEATURES The Texture features determine local intensity variations 1n 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 BRIGHT DETAIL INTENSITY R3 AND BRIGHT DETAIL INTENSITY R7 FEA TURES The Bright Detail Intensity R3 and Bright Detail Intensity R7 features compute the intensity of localized bright spots within the masked area in the image Bright Detail Intensity R3 computes the intensity of bright spots that are 3 pixels in radius or less while Bright Detail Intensity R7 computes the intensity of bright spots in the image that are 7 pixels in radius or less In each case the local background around the spots is removed before the intensity computation The figure below shows the process of obtaining the localized bright spots in the image Original Image Detail Eroded Image Bright Detail Image The graph below illustrates the use of the Bright Detail Intensity R3 feature on a nuclear image to separate apoptotic cells from non apoptotic cells UNDERSTANDING T
23. 7 AAD NFKB 7 AAD Mean Mean Population Gated E fe coas irc08 NFKBFAAD CD45 1 CD8 NFKBFAAD CD45 1 CD8 NFkB AAD ae i EIN al 4 2499 13 24 0 084 0 7516 Statistics Area H3 amp Focus 6 453 Aspect Ratio Intensity_ 7 44 Population I gt Focus amp Ail amp R3 amp Focus amp f Intensity_CD8 o8 R M a CCR PC E 1e3 0 163 1e4 1e5 1e6 Intensity _CD45 1 SS eye SIS x 0 OVA En EE 2 le Taat e m Cr i 1ay a 2 12 BD o N in Normalized Frequency Oo tn o iy I gt I no a g o oc oO as Q a a E o iy i I 1 I j 1 0 I 0 100 200 300 400 500 600 700 1 0 1 Area comme Analysis Area mere PQRHHERA COT Pree ll e The Image Gallery displays the images of populations of cells segmentation masks and composite images For more information refer to Overview of the Image Gallery on page 61 e The Statistics Area displays feature values for objects and populations in tabu lar form For more information refer to Overview of the Statistics Area on page 389 e The Analysis Area displays plots and distributions of cellular feature values Individual images and text panels For more information refer to Overview of the Analysis Area on page 73 OVERVIEW OF THE DATA FILE TYPES Data from the ImageStream cell analysis system are collected and managed using three types of data files raw image
24. 94 ese A SS Seed OS ee SSR eens 200 Os eda TE PEPEE PEPEE EEE Mee EEE SEEE eae dean 201 Sr M ear a Ne a aes reg ors eg ea A aerogenes A E T 203 Tue bokt Mek cca iat 8 ah 2h ace A tacts E E ETE E E EATE RE EE 204 V ESNIE aeaaea ky Bata a ev e a a T ea Blo pea ET 205 ROUBLESHOOTING 207 Apphcidon Hangino serre trr roren EEEIEE EROGENE SERNER SS 207 COMP EMSA OM paean aa Se E EN GOERS GORA O 207 Ea ie EE E E EELEE ETENEE 209 Deleting a Population and Region n on a naana 209 Dely in CO py PIE a a a a eee a es a a ee ee 209 Object N mber set tO Zero rsrsr as eat aea SES E S 209 Buttons or options in windows are not appearing 20008 209 Images and brightfield channel appear uniformly bright 210 GLOSSARY 211 vi CHAPTER 1 Preface Welcome to the IDEAS version 4 application documentation IDEAS 4 0 or later versions are required to open ImageStream data Many new improvements have been added to the application How to use this manual on page 1 Whats New in IDEAS 4 0 on page 2 HOW TO USE THIS MANUAL This manual provides instruction for using the Amnis IDEAS application to analyze data files from the Amnis ImageStream cell analysis system The intuitive user interface of the IDEAS application makes it easy for you to explore and analyze data The application contains powerful algorithms that allow you to create an unlimited number of tailored features for a specific experiment The applica
25. Axis 6 Set the scaling for each axis of the graph The default is Auto which allows the application to automatically scale the graph To set minimum and maximum values for an axis select Manual Select Linear or Log and enter Maximum and Minimum limits If you selected Log enter the X gt value Note You can scale the X Axis of a graph or the Y Axis of a scatter plot in one of two modes Linear or Log The Linear mode is the default The Log mode allows you to logarithmically scale a section of the graph or scatter plot Selecting this mode causes the IDEAS application to perform bi exponential plotting The gt X value defines the linear portion of the graph as X through X The application plots the values outside of these limits on a logarithmic scale You can plot negative values as well as positive ones on a logarithmic scale by adjusting the limits Take care not to split a population such that it appears to be two separate popula tions This splitting is especially likely when negative values exist due to compensa tion 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 logarith mic 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 population The IDEAS application automatically chose 1000 for the scale of the graph that is in the center
26. DATA Add files to the list on the left to export values for multiple files In the Select a population drop down menu select the population that you want If you haven t defined any populations All is the default To make a new popula tion refer to Creating Tagged Populations on page 71 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 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 On the Tools menu click Export Image Pixel Values The Export Image Pixel Values window appears Export Image Pixel Values Sele Select object to export Export to f Clipboard File OK Cancel 2 Select the object to export in the drop down menu 3 Select to Export to either the Clipboard or File 4 Click OK CREATING TIFS FROM POPULATION FOR EXPORT The IDEAS application allows users to create separate TIF files for channel images for every
27. E a ee 33 penine d E n E T deeteacaeai 35 IVI Gresik Alessio E E E S 36 Viewing Sample Information jx eso co cd aod wee nade accep acd one Ip eee 37 Overview Ol Compensi ci preat e tad hea eS es WE eh aes lt 38 Creating a New Compensation Matrix File 45 40 Preview a compensation Matrix geo Uwe oe Ra Se hae Stee BESS 48 Merine Raw Image Files edits logins sdam a hid ee As Wake Ae 49 Merging Compensated Image Files 3229042985 oe Se e298 Ged Bt 50 Saving Data Pless 24 4 ce peo ee hin dep kha Reh ES BR 51 Savin ra Data Analysis File Cdan eenaa a teen anareate ia 51 Saving a Compensated Image File CE secectc nn iiie n 52 Savine a Template aS a a EE TN 52 Creating Data Files from Populations 0 ccceeccccceeeceeeeeeeeeeeeeeeeeenees 52 TAC OCS SSIS Danera nerea tay Paw cat A aac ete ce Pace cas 54 USING THE DATA ANALYSIS TOOLS 59 Overview of the Data Analysis Tools 2 0 2 2 00 00 00 cece eee 59 Wsine thedlmace GUEN eei sc 5c ath aie hn hehe Da ep bee are 8 tg oe 5 60 Overview of the Image Gallery ce saiat behok CoS ow cee eet abees x 61 Setting the Image Gallery Properties 20 000 eee eee 64 Working with Individual Images 2 00 00 8o one hoo de saeeag ies Sa OSes 70 Creating Tagged Populations 5 c c 20 6606 ere Genki eexeS eevasde 75l Usnoethe Ani Aret lt n stance aaa E a TA a a 73 Overview of the Analysis ATCT e rena o es a E ens ene weet 73 Creatina GTAP seeria ea a e a ati a e a
28. Merge cif Files The Load Multiple cif Files window appears Fei Load Multiple cif Files Files to Load Select cif files to load Enter the number of objects to load from each file A population will be created for each file Specify the population name File eects Population Name the output files to be created To use a custom template for analysis Compensated image file cif Select a template or data analysis file ast daf OOOO a Data analysis file dat 2 Click Add Files and select the cif files that you want Click Remove Files to remove a file from the list The file names appear in the Files to Load list 3 For each file type the number of objects to load By default all objects will load unless specified For each file the IDEAS application creates a population using the file name as the population name 4 Type or select the resulting cif and daf file names If you type or select an existing file name a warning appears that asks you to verify the overwriting of the file 5 Browse to select a template 6 Click OK The IDEAS application loads the cif files and creates a single cif and daf file 36 GETTING STARTED WITH THE IDEAS APPLICATION VIEWING SAMPLE INFORMATION All of the information associated with an IDEAS file such as the collection information camera settings and corrections 1s saved within IDEAS and can be viewed in the Sample Information window TO OPEN THE
29. Translocation Dose and Time 4 0 rifs 10ng 60_18_1 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 75_23_6 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 90_28_11 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 15_5 12 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 30_10_17 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 45_15_22 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 60_20_3 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 75_25_8 cif li C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 90_30_13 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 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 sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 1ng 30 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 rfs 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 sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_26 _9 daf C Users sfriend Desktop IS100 NFkB Translocation
30. Y Intensity Features Delta Centroid X and Delta Centroid Y Features Delta Centtoid XY Feites aeriana BS ee Ew a Se SS Max Contour Position Feature 00 cee e eee eee Raw Centroid X and Raw Centroid Y Features Spot Distance Min Fearute sa toy a he hee Boo Sheng ees Bes Valley X and Valley Y Peatires ccu oa Mack Se iach pe euE EEn Ro Understanding the Shape Features 113 113 116 118 119 120 121 T21 123 124 125 126 128 134 134 134 135 136 136 137 138 139 140 141 141 142 143 143 143 144 145 146 147 149 150 151 152 154 11 Aspect BR Atlon FEIE 4 6 ji ed oye AE dh hada E ahonte b ahatce a eters 154 Aspect Ratio Intensity FeAtire sses 2 Kab aie ee ewe we we Ge I E 156 Circulare Pen ure 15 amp eo ee ee ee a ee 156 Compactes Peature a egie eden E pE E r p bed Adee aes aes 158 Eloieatedness Feature sitiari e a a he eee ce 159 kobe Count Peace riea y edee t ede eS ecient sevens Geeta es 160 Shape RAO FENU sanirane Cere he Pe a e ee eee oe 161 Symmetry 2A o p Fere rnia e ea he aS a a a Pare a 162 Understanding the Texture Features n a naaa aaa 163 Bright Detail Intensity R3 and Bright detail Intensity R7 Features 163 Contrast Feature st cee oe ee ened 6 ae Se ee othe a aa 6 eed 165 Gradene Max Feti C 0 Sas toh hee de are amp ila amp oho amp hake E 166 Gradient RMS Feature si 6 Stn ie yew ets on es Ba Mae nd W
31. also deletes any populations that are dependent on that feature Your feature list may become large and unwieldy You can narrow down the list without deletions by sorting the list See Sorting Features on page 102 for more information USING THE DATA ANALYSIS TOOLS 107 USING THE POPULATION MANAGER A population is a group of objects You create populations by drawing regions on graphs by hand selecting tagging objects in the Image Gallery or on plots or by combining existing populations After a population has been defined you can view it in the Image Gallery or on a plot and you can use it to calculate statistics The Population Manager provides a central place for maintaining the display properties of existing populations and for creating new combined populations TO OPEN THE POPULATION MANAGER AND VIEW THE POPULATION DEFINITIONS 1 Select Analysis gt Populations or right click a graph and select Populations The Population Manager window appears z Population Manager Properties Name All Dark Mode Color Light Mode Color a m A5 amp A4 amp A3 amp A1_2 R5 Symbol Simple Dot Definition All Note The list of populations is presented as a hierarchy that shows the dependen cies of the populations on each other The icon associated with a population indi cates how the population is defined e The tagged icon indicates a tagged population e A population defined by a region is in
32. and analyze your file The analysis includes e Opening the data file e Setting the display properties e Gating on single cells e Gating on focused cells e Gating on fluorescent positive cells e Creating and graphing the feature Bright Detail Similarity for measuring co localization e Creating a statistics report Cells with colocalized probes are identified in the final graph presented in the wizard In this example cells with high Bright Detail Similarity values have co localization of the two probes CD107a green and CpG red Horriunlignkd rep ph Li i Pj a Bega Coda Seriy CO a Cpe Low co localization High co localization For a more thorough explanation of the Bright Detail Similarity feature see Bright Detail Similarity R3 Feature on page 184 GETTING STARTED WITH THE IDEAS APPLICATION 23 INTERNALIZATION This wizard will create an analysis template for measuring the internalization of a probe in any population of cells you identify TO BEGIN DOUBLE CLICK ON INTERNALIZATION Follow the instructions to open and analyze your file The analysis includes e Opening the data file e Setting the display properties e Gating on single cells e Gating on focused cells e Gating on fluorescent positive cells e Creating and graphing the feature e Creating a statistics report cells with internalized probe are identified in the final graph presented in the wizard The example below shows the
33. cells 62 FAAL Ie pl FY Value alley Skeleton h5 7440 Think 43 alley Skeleton M5 440 Think 6 r om T l H te H a iy m Tre i m i F i d E a fa Ang Pm d irp ee i re H l H TAM Speen n r pi A LI it ag rAAD Magg Composite Fisel 43 6r Intensity 38 These features define the origin of the Valley mask 152 UNDERSTANDING THE IDEAS FEATURES AND MASKS APPLICATION EXAMPLE Measure the exact center of where a synapse between two cells is located UNDERSTANDING THE IDEAS FEATURES AND MASKS 153 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 Elongatedness 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 on page 137 Aspect Ratio Minor Axis Major Axis inor Axis ajor Axis See also Elongatedness Feature on page 159 and Shape Ratio Feature on page 161 for other shape ratios 154 UNDERSTANDING THE IDEAS FEATURES AND MASKS Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightWidth Thickness min Length ess min inor Axis ajor Axis APPLICATION EXAMPLES Quantify the roundness of the mask
34. 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 notices that are on the original copy of the Softwar
35. e es 73 Creatine Reecions On Graps mirei Ti e aoa E T oe re ee eee 80 PUVA 2AM Mapes i ye th Ya de ta ete esta uted al gp nd ET A T he erte ee 85 Adding Text tothe Analysis Area asoceto hee eee ee ee se ee eS 88 eiiiosthe Statistics Miveass 2 bene eo 8S oo SS oe RS Sed Coed Saas Soe 89 Overview Or the S tistics Area utege ee ch ay ai a aa a aaa eles 89 Viewing the Popu lation Statistics oces sesir ae tal Geeey eeeay eee ue 89 Viewing the Object Feature Values o3 6563424442 644 24402454044 J2 Usine the Vlas UM ana Or escrire re ea kna eet we e eE 94 Overview or the Mik Manager Gudrs naisao E ih aed EN A 94 Creating New Masks with the Mask Manager 204 94 Esap ol Create a Mask reee a aa Steals Se ns ER E ee 98 Viewing and Editing a Mask rouvi ticia bea bee a ee ee 100 Usine tihe Pedane Manag eiie ean ie e E wo a dk a a E E e E 101 Overview of the Feature Manager lt a ie nes Sint ee at BO ee 101 Creating New Features with the Feature Manager 103 Usingthe Population Manis seseina BEG SA Se ee eee 108 CHAPTER 6 CHAPTER 7 Using the Region Manager CREATING REPORTS AND EXPORTING DATA Printing Reports Creatine a Statistics Report Template 6 ccd bot Boh ie de i ea Generating a Statistics Report using daf Files naana anaa Exporting Dii a baste k ted Exporting Feature Data Exporting Pixel Data Creating TIFs From Population for Export
36. file A compensation matrix performs fluorescence compensation which removes fluorescence that leaks into other channels See Overview of Compensation on page 38 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 application 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 analysis for the daf The default template includes over 200 calculated features per object An expanded template is available that includes over 600 calculated features per object 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 specified by the selected template Once a data analysis file daf file or compensated image file cif file is saved it can be opened directly for data analysis You would only open a cif if you wanted to change the template or a rif file to change the compensation The diagram on the next page displays this workflow
37. file Multiple cif files can be created from a single rif file by applying a different fluorescence 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 14 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 The daf file contains e Feature definitions e Population definitions e Calculated feature values e Image display settings e Definitions for graphs and statistics Loading a daf file restores the application to the same state it was in when the file was saved 1 e with the same views graphs and populations In IDEAS versions 3 0 or later a daf file may be used as a template OVERVIEW OF THE IDEAS APPLICATION 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 defini
38. image of cells in anaphase or telophase 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 198 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 S 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 Spot Mask into individual components 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 minimum and maximum aspect ratio To select pixels within a range of intensity values see Intensity Mask on page 196 lt 4 gt selecting small aspect ratio object 3 object 4 selecting small size object 1 object 3 1 O Selecting small intensity object 1 object
39. imagery is displayed bivariate plots of adjacent channels Intensity are added to the analysis area and the compensation matrix values are computed and displayed in a table 42 GETTING STARTED WITH THE IDEAS APPLICATION Create Compensation Matrix el Step 3 Validate the compensation matrix Double click each matrix coefficient to validate the fit of the positive control population The resulting graphs can be added to the analysis area to refine the positive control populations P Q D D ajaj naaalala lA a aje A AJA Aaaa Aa aal 0 0 0 0 0 1 0 0 0 0 0 0 Best Ft Means Preview Images Restore Matrix Positive Control Populations None ChO1 None 7_Positive ChO2 2 Positive 8 Positive Ch03 3 Positive None ChO4 4 Positive None i 5_Positive 11_Positive xl xl 12_ Positive The Positive Control Populations are shown in the graphs below Al qamna 4e6 3e6 5 3 3e6 1206 ef oO oO 2e6 gt gt 1e6 E T u 1e6 w o l 0 1e5 2e5 3e5 0 2e5 4e5 6e5 8e5 1e6 0 5e5 1e6 1 5e6 2e6 Intensity_MC_ChO1 Intensity_MC_ChO3 Intensity_MC_ChO5 Intensity_MC_ChO8 Intensity_MC_Ch10 I 2e6 4e6 6e6 Se Intensity_MC_Ch11 3e5 6e5 9e5 Intensity _MC_Ch09 0 3e5 Bed 9e5 1 2e6 Intensity_MC_ChO GETTING STARTED WITH THE IDEAS APPLICATION 43 6 In Step 3 choose one
40. intensity measurements 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 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 calculations so the default template does not include them The reason 1s to save time when you load files However you can add these features to the feature list USING THE DATA ANALYSIS TOOLS 101 TO VIEW EXISTING FEATURES 1 Click Analysis gt Features or select a graph panel context menu The Feature Manager window appears fei Feature Manager 0 0ng_2_9 daf Features Area_M1 Feature Type Area_M2 Area_M3 Area_M4 Area_M5 Area_M6 Area_MC Aspect Ratio Intensity_M1_Ch1 Aspect Ratio Intensity_M2 Ch2 Aspect Ratio Intensity_M3_Ch3 Aspect Ratio Intensity_M4_Ch4 Aspect Ratio Intensity_M5 Ch5 Aspect Ratio Intensity_M6_Ch6 Aspect Ratio_M1 Aspect Ratio M2 Acmact A stin hi v Sort features by Add Multiple Features 2 Click a feature in the Features list to view its definition 3 Choose an icon to sort the features TABLE 4 SORTING FEATURES FEATURE DEFINITION ICON Sorts features alphabetically Sorts f
41. internalization of labeled CpG red Leh bid dell Fripe yp W b a e CTT N Ed Cpe Low Internalization CEE E High Internalization _ For a more thorough explanation of the Internalization feature see Internalization Feature on page 187 24 GETTING STARTED WITH THE IDEAS APPLICATION NUCLEAR LOCALIZATION This wizard will create an analysis template for measuring the nuclear localization of a probe in any population of cells you identify TO BEGIN DOUBLE CLICK ON NUCLEAR LOCALIZATION Follow the instructions to open and analyze your file The analysis includes e Opening the data file e Setting the display properties e Gating on single cells e Gating on focused cells e Gating on fluorescent positive cells e Creating and graphing the feature e Creating a statistics report 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 DRAQ5 red and NFkB green to measure the nuclear localization of the NFkB Untreated stimulated aiaga i gd od TE zaj Pe res pice pie For a more thorough explanation of the Similarity feature see Similarity Feature on page 188 GETTING STARTED WITH THE IDEAS APPLICATION 25 SHAPE CHANGE This wizard will create an analysis template for measuring the shape circularity of any
42. of two methods for calculating the coefficients The Best Fit method is used for objects such as cells where intensities vary The Means method is used for objects such as beads that have only slight vari ations in intensity For each fluorochrome the application automatically identifies a positive con trol population excluding the brightest and dimmest objects and assigns it to the proper channel 7 Inspect the matrix values in the table of coefficients Coefficients should always be less than 1 and decrease from the assigned channel In other words leakage should be greater in the channel nearest to the assigned channel Fluorescence always extends in the long wavelength direction from the exciting light Verify that no coefficients are larger than 1 Verify that in a column corresponding to a fluorochrome the coefficients decrease from the assigned channel Verify that the coefficient is greater in the channel below the 1 in the table than the value above the 1 in the table Verify that these coefficients also decrease in subsequent channels below the 1 Verify that there are no changes from the identity matrix in the columns where there are no fluorochromes including the scatter and brightfield channels If necessary the column can be set to the identity values by double clicking on the heading Inspect the coefficients in the matrix by double clicking on the coefficient Coefficients highlighted by red have err
43. overview 101 tasks 106 tools 102 features angle 143 angle intensity 143 aspect ratio 154 aspect ratio intensity 156 Bkdg mean 172 Bkgd std dev 172 bright detail intensity 163 camera line number 191 camera timer 191 centroid delta x and y 146 centroid delta xy 147 centroid x and y 144 circularity 156 compactness 158 contrast 165 create new 103 104 delete 107 diameter 135 elongatedness 159 flow speed 191 gradient max 166 gradient RMS 167 height 136 142 intensity 173 internalization 187 lobe count 160 major and minor axis intensity 138 major axis and minor axis 137 max contour position 149 min pixel 176 modulation 169 object number 191 object per mL 191 object per sec 192 overview 124 perimeter 139 raw intensity 177 raw max pixel 177 raw mean pixel 179 raw median pixel 179 raw min pixel 180 saturation count 181 saturation percent 182 shape ratio 161 similarity 188 similarity texture R3 184 spot area min 140 spot count 170 spot distance min 151 spot intensity min 183 std dev 171 symmetry 2 162 table 128 thickness max 141 time 192 valley x and y 152 viewing 102 width and height 136 142 file name extensions 6 fluorochromes table 41 42 G graphs apply or remove region 84 copy and paste 82 creating 75 creating regions 80 legend 79 moving 79 printing 114 resizing regions 82 statistics 78 zoom 82 INDEX 215 H hardware requirements 5 IDEAS
44. 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 spectral bandwidth 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 fluorochrome Emmiussion Spectra for two fluorochromes Channel 3 Channel 4 Percent Emission 475 500 525 550 575 600 625 650 675 700 725 Wavelength in nm 38 GETTING STARTED WITH THE IDEAS APPLICATION Below is an example of cells stained with two fluorochromes independently and run together as one sample Intensity scatter plots and images are shown uncompensated and compensated Un compensated Compensated le6 le6 165 1e5 Pa 5 E led 1e4 T wT 1000 0 1000 L 111 tti r 1 ttt 1000 i L o e 1000 le4 1e5 1000 0 1000 le4 165 3 Intensity 3_Intensity Uncompensated Compensated SSC Brightfield FITC PE PE Alexa610 ssc Brightfield FITC PE PE Alexa610 Draq 5 Channel 1 Channel 2 Channel 3 Channel S 7 Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Channel 1 Channel 1 3 Channel 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 You can mix singly stained cells and
45. 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 Coalacalized Colocalized 0 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 1s subtracted from the original image resulting in the bright detail image 184 UNDERSTANDING THE IDEAS FEATURES AND MASKS Original Image Detail Eroded Image Bright Detail Image The figure below shows the correlation analysis between an image pair Hon specifically Localized Cell ADC Image Endosomes image Codocalized Cell PE ant RTX H BA ADC Image Endosomes image APPLICATION EXAMPLES Quantify the degree of colocalization between two probes Track internalization and intracellular trafficking of antibody drug conju gates to either the endosomes or the lysosomes Colocalization of Rituxan and compliment C3b UNDERSTANDING THE IDEAS FEATURES AND MASKS 185 INTENSITY CONCENTRATION RATIO FEATURE
46. statistics Tools for drawing regions are found on the Analysis Area toolbar See Creating Regions on Graphs on page 80 TO OPEN THE REGION MANAGER AND VIEW THE REGION DEFINITIONS 1 Select Analysis gt Regions or right click a graph and select Regions The Region Manager window appears ree Region Manager Regions Hame 3 spot Dark Mode Color E Light Mode Color G Use for statistics only Shape Line Vertices a Delete Revert Close 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 COND OO HB WO N FR Note When a region 1s deleted all populations that are defined by that region will be deleted A warning dialog box appears listing the populations that will be deleted 111 USING THE DATA ANALYSIS TOOLS 112 USING THE DATA ANALYSIS TOOLS CHAPTER 6 Creating Reports and Exporting Data The following subsections describe how you can print data directly from the IDEAS application or export data to other applications such as those in Microsoft Office Printing Reports on page 113 Creating a Statistics Report Template on pag
47. this case Nuclear morphology measurements with lobe count feature for cell classifi cation 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 processing 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 background 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 201 UNDERSTANDING THE IDEAS FEATURES AND MASKS Original Image Detail Eroded Image Bright Detail Image The image pairs below show objects in grayscale next to their corresponding Spot Masks in cyan Spot masks can be further refined using the Peak and or Range masks See Peak Mask on page 199 Range Mask on page 199 APPLICATION EXAMPLES Used with the Spot Count feature to enumerate spots in images such as for FISHIS Used with Intensity features to quantify intensity in spots
48. to Standard on 6 Click OK to save the changes or Cancel to exit 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 Copy Graph Select options for copying Graph E Legend Statistics E Cursor Show Sample Name in Title Size sealing factor s 50 100 200 300 2 Select the checkboxes Graph Statistics Legend Cursor Show Sample Name in Title to include the elements in the report 3 If necessary adjust the size scaling factor Recomended setting is 100 114 CREATING REPORTS AND EXPORTING DATA 4 Click OK to print the graph CREATING REPORTS AND EXPORTING DATA 115 CREATING A STATISTICS REPORT TEMPLATE A statistics report template is a separate template 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 applied during batching if it is part of the template used It may also be applied to preexisting daf files from the Reporting menu In this case the rest of the template is not processed only the report The statistics report 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 TEMPLATE 1 Select Reports gt Statistics Report Template The Statistics Report Template appears Fes Statistics Re
49. 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 66 USING THE DATA ANALYSIS TOOLS Tip For fluorescence channels set the vertical green line that appears on the left side to the dimmest pixel in the image and set the right vertical green line to the brightest pixel To get a good mapping range adjust the same line so that the yel low cross is centered among the pixel intensities on the X Axis For the brightfield channel set the vertical lines to about 50 counts to the right and left of the histo gram to produce an image with crisp brightfield contrast e To change the mapping curve to be logarithmic or exponential click drag the yellow cross e To restore the mapping to a linear curve Click Set Linear Curve e To see the full scale for the X Axis Click Full Scale e To set 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 Autoscale e You may enter values manually by selecting the Manual tab Automatic Marc
50. 136 Based on a bounding rectangle the Width is the smaller side and the Height is the longer side of the rectangle Length Feature on page 136 Measures the longest part of the mask Major Axis and Minor Axis Features on page 137 Describes the widest part of the mask and the narrowest part of the mask respectively Major Axis Intensity and Minor Axis Intensity Features on page 138 Based on a bounding ellipse the Minor Axis is the narrow part and the Major Axis is the widest part Minor Axis Major Axis and Minor Axis Features on page 137 Perimeter Feature on page 139 Describes circumference of the mask Spot Area Min Feature on page 140 The Area of the smallest spot in the mask See also Raw Centroid X and Raw Centroid Y Features on page 150 Spot Intensity Min and Spot Intensity Max Features on page 183 and Spot Count Feature on page 170 Thickness Max Feature on page 141 Describes the longest width of the mask Thickness Min Feature on page 141 Describes the shortest width of the mask 128 UNDERSTANDING THE IDEAS FEATURES AND MASKS IN DEFAULT TEMPLATE No No IN EXPANDED DEFAULT TEMPLATE Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes MASK_IMAGE USED IN DEFAULT TEMPLATE M1 M6 MC M1 Mo6 M1 Mo6 M1 Mo6 M1 Mo6 M1_Ch1 Mo6_Ch6 M1 Mo6 M1 Mo6 M1 Mo6 M1 Mo6 TABLE 2 LIST OF FEATURES BY
51. 6 Repeat the previous 2 steps until finished adding columns to the view A column will be added under the column currently selected To insert a column click on the 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 9 If you want to delete a view click the view to select it and then click Delete A confirmation window appears 10 If you want to preview any new changes in the Image Gallery return to the Image Gallery and choose your new view in the View drop down menu Then return to the Image Gallery Properties window and click Preview Changes in Gal lery 11 Continue customizing the Image Gallery display properties with another proce dure in this section or click OK to finish and save changes or Cancel to finish and discard changes TO CREATE A COMPOSITE 1 Within the Image Gallery Properties window click the Composites tab 68 USING THE DATA ANALYSIS TOOLS T Image Gallery Properties Display Properties Views Composites NFkB DRAQS i Translocation Name NFkB DRAGS Object 0 NFkB DRAGS NFkB 100 DRAQS 100 Percent Preview Changes in Gallery 2 Type a name for the composite or leave blank to allow the name to be built auto matically from the image names added to the composite 3 Click Add Image The selected image appears in the Object box Chang
52. A ANALYSIS TOOLS 79 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 histogram 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 oraphs To change the attributes of a region or delete a region and the populations dependent on that region see Using the Region Manager on page 111 TO DRAW A REGION ON A SCATTER PLOT On the Analysis Area toolbar click either the e Rectangle Region or e Oval Region or e Polygon Region button on the Analysis Area toolbar Refer to Polygon Tool Option on page 81 for more details T 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 Regi
53. ASKS 143 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 144 UNDERSTANDING THE IDEAS FEATURES AND MASKS CENTROID X INTENSITY AND CENTROID Y INTENSITY FEATURES Centroid X Intensity is the intensity weighted X centroid and 1s 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 af Centroid X Y PE Perro se Pe Versyse oe 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 UNDERSTANDING THE IDEAS FEATURES AND MASKS 145 DELTA CENTROID X AND DELTA CENTROID Y FEATURES Both the Delta Centroid X and Y features measure the distance between the Centroids X or Centroi
54. CATEGORY FEATURE CATEGORY LOCATION SHAPE IN FEATURE NAME DEFAULT TEMPLATE Width Feature on page 142 Yes 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 ori gin in the upper left corner pixels or contour Angle Feature on page 143 No The angle of the major axis from a horizontal plane in radians Angle Intensity Feature on page 143 No The angle of the major axis intensity from a horizontal plane in radi ans Centroid X and Centroid Y Features on page 144 No The central tendency of the pixels along the X Axis and Y Axis respectively Centroid X Intensity and Centroid Y Intensity Features on No page 145 The central tendency of the pixels along the X Axis and Y Axis respectively with the pixel intensities weighted Delta Centroid X and Delta Centroid Y Features on page 146 No The distance between the X or Y Centroids of two images Delta Centroid XY Feature on page 147 No The distance between the Centroids of two images Max Contour Position Feature on page 149 No The location of the contour in the cell that has the highest intensity concentration Raw Centroid X and Raw Centroid Y Features on page 150 Yes The shortest distance between two components spots See also Spot Area Min Feature on page 140 Spot Intensity Min and
55. CQUISITION AND COMPENSATION Data are first acquired from the ImageStream using the Amnis INSPIRE instrument control 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 flowspeed variations spatial alignments illumination irregularities and camera background Compensation for spectral crosstalk is calculated from control files and applied to experimental files After the preprocessing completes the IDEAS application allows for the interrogation of the image data segmenting out cells nuclei cytoplasm FISH spots beads and other objects of interest Using a default template the application calculates the values for over 200 standard features per object to be used in subsequent analyses Guided analysis for many common applications 1s 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 histograms 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 histograms or scatter plots or by tagging individual objects The IDEAS application provides standard distribution statistics for all defined populations
56. Dose and Time 4 0 rifs 10ng 15_3_10 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 30_8_15 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 45_13_20 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 60_18_1 daf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 75_23_6 daf C Users sfriend Desktop 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 rifs 1000ng 30_10_17 daf 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 rifs 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 rfs 1000ng 90_30_13 daf Start time 2 1 2011 11 53 28 AM End time 2 1 2011 12 00 41 PM Result All files were processed successfully Statistics Report C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 0ng_2_9 _4 Default daf_stats b4 57 GETTING STARTED WITH THE IDEAS APPLICATION 58 GETTING STARTED WITH THE IDEAS APPLICATION CHAPTER 5 Using the Data Analysis Tools This section describes how to view imagery graph d
57. Edit Statistic Column opens a Statistics Properties window to enable changes to the statistic Clone Statistic Column opens a Statics Properties window to create a new statistic with the same parameters This is a quick way to make a statistic that shares some parameters with the original by changing the desired parameters Clear All removes all of the statistics from the table Copy Statistics to Clipboard copies the table in a text format that can be pasted into other programs such as Excel USING THE DATA ANALYSIS TOOLS 91 VIEWING THE OBJECT FEATURE VALUES The Object Feature Values tab 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 Population Statistics Object Feature Values Current object number Tobe TO VIEW AND CUSTOMIZE THE FEATURES SHOWN ON THE OBJECT DATA TAB 1 Click the Object Feature Values tab in the Statistics Area 2 Right click anywhere in the tab 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 Select Object Features BHA Features Aspect Ratio MC Intensity_MC_Ch1 Intensity_MC_Ch2 Intensity_MC_Ch3 Intensity_MC_Ch4 Intensity_MC_Ch5 Intensity_MC_Ch6 Object Number Raw Max Pixel MC
58. FINITION 1 To Select Analysis gt Masks The Mask Manager window appears E Mask Manager Name M1 Definition M1 Not Combined Mask Click a mask in the Masks list to view the definition in the Definition area Click Close EDIT A MASK FUNCTION In the Mask Manager window select the mask that contains the function you want to edit Click Edit Remove the definition for the combined mask using the back arrow tool as needed Refer to To create a new combined mask on page 96 for more informa tion on the definition tools el Or click the Function button on the toolbar for a function mask The Define Mask Function window appears Refer to To create a new mask using Func tions on page 94 for more information Function Click OK when finished 100 USING THE DATA ANALYSIS TOOLS USING THE FEATURE MANAGER This section describes how to create and delete features and it provides a high level description of the base features that are provided by the IDEAS application The following subsections cover this information OVERVIEW OF THE FEATURE MANAGER The IDEAS application defines a set of base features that you can use to create features for each object To do so you use the object s mask 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 descriptions of all the base
59. Gated Plotted Maximum To display the statistics for the graph select Show statistics To close the Statistics Area for the graph select Hide statistics Select the statistics that you want to display The selected statistics will be displayed for each population on the graph The statistics that are supported are the Count Percent Total Percent Gated Percent Concentration count sample volume Mean Median Standard Deviation MAD Median Average Deviation RD Mean RD Median CV Minimum Maximum Geometiric Mean Mode vari ance and NaN not a number When finished click Close SHOW THE LEGEND FOR A GRAPH 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 disap pears 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 popu lation name associated color and line type e To move the legend click and drag it You cannot drag the legend past the boundary of the graph panel MOVING A GRAPH e With any graph in the Analysis Area you can move it to another location by clicking in the upper section of the graph and dragging it All N alel EHH x mm USING THE DAT
60. HE IDEAS FEATURES AND MASKS 163 single cells GAY oe S Frequency p mm i Low Bright Detail Intensity R3 values identity non apoptotic cells ra 2 High Bright Detail Intensity R3 values identify apoptotic cells l 0 Jed zed 1 5e4 bes Jez le Bright Detail Intensity AS Threshold M5 Ch 30 ChS APPLICATION EXAMPLE Identify cells that have bright specks such as Apoptotic cells 164 UNDERSTANDING THE IDEAS FEATURES AND MASKS CONTRAST FEATURE The Contrast feature measures the sharpness quality of an image by detecting large changes of pixel values in the image and 1s useful for the selection of focused objects or apoptotic brightfield images For every pixel the slopes of the pixel 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 ies a PO single cells re Contrast_BF Gradient RMS BF APPLICATION EXAMPLES Find apoptotic images with high contrast in brightfield imagery Determine overall focus quality of images Use with Gradient RMS to determine focus quality Characterize texture See also Gradient Max Feature on page 166 and Gradient RMS Feature on page 167 UNDERSTANDING THE IDEAS FEATURES AND MASKS 165 GRADIENT MAX FEATURE The Gradie
61. IDEAS IMAGE DATA EXPLORATION AND ANALYSIS SOFTWARE USER S MANUAL VERSION 4 0 DECEMBER 2010 Amnis Corporation 2505 Third Avenue Suite 210 Seattle WA 98121 Phone 206 374 7000 Toll free 800 730 7147 Wwww amnis com 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 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 AGREEMENT GOVERNING USE OF THE SOFTWARE Amnis is willing to license the Software to you only upon the
62. If 4 SepeRI Ratio 0 57 Major 4 12 76 Minor rad Aspect Ratio Intensity Major Axis Intensity 14 22 APPLICATION EXAMPLES Quantify and compare the image fluorescence shape Identify single cells 138 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 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 UNDERSTANDING THE IDEAS FEATURES AND MASKS 139 SPOT AREA MIN FEATURE The Spot Area Min feature provides the area of the smallest spot connected component 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 Min Spot Distance Min and Spot Intensity Min or Max features measure properties of different spots in an image and are often used with the Spot Count feature under Texture For more information see Raw Centroid X and Raw Centroid Y Features
63. In addition users can further define images by creating features a mathematical expression that contains quantitative and positional information 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 graphically and the population distribution statistics include a variety of calculations such as the mean standard deviation and coefficient of variation CV 12 OVERVIEW OF THE IDEAS APPLICATION 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 three sections the Image Gallery the Statistics Area and the Analysis Area The placement and size of these areas are adjustable ES IDEAS 4 0 File Guided Analysis Advanced Analysis Compensation Tools Options Reports Windows Help E55 No pentide5 Il Batch2 daf f_ fos ES 6 OVA 50 nM6_Batch2 daf e amp o Population Selected Bin Population Statistics Object Feature Values Similarity Similarity bees z NFKB
64. L FEATURE The Raw Max Pixel feature is the largest value of the pixels contained in the input mask RTX FITC CD45 PE Brightfield This image is saturated in the FITC channel but not in the PE channel oo S e Ja Raw Max Pixel Ch3 ma 200 A400 600 800 Raw Max Pixel Ch4 UNDERSTANDING THE IDEAS FEATURES AND MASKS 177 APPLICATION EXAMPLES Determine the presence of saturated events May also be used to estimate the peak fluorescence activity though the Max Pixel feature is recommended for this application Measure the maximum pixel value within the mask Identify cells that saturate the CCD Saturation Count feature can also be used for this application 178 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 relevant that the Mean Pixel feature 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 UNDERSTANDING THE IDEAS FEATURES AND MASKS 179 RAW MIN PIXEL FEATURE The Raw Min Pixel feature is the
65. Measures the correlation of the bright details between image pairs Intensity Concentration Ratio Feature on page 186 No No Given two masks the ratio of the intensity in one mask to the total intensity in both masks Internalization Feature on page 187 No No The ratio of the intensity inside the cell to the intensity of the entire cell Similarity Feature on page 188 No No The Similarity is a measure of the degree to which two images are lin early correlated pixel by pixel within a masked region XCorr Feature on page 190 No No The XCorr is a measure of the degree to which two images frequen cles are cross correlated Sere System features do not require a mask and tend to deal with system wide metrics Camera Line Number Feature on page 191 No Yes An incremental count of objects 132 UNDERSTANDING THE IDEAS FEATURES AND MASKS TABLE 2 LIST OF FEATURES BY CATEGORY i MASK_IMAGE FEATURE ty EXPANDED USED IN FEATURE NAME DEFAULT DEFAULT CATEGORY TEMPLATE TEMPLATE DEFAULT i TEMPLATE Camera Timer Feature on page 191 No Yes The clock rate in KHz This is relative time Flow Speed Feature on page 191 Yes Yes The calculated flow speed in mm sec Object Number Feature on page 191 Yes Yes The sequence of objects Objects ml Feature on page 191 No Yes A local concentration of all objects per ml Note to get objects per ml of a population use the statistic Concen tration Ob
66. Ohta eae tee Reames es O EA lnteriace OF the IDEAS Applicaton 4 22 2 ere oe es Sees Overview of the Data rile Types ses nheue thee dg eho gebiotede awed Raw baee File iil S252 ite ie ee ee eho lM G Compensated Image Fie Cem snidu eite a an E E aT E Patr Analysis File Cda acct ht iets ihe yd sell ohare Tein plate aS see sha nceree or doew E nena even eo ace Compensation Matrix File etm icce cl tee a a a PeeViewoOr Data Pile Types saninin ens Wehte alee ie decctve GETTING STARTED WITH THE IDEAS APPLICATION Coded Analysis ake dilate kt dane hath A a tate he ea ae eae wane Appueation Wizards sf acc0haueehcuawe tick tale hawaii eee oe CDG ty NG a eee Sahat ah Bat caiirien cet beet et oalaa te gdasrcati eden ateuhh noes netacydareieats Display Propetes iir a E E eo cae eee ADOPTOS e enaa stom emt case E gt Aa eanseeeh anata tien Renae nate tare Well Cy clese IMMtOSIS c3 Acteteiedl oead ove cape a e doeelate COs OCT Z AONE anar a a r erna AON quis e ae ie ee ote oe IN iClear IO CAN ZA OI reie is Siataen cde A S Snape Canoe neria a teed ee decae woes Sieranta adeetep ened dso Buukna Block eis dois s dost a ee neta ween ee ee Re eek 10 12 12 12 13 13 14 14 14 15 15 16 17 18 18 19 20 21 22 23 24 29 26 27 CHAPTER 5 li Advanced Analys aaa a ae tah oan a wae eh eon Bert eae ee lt 29 Ere TleMeni sordrteent icen Sauces each ata ae ates A A 29 Openin Era E e er a a a OS 29 Openmo ane E ea aa
67. Populations Statistics Bs 0 1ng 30_6_ 13 cif Count Be All Percent Total 5 0 R Percent Gated Percent Era R2 Cone obj ml Reference population ff required All Features if required M0 M02 MO MH _M05 MOS ME E Aspect Ratio Intensity_M02_Ch02 mml a ari e bo 4 mt aes A mr Select the populations from the list under Rows Select the statistics from the list under Columns If a statistic requires a reference population percent RD choose one 6 Select the Features for the statistics in the Features list Hint Sort features by cate gory mask or image to select an entire set of features Click Add Statistics The Statistics are added to the statistics table for each population Click Close Population Statistics Object Feature Values Bright Detail Bright Detail Bright Detail ee Intensity Intensity Intensity rage R3_MO4_Ch4 R3_MC_Ch4 R7_MO4_Ch4 Median Median Median 131 1982 45 l 125 5 1958 5 45 131 1963 5 47 10 Rows can be deleted by highlighting the row and using the delete key 11 Rows or columns can be moved by click dragging 12 Right click anywhere in the table to open the menu 90 USING THE DATA ANALYSIS TOOLS 13 14 15 16 Add Statistics Edit Statistic Column Delete Statistic Column Clone Statistic Column Clear All Use Short Population Mames Copy Statistics to Clipboard
68. Properties Name NewPopulationN amg Dark Mode Color Light Mode Color E symbol Simple Dot Definition f Combined G o lt ole Ok Cancel 6 Use the toolbar to build the population definition as described in the table and click OK when done TABLE 6 POPULATION TASKS AND TOOLBAR TASK Add a population to the definition Combine two populations Select all pixels that are not in the original popula tion Affect the order of operations Remove an item from the end of the definition 110 USING THE DATA ANALYSIS TOOLS TOOLBAR Double click the population Or single click the population and select the down arrow button on the toolbar Use the Boolean AND or OR operator ml Use the AND operator to include only the pixels that are in both of the original populations Use the OR operator to include the pixels that are in either one of the original populations Use the Boolean NOT operator in The NOT operator specifies which population will not be used Use the parentheses toolbar buttons J Click the left arrow button on the toolbar 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 Manager tool Regions are drawn on graphs to create new populations based on the physical location of objects on a graph and to compute
69. RTED WITH THE IDEAS APPLICATION Y AXIS FEATURES Intensity_MC_ChX for all channels Aspect Ratio_brightfield default Aspect Ratio Intensity_MX_ChX for all fluorescence channels Intensity_scatter Aspect Ratio_brightfield ADVANCED ANALYSIS The File Menu on page 29 Viewing Sample Information on page 37 Overview of Compensation on page 38 Creating a New Compensation Matrix File on page 40 Merging Raw Image Files on page 49 Saving Data Files on page 51 Creating Data Files from Populations on page 52 Batch Processing on page 54 THE FILE MENU Use the File menu which is shown in the following figure to open save and close image and analysis files and to quit the IDEAS application Alternatively you may open a data file by drag and drop into an open IDEAS window Muliple data files can be open in one instance of the IDEAS application File Open Save Data Analysis File daf Save as Data Analysis File Used Feature Save as Template ast Save All Close Em a OPENING A RIF FILE A rif file is opened when there 1s 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 c
70. SAMPLE INFORMATION WINDOW 1 Information for th Go to Tools gt Sample Information to open the window e open data file will be loaded otherwise 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 wes Dosa Sample Information an t x EEE Select Data File NFkB Fite Dq5 No LPS Analyzed _2 daf Comections Focus Fluidics Detection Camera Settings illumination EDF Compensation Channels Raw Data File Name rf Aca Date Name cif Version Sample Processed Data File NFkB Fitc Dq5 No LPS_2 nf 3 31 2010 Version No Objects C Users sfriend Desktop 2Cam DEMO NFkB Translocation NFkB Fite Dq5 No LPS Analyzed _2 40 427 0 No Objects 4721 NFkB Fite Dg5 No LPS Analyzed _2 Show Sample Name in Graph Titles GETTING STARTED WITH THE IDEAS APPLICATION 37 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
71. SAVE A TEMPLATE 1 On the File menu click Save As Template File ast A Save File dialog box appears Enter the name of the file to save 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 Application Defaults CREATING 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 contains 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 on page 80 Note that you cannot create a new cif or rif when multiple data files are open TO CREATE DATA FILES FROM POPULATIONS 1 On the Tools menu click Create Data File from Populations The Create cif and or rif From Populations window appears 52 GETTING STARTED WITH THE IDEAS APPLICATION fet Create cif and or rif From Populations Sele Select populations Mew Raw Image File rif a l New Compensated Image File cif a E Cancel 2 Inthe Select populations list select the populations that you want to include in the new data file s Ctrl click to select multiple populations 3 To create a rif file select the New Raw I
72. Spot Intensity Max Features on page 183 and Spot Count Feature on page 170 Valley X and Valley Y Features on page 152 No The X Y coordinates of the minimum intensity within the skeletal lines that are used when creating the Valley Mask Shape Features define the mask shape and have units that vary with the feature Aspect Ratio Feature on page 154 Yes The ratio of the Minor Axis divided by the Major Axis IN EXPANDED DEFAULT TEMPLATE Yes No No Yes Yes No No No No Yes UNDERSTANDING THE IDEAS FEATURES AND MASKS 129 MASK_IMAGE USED IN DEFAULT TEMPLATE M1 Mo6 M1 Mo6 M1_Ch1 Mo6_Ch6 M1 Mo6 TABLE 2 LIST OF FEATURES BY CATEGORY FEATURE CATEGORY TEXTURE FEATURE NAME Aspect Ratio Intensity Feature on page 156 The ratio of the Minor Axis Intensity divided by the Major Axis Intensity Circularity Feature on page 156 The degree of the mask s deviation from a circle Compactness Feature on page 158 Describes the density of intensities within the object Elongatedness Feature on page 159 The ratio of the Height Width which use the bounding box Lobe Count Feature on page 160 The number of lobes in a cell Also see Symmetry Shape Ratio Feature on page 161 The ratio of Thickness Min Length features Symmetry 2 3 4 Features on page 162 These three features measure the tendency of the object t
73. TED TO 100 IN NO 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 LIABILITY 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 AGREEMENT IS FOUND TO HAVE FAILED OF ITS ESSENTIAL PURPOSE 8 U S Government End Users The Software and Documentation are commercial items as that term is defined in FAR 2 101 consisting of commercial computer software and commercial computer software documentation respectively as such terms are used in FAR 12 212 and DFARS 227 7202 If the Software and Documentation are being acquired by or on behalf of the U S Government then as provided in FAR 12 212 and DFARS 227 7202 1 through 227 7202 4 as applicable the U S Government s rights in the Software and Documentation will be only those specified in this Agreement 9 Export Law You agree to comply fully with all U S export laws and regulations to ensure that neither the Software nor any technical data related thereto nor an
74. TH FEATURE NAME Modulation Feature on page 169 Measures the intensity range of an image normalized between 0 and 1 Spot Count Feature on page 170 Enumerates the number of spots See also Raw Centroid X and Raw Centroid Y Features on page 150 Spot Area Min Feature on page 140 and Spot Intensity Min and Spot Intensity Max Features on page 183 Std Dev Feature on page 171 Describes the overall distribution of pixel intensities Signal Strength Features are measured in pixel values Bked Mean Feature on page 172 The average intensity of the camera background Bked StdDev Feature on page 172 The standard deviation of the background intensities Intensity Feature on page 173 The sum of the pixel intensities in the mask background subtracted Max Pixel Feature on page 174 The largest pixel value within the mask background subtracted Mean Pixel Feature on page 175 The average pixel value within the mask background subtracted Median Pixel Feature on page 176 The median pixel value within the mask background subtracted Min Pixel Feature on page 176 The smallest pixel value within the mask background subtracted Raw Intensity Feature on page 177 The sum of the pixel intensities within the mask Raw Max Pixel Feature on page 177 The smallest pixel intensity Raw Mean Pixel Feature on page 179 The average pixel intensi
75. The intensity concentration ratio 1s defined as the ratio of the intensity inside the first input mask to the intensity of the union of the two masks the higher the score the greater the concentration of intensity inside the first mask All pixels are background subtracted The ratio is invariant to cell size and can accommodate concentrated bright regions and small dim spots The ratio is mapped to a log scale to increase the dynamic range to values between inf inf This feature is a generalization of the Internalization feature See Internalization Feature on page 187 for more information APPLICATION EXAMPLE Quantify relative intensity concentrations between different cellular com partments Internalization 1s a special case of this where the first mask is the internal compartment and the second is the membrane region 186 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 intensity 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 on page 193 and Overview of the Mask Manager on page 94 The feature is invariant to cell size and can accommodate concentrated bright regions and small dim spots The ratio is mapped to a log
76. ULATION FEATURE The Modulation feature measures the intensity range of an image normalized between OQ and 1 The formula is Modulation Max Pixel Min Pixel Max Pixel Min Pixel The following example illustrates Modulation on brightfield images and Intensity of scatter in channel 1 Single focused cells Qs E x mam T pen l Intensity SSC ded a Modulation _BF Low Modulation APPLICATION EXAMPLE Quantify image quality and characterize contrast and texture in cells UNDERSTANDING THE IDEAS FEATURES AND MASKS 169 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 on page 201 Peak Mask on page 199 and Range Mask on page 199 for information on masking spots See also Spot Area Min Feature on page 140 Raw Centroid X and Raw Centroid Y Features on page 150 and Spot Intensity Min and Spot Intensity Max Features on page 183 for more information The following figure illustrates the application of Spot Counting to quantify parasitic infection of Babesia in erythrocytes by staining nuclei with YOYO green Brightfield Babesia YOYO1 Single Parasite T
77. 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 Populations window appears Tagged Populations Tagged Populations e A Image viewing mode C Update existing 2 All Channels A Create new 2 Select either Update existing or Create New e To Create 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 To customize the Image Gallery views images and masks on page 67 for more information 5 To add or remove an image from the tagged population double click either the image in the Image Gallery or a dot in a bivariate plot The selected channel image for each tagged cell is displayed in the viewing area of the Tagged Populations window In the Image Gallery a small smiley face icon appears on the left side of each tagged image Each tagged object is also displayed as a yellow star in a graph in the Analysis Area 6 Ifyou are updating an existing population click the Update button in the Tagged Populations window 7 When you are finished click Clos
78. _Ch1 Raw Max Pixel MC_Ch2 Raw Max Pixel MC_Ch3 Raw Max Pixel MC_Ch4 be Sort features by NI E H Cancel 4 Select the features to view 92 USING THE DATA ANALYSIS TOOLS 5 Click OK The features appear on the Object Data tab To add selected objects to the table nght click and choose Add Current Object Rows and columns can be moved by click dragging TO EXPORT OR COPY STATISTICS e Right click the statistics or features shown and then click Copy feature val ues to clipboard For more information see Exporting Data on page 119 USING THE DATA ANALYSIS TOOLS 93 USING THE MASK MANAGER This section contains the following subsections which describe how to create edit and delete a mask Overview of the Mask Manager on page 94 Creating New Masks with the Mask Manager on page 94 OVERVIEW OF THE MASK MANAGER A mask defines a specific area of an image to use for displaying feature value calculations The IDEAS application contains a Mask Manager for viewing existing masks and creating new ones 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 M1 through M6 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 mas
79. 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 9 INTENSITY MASK The Intensity mask masks pixels between the lower and upper raw intensity thresholds not background subtracted See also Threshold Mask on page 204 In the example below cell 10678 is bright and cell 11992 is dim The 50 Threshold mask is similar for both images whereas the Intensity mask 250 is quite different 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 Threshold 50 Intensity 250 1023 196 UNDERSTANDING THE IDEAS FEATURES AND MASKS INTERFACE MASK The interface mask identifies pixels in an object where the object is in contact with a second object Three input parameters are defined First the mask of one of the objects cell of interest Next the mask that covers both objects conjugate A close fitting mask using another function mask such as Object tight can be used for the cell of interest mask A brightfield mask can be used for the conjugate Finally the width of the interface mask from the contact point towards the cell of interest is entered Examples are shown below Interface bask shown on Bnoghttield Image
80. acent chan nels A type of illumination in which the sample is illuminated at angles that do not directly enter the objective On the ImageStream cell analysis sys tem 90 degree angle side scatter from the 488 nm laser provides the darkfield imagery See fluorescent in situ hybridization FISH A fluorescent dye used to label cellular constituents or specific probes of cellular constituents Light emitted by a fluorescent dye following excitation The adjustments made to remove the fluorescence emissions of a fluoro chrome into adjacent channels A physical mapping approach that uses fluorescent tags to detect the hybridization of probes with metaphase chromosomes or the less con densed somatic interphase chromatin The amplification of a detector signal The brightness level ranging from black to white of a pixel or group of pixels A pixel is equal to a half micron in length with the 40X objective 1 micron with the 20X objective and 0 33 microns with the 60X objec tive Note that 1 pixel x uum The state of a pixel that has a value at or above 1023 for the IS100 or 4095 for the ImageStream TABLE 1 GLOSSARY OF TERMS TERM DEFINITION segmentation The process of discriminating an object from its background spectral decomposi A custom set of longpass dichroic filters arranged in an angular array tion element The spectral decomposition element directs different spectral bands to laterally distinct cha
81. aken from IDEAS for a file with this problem The user must disable flow speed normalization when loading these older problematic files If this problem occurs on recently acquired files call Amnis customer support for help TO LOAD A FILE WITH INCORRECT FLOW SPEED VALUES WHEN LOADING A RIF FILE 1 Click Advanced in the opening a rif window 2 Uncheck the Perform Normalizaation checkbox in the Flow Speed section and proceed loading the file WHEN BATCHING FILES 1 Uncheck the Flow speed normalization checkbox in the Corrections section 210 TROUBLESHOOTING CHAPTER 9 Glossary TABLE 1 GLOSSARY OF TERMS TERM acquisition brightfield brightfield image brightfield calibration CCD channel charge coupled detec tor CCD coefficient of variation CV CV DEFINITION The process of collecting data from the ImageStream cell analysis system A type of illumination that uses transmitted light On the ImageStream cell analysis system this light is provided by a halogen lamp An image that 1s produced by transmitted light On the ImageStream cell analysis system this light is provided by a halogen lamp The camera channel that the brightfield image appears in The precise adjustment of instrument components based on test results for the purpose of optimizing functionality See charge coupled detector CCD One of the six physical partitions on the camera Each camera channel collect
82. ata create populations by drawing regions in graphs or by tagging objects perform Statistical analysis of data and create new features Overview of the Data Analysis Tools on page 59 Using the Image Gallery on page 60 Using the Analysis Area on page 73 Using the Statistics Area on page 89 Using the Mask Manager on page 94 Using the Feature Manager on page 101 Using the Population Manager on page 108 Using the Population Manager on page 108 Using the Region Manager on page 111 OVERVIEW OF THE DATA ANALYSIS TOOLS The IDEAS application provides a powerful tool set that allows you to explore and analyze data The rich feature set lets you create hundreds of your own features to differentiate objects and statistically quantify your results As shown in the following figure the application window is divided into three panels Image Gallery Statistics Area and Analysis Area which each provide the corresponding tools that you can use for data analysis There are muliple window layouts with resizable panels USING THE DATA ANALYSIS TOOLS 59 E IDEAS 4 0 File Guided Analysis Advanced Analysis Compensation Tools Options Reports Windows Help P 5 No pentide5 Il Batch 2 daf f Vol FS 6 OVA 50 nM6_Batch daf DER Ro Population Selected Bir z Population Statistics Object Feature Values View Similarity Similarity double comp x NFkB 7 AAD NFkB 7 AAD
83. ation The slope of the line on the plot is the coefficient in the matrix 8 If objects in the population exist that are outliers they should most likely be removed from the positive population within the compensation matrix by the fol lowing optional steps 3 Positive l l l l 20000 40000 60000 80000 Intensity MC _ChoO3 Coefficient value 9 749 Coefficient error 0 00036 Add Graph to Analysis Area The slope of the line on the plot is the coefficient in the matrix If objects in the population exist that are outliers they should most likely be removed from the positive population within the compensation matrix by the following optional steps OPTION REMOVE OBJECTS FROM THE POPULATION 1 Within Step 2 of the compensation wizard double click the coefficient to display the intensity plot 45 GETTING STARTED WITH THE IDEAS APPLICATION Matrix Coefficient Intensity Plot 12 Positive m a a A fen Ea am 2 l l l 1E 05 2 4E 05 Intensity MC Chi Coefficient value 9 071 Coefficient error p 012 Add Graph to Analysis Area 2 If you notice outliers click Add Graph to Analysis Area The plot populates in the Analysis Area 3 Return to the Analysis Area and use the region tools to draw a new region on the plot that defines a new positive control population excluding the outliers Refer to Creating Regions on Graphs on page 80 for more information e Create a new region to exclude ou
84. aw Centroid Y Features on page 150 and Spot Count Feature on page 170 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 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 UNDERSTANDING THE IDEAS FEATURES AND MASKS 183 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 BRIGHT DETAIL SIMILARITY R3 FEATURE The Bright Detail Similarity R3 feature is designed to specifically to compare the small bright image detail of two images and can be used to quantify the co localization 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 correlated in the
85. ble 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 This is for display only and does not effect the pixel values For more information on image display see Setting the Image Gallery Properties on page 64 KJ Display Properties Wizard Step 1 Set image display properties Step Progress 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 v v 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 20 GETTING STARTED WITH THE IDEAS APPLICATION APOPTOSIS The apoptosis wizard will guide you through the process of creating the features and graphs to measure apoptosis using the images of the nuclear dye and brightfield TO BEGIN DOUBLE CLICK ON APOPTOSIS Follow the instructions to open and analyze your file The analysis includes e Opening the data file e Setting the display properties e Gating on single cells e Gating on focused cells e Gating on fluorescent positive cells e Creating and graphing the features that measure apoptosis e Creating a statistics report Apoptotic cells are identif
86. bounding box See also Width Feature on page 142 See also Aspect Ratio Feature on page 154 and Shape Ratio Feature on page 161 for other shape ratios Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightWidth Thickness min Length ess min inor Axis ajor Axis APPLICATION EXAMPLES Measure object shape properties to differentiate between long and narrow versus short and thick objects Quantify the roundness of the morphology mask Identify single cells and doublets Cell classification based on shape change Identify recently divided cells in mitosis UNDERSTANDING THE IDEAS FEATURES AND MASKS 159 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 on page 162 Symmetry Lobe Count Low Low Low Low Low Low Lobe Count a Single Focused Cells APPLICATION EXAMPLE Used in cell classification studies Also used to differentiate small round cells from small square cells of similar area 160 UNDERSTANDING THE IDEAS FEATURES AND MASKS SHAPE RATIO FEATURE The Shape Ratio is Thickness Min divide
87. c 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 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 s 2 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 a 3 Click the Zoom Out Scaling toolbar button to automatically scale the graph The Zoom Out Scaling toolbar button is removed from the graph panel toolbar TO RESIZE A GRAPH e Click the sizing buttons on the graph panel toolbar A graph may be resized from small the default to medium or large The two options that are not cur rently in use are available on the toolbar TO COPY AND PASTE A REGION TO ANOTHER GRAPH 82 USING THE DATA ANALYSIS TOOLS 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 a
88. cence compensation Fluorescence compensation matrix cif daf or ctm O IDEAS Compensation Matrices 2009 ctr Cho ChO2 Ch03 0 0 042 1 22 l 1 J 0 238 0 100 200 300 400 0 09 Minimum 22 03 Maximum 24 63 0 047 Change Correction Offsets Select Change Compensation Matrix EDF C Perform deconvolution Choose EDF Kernels Spatial Alignment Perform correction Excitation Kernels Vertical 0 12107 0 03008 0 04692 0 01151 0 0015 0 Offsets Horizontal 0 05694 0 08029 0 02957 0 05552 0 07601 0 Offsets Change Alignment Offsets Camera Gains C Perform correction Corrected during acquisition 1 015 Flow Speed Number of objects to load THR Perform normalization 1000 of 1000 naa 14 Output Options Apply cell classifiers Separate single objects 0 995 4 0 39 l T l l Erase non framed objects Remove clipped objects 0 100 200 300 400 Minimum 0 9914 Maximum 1 0103 Change Brightfield Gains Save Corrections to Raw Image File Make any changes to the corrections that you need and then click OK Refer to the Troubleshooting chapter Troubleshooting on page 207 for more information about these options OPENING A CIF FILE A cif file is generated when corrections are applied to a rif file as described in Compensated Image File cif on page 14 When opening a cif file the IDEAS application calculates feature values and creates a daf file to display images and graphs When open
89. ch provide a short cut method to building commonly used graphs GETTING STARTED WITH THE IDEAS APPLICATION 17 GUIDED ANALYSIS Guided analysis consists of Application Wizards that help you to analyze your data for specific applications and Building Blocks on page 27 to define specific parameters for common graphs APPLICATION WIZARDS Application wizards are available to guide you through an analysis The wizard window is organized so that the instructions for each step are written at the top of the window the 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 Follow the instructions in the wizard to complete an analysis The following wizards are available General e Open File on page 19 Guides you through the process of opening a data file e Display Properties on page 20 available only after a file is open Automatically optimizes the display of the pixel intensities Application specific e Apoptosis on page 21 Guides you through the process of creating the features and graphs for ana lyzing apoptosis e Cell Cycle Mitosis on page 22 Guides you through the process of creating the features and graphs for ana lyzing the cell cycle and enumerating mitotic events e Co localization on page 23 Guides you through the process of creating the features and graphs for ana lyzing the co localizatio
90. 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 1023 10 bits for the first generation ImageStream or 0 4095 for the Imag eStream or 0 32 767 for EDF mode collection The limits of the graph enable you to use the full dynamic range of the display to map the pixel intensities of the image BOO agi 1000 Pixel Value l 400 l 0 200 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 inten sities to the display intensities which are in the 0 255 range Manual setting is done by Click dragging the vertical green line on the left side crossing the X Axis at 0 allows you to set the display pixel intensity to O 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 display pixel intensity to 255 for all intensities that appear to the right of that line 2 From the Image Gallery window select the object
91. ckground subtracted pixels contained 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 single focused cells q oly 1e6 He5 M4 CD71 Intensity 00 600 Max Pixel_M4_CD71 Intensity 230 000 230 000 APPLICATION EXAMPLES 174 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 FITC Cell A Cell B a Median Pixel MinPixel oos 0 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 su
92. cks converted to secs with a formula APPLICATION EXAMPLE Obtain the time taken to collect a sample 192 UNDERSTANDING THE IDEAS FEATURES AND MASKS ABOUT MASKS The set of pixels that contains the region of interest is called the mask In the following picture the mask consists of the set of pixels in the nght image that are colored 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 intensity values There are three types of masks Default masks Combined Masks and Function Masks 1 2 Default masks named M01 through M12 are created when a rif file is opened The default masks obtain a region of interest corresponding to objects in the imagery using the Object function default option described below 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 old and new default masks Image System 5 Object default 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
93. count Note that the default data directory is installation directory ImageStreamData where installation directory is the directory that you installed the IDEAS application in For example the default data directory might be C Program Files AmnisCorporation IDEAS ImageStreamData TO COPY THE EXAMPLE DATA FILES 1 Copy the data files 2 Right click the directory that contains the data files and click Properties 3 Clear the Read only check box 4 Click OK SETTING UP THE IDEAS APPLICATION 7 VIEWING AND CHANGING THE APPLICATION DEFAULTS Files are automatically saved to the specified default directory e To view or change these defaults click Application Defaults on the Options menu and the Directories tab will be displayed as shown in the fol lowing figure e To view or change the default color or symbol for populations click the Pop ulations tab Application Defaults Defaut Data Files Directory Customer Training and Application Support_1270309 5X Training 04071 Update automatically when file is selected Defaut Template Files Directory C Users stiend App Data Roaming Amnis Comoration templates E Update automatically when file is selected E Use default data directory Defaut Batch Report Files Directory C Users stiend App Data Roaming Amnis Comoration batches E Update automatically when file is selected Defaut Compensation Matrix Files Directory CAUsers sfiend App Data RoamingAmnis Corpora
94. d 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 Thickness max shape ratio Thickness min Length Thickness min See also Aspect Ratio Feature on page 154 and Elongatedness Feature on page 159 for other shape ratios Aspect Ratio Elongatedness Shape Ratio Minor Axis Major Axis HeightWidth Thickness min Length ess min APPLICATION EXAMPLE Measure object s elongatedness to provide shape classification UNDERSTANDING THE IDEAS FEATURES AND MASKS 161 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 on page 160 for more information 2 lobe symmetry 3 lobe symmetry 4 lobe symmetry Single Focused Cells aE X Single Focused Cell lE X Single Focused Cells fet E x 18 10 E i MJ T Frequency OG Frequency ns 0 i i i i i i 1 I 0 10 20 3 4 30 OO g Symnmeby 2 Ch 20 Synmeby
95. d cells Used to measure shifts in X or Y direction between two images DELTA CENTROID XY FEATURE The Delta Centroid XY feature measures the distance between the Centroid feature of two images using the user provided masks for each image Either one or both the centroids of the images may be intensity weighted X and Y pixel coordinates are calculated from an origin in the upper left corner to obtain the centroid positions and the distance between the centroids is converted to microns In the example below an image pair is shown stained with the nuclear dye Draq 5 and a PE labeled antibody that is differentially expressed two cells either uniformly or in the pseudopod The two cells are identified by their different Delta Centroid XY values UNDERSTANDING THE IDEAS FEATURES AND MASKS 147 PE Podo Composite gt pz O Soe QO Composite Delta Centroid Y Delta Centroid X Centroid X Delta Centroid XY v Delta Centroid X Delta Centroid Y Below is an example of using the Delta Centroid XY A bivariate graph of a shape ratio versus Delta Centroid XY can identify cells with caps as shown here Shape Ratio BF 6 9 12 Delta Centroid XY_PODO DRAGS APPLICATION EXAMPLES 148 UNDERSTANDING THE IDEAS FEATURES AND MASKS Quantify the spatial relationship between two fluorescent probes Identify false apoptotic positive cells in the TUNEL and Annexin V assays Quantify shape change
96. d 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 BKGD MEAN FEATURE The Bkgd Mean feature estimates the average camera background level in an image by taking the mean of the background pixels APPLICATION EXAMPLES Obtain estimate of the mean camera background level Compute background subtracted pixel values in other feature computa tions BKGD STDDEV FEATURE The Bked Std Dev feature estimates the standard deviation of the camera background level in an image computed using the background pixels APPLICATION EXAMPLE Obtain estimate of the camera background noise 172 UNDERSTANDING THE IDEAS FEATURES AND MASKS INTENSITY FEATURE The Intensity feature is the sum of the background subtracted pixel values within the masked area of the image Brightfield Scatter CD45 FITC CD14 PE DRAGS O wu tz k TH A _ vy Cc a Intensity CD45 APPLICATION EXAMPLES Quantify relative levels of fluorescence between cells and within different regions of the same cell Immunophenotyping Cell cycle analysis Protein expression Protein activation UNDERSTANDING THE IDEAS FEATURES AND MASKS 173 MAX PIXEL FEATURE The Max Pixel feature is the largest value of the ba
97. data files into an open instance of IDEAS Open data files containing up to 12 channels of imagery daf files can be used as a template for loading data files 3 Guided Analysis New application wizards Open File Display Properties Apoptosis Cell Cycle Mitosis Co localization Internalization Nuclear localization Shape change New building blocks to generate graphs with recommended feature choices and default scaling Single cells Focus Fluorescence positives 4 Statistics and Reporting New statistic Concentration Reports the concentration of a population View and export statistics for multiple populations or feature values for multiple objects in the statistics area New mean and median RD statistics available in the statistics area View and export graphs in the analysis area with a white light mode or dark dark mode background 5 Image Display Display settings accommodate 10 and 12 bit imagery as well as higher pixel intensities that result from EDF deconvolution Multiple objects in an image are now separated into individual objects Objects in the image gallery are vertically and horizontally centered Object numbers appear in the upper left corner of an image and may be turned off for reporting Zoom option to enlarge images in the image gallery and in the analysis area Unlimited columns in Viewing modes in the image gallery Any mask can be as
98. dicated by one of the following icons j O 6 The definition of a selected population is shown in the Definition area TO EDIT THE DISPLAY PROPERTIES OF A POPULATION 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 1 2 3 4 Click a display symbol in the Symbol drop down menu 5 Click Close to save the population changes 6 Click Revert to reject the changes 108 USING THE DATA ANALYSIS TOOLS TO DELETE A POPULATION 1 Within the Population Manager click a population in the Populations list 2 Click Delete A confirmation warning message appears indicating all the dependent populations that will also be deleted 3 Click Yes to confirm TO CREATE A NEW COMBINED POPULATION 1 Within the Population Manager Analysis gt Populations click New The right side of the Population Manager window changes to allow you to define a new population Properties Mame Dark Mode Color Light Mode Color g symbol Simple Chat Definition Combined Enter a unique population name in the Name box 2 3 Click a Color square to select a new color on the color palette and click OK 4 Click a display symbol in the Symbol drop down menu 5 Select Combined for this combined population The toolbar for creating a combined population appears USING THE DATA ANALYSIS TOOLS 109
99. ds 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 1s converted to microns An example is shown below PE Podo Composite PE Podo gt O O p au o QO Delta Centroid Y Delta Centroid X Centroid X Delta Centroid XY v Delta Centroid X Delta Centroid Y The graph below illustrates using the Delta Centroid X versus Delta Centroid Y to identify cells with a variation of location of a protein with respect to the nucleus Cells with no spatial shift of signal between the nuclear stain Ch6 and protein of interest Ch4 have a low Delta Centroid X and Y and are found in the lower left corner Cells with a large shift between the images in both the X and Y direction are found in the upper right section and those with a large shift in X but not Y are found in the lower right Similarly a cell with a large shift in the Y direction and not X are found in the upper left See Delta Centroid XY Feature on page 147 to measure the X and Y shift together 146 UNDERSTANDING THE IDEAS FEATURES AND MASKS Single Focused cells w a 5 v 2 oO s a a 4 6 Delta Centroid _Ch4Ch6 APPLICATION EXAMPLE Used to identify capped versus not cappe
100. e 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 limited to adding new features or otherwise making adaptations that alter the functioning of the Software b transfer sublicense lease lend rent or otherwise distribute the Software to any third party or c make the functionality of the Software available to multiple users other than the users of the single computer for which it is licensed through any means including but not limited to uploading the Software to a network or file sharing service or through any hosting application services provider service bureau software as a service SaaS or any other type of services You acknowledge and agree that portions of the Software including but not limited to the source code file formats and the specific design and structure of individual modules or programs constitute or contain trade secrets of Amnis and its licensors Accordingly you agree not to disassemble decompile or reverse engineer the Software or data files in whole or in part or permit or authorize a third party to do so except to the extent
101. e 116 Generating a Statistics Report using daf Files on page 118 Exporting Data on page 119 PRINTING REPORTS 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 the light mode 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 The IDEAS application prints all the images that are visible in the Image Gal lery CREATING REPORTS AND EXPORTING DATA 113 TO MAP THE DARK MODE COLORS TO LIGHT MODE COLORS 1 Select Options gt Manage Color Schemes The Modify Reporting Color Scheme window appears E Modify Reporting Color Scheme EER Select Dark Mode Color Select Light Mode Color Mapping O Blue Update All Populations Reset To Standard 0R 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 If you want to return the settings to the IDEAS defaults click Reset
102. e 64 to optimize the image display before copying images TO COPY THE IMAGE GALLERY DATA TO THE CLIPBOARD e Right click anywhere in the Image Gallery and then click Copy Displayed Images to Clipboard The IDEAS application copies all the images that are visible in the Image Gal lery to the Clipboard 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 TO COPY A GRAPH AND OR STATISTICS TO THE CLIPBOARD 1 Select light or dark mode graphs in the analysis area using the tool or selecting Use Light Mode Graphs in the Reports menu 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 Legend Statistics Cursor Show Sample Name in Title Size scaling factor 2 i Cancel 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 Click OKto copy the graph and or the statistics to the Clipboard CREATING REPORTS AND EXPORTING DATA 119 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 TO EXPORT GRAPH STATISTICS TO THE CLIPBOARD e Right click a graph and then cl
103. e created Data analysis file 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 templates for specific experimental procedures 34 GETTING STARTED WITH THE IDEAS APPLICATION 4 Change the Data analysis file name if necessary The default name matches the name of the cif 5 Click OK During the opening ofa 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 1s saved See also Saving a Compensated Image File cif on page 52 OPENING A DAF FILE A daf file contains the calculated feature values so that they will not need to be recalculated as described in Data Analysis File daf on page 14 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 on page 19 otherwise 1 From the File menu choose Open or drag the file in
104. e in the Masks list and click the leftmost down arrow button on the toolbar Combine two Use the Boolean AND or OR operator masks ml 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 Use the Boolean NOT operator that are not in the g The NOT operator specifies which mask will not be original mask used Aftect the order Use the parentheses toolbar buttons of operations OCI Remove an item Click the left arrow button on the toolbar from the end of D the definition ft Mask Manager Mame ME nd M3 Or Combined Mask Definition Function A Oy OO 3 ee ME And M3 Or Combined Mask Combined Mask Not Combined Mask Dilate M1 14 4 Add masks and Boolean logic to the definition as needed 5 Click OK to add the definition to the Masks list 6 Click Close USING THE DATA ANALYSIS TOOLS 97 EXAMPLE OF CREATING A MASK Here is an example of creating a mask of the cytoplasmic membrane In this example cells were stained with a green intracellular marker in Channel 3 and a red nuclear dye in Channel 5 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 Grayscale Images System Masks Turquoise Overlay Channel 3 Channel 5 Mask M3 Mask M5
105. e in the Tagged Populations window USING THE DATA ANALYSIS TOOLS 71 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 72 USING THE DATA ANALYSIS TOOLS USING THE ANALYSIS AREA This section contains the following subsections which describe how to create graphs analyze images and use text panels in the Analysis Area of the IDEAS application Overview of the Analysis Area on page 73 Creating Graphs on page 75 Creating Regions on Graphs on page 80 Analyzing Images on page 85 Adding Text to the Analysis Area on page 88 OVERVIEW OF THE ANALYSIS AREA The Analysis Area provides display space for individual images plots of cellular feature values visualizations of population distributions and statistics and text annotations You can select different layouts for the IDEAS window and placement of the analysis area and expand the Analysis Area by dragging it s boundaries The Analysis Area is divided into panels of a fixed size The size 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 As illustrated by the following figure the Analysis Area can contain seven types of panels histogram histogram overlay scatter plot channel image composite image and
106. e statistics for each required data 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 fei Generate Statistics Report using daf Files Report title Report template Ea doube otek Mere fo add ies Add Files Remove Files OF Cancel 2 Change the Report title or Report template if necessary The template may be obtained from a daf or ast file 3 Additional daf files can be added or removed with the Add Files or Remove Files buttons 4 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 gener ated 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 118 CREATING REPORTS AND EXPORTING DATA EXPORTING DATA The IDEAS application allows users to export feature data pixel data or TIF files for separate analyses You can export graphs statistics and images to other applications See Setting the Image Gallery Properties on pag
107. e 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 Gal lery Continue to add images as desired To remove and image from the composite Click Remove Image The composite is automatically added to the list A composite can be removed from the list by clicking Delete CON DN a A Continue customizing the Image Gallery display properties with another proce dure in this section or click OK to finish and save changes or Cancel to finish and discard changes USING THE DATA ANALYSIS TOOLS 69 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 Display Single Image Show Masks Color On Hide Saturation Color Copy Image to Clipboard Copy Column to Clipboard Copy Column Image to Clipb
108. ean Mean CD45 1 CD8 NFkB AAD CD45 1 CD8 NFkB AAD CD45 1 CD8 NFkE7 AAD oO a Q Q Q Aspect Ratio Intensity_7 44 Population b Focus amp R1 amp R3 amp Focus amp eie e E L L E 1e3 0 1e3 1e4 1e5 Intensity_CD45 1 gt o o 9 o W i O z i Bis oo 2 oon tf o Als s Hie ale wo E 2 we R You can create populations of objects by tagging hand selected images drawing regions on graphs and using Boolean logic to combine existing populations Another way to create a population of objects is by basing it on the similarity of a set of feature values to one or more cells in the data set 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 the Statistics Area Graphs show data plotted with one or two feature values and tools are provided that allow you to draw regions for the purpose of generating new populations You can show any population on a plot 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 USING THE IMAGE GALLERY This section contains the following subsections which describe how to view populations of objects in various ways view masks customize the Image Gallery dis
109. eatures based on the images used Sorts features based on the masks used Sorts features by category such as size location shape texture signal strength and system Sorts by base features such as area aspect ratio intensity and object number 102 USING THE DATA ANALYSIS TOOLS 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 1s enabled Feature Type Single Angle r C Combined Mame FO Mask Combined Mask be Set Default Hame OF Cancel 2 Select Single as the Feature Type The Mask and Image lists become visible depending on the single feature selected Feature Type Single M C Combined Name Mask Combined Mask Image 1 Channel 1 ad Image 2 Channel 1 bt Select the mask and or image that you want Enter a unique feature name or click Set Default Name The default name is the name of the base feature followed by the name of the mask and name s of the image s 5 Click OK to add the new feature It appears in the Features list on the left side of the Feature Manager 6 Click Close Note When you close the Feature Manager the IDEAS application calculates val ues for the new features These calculations may take several minutes depending on the number and complexity of the new
110. ecn a aac 186 Internalization Feature 44 wee cosh Med Bed ae a ee 187 Sian aide Tee uor asa Maree he eal tak See Sch hess hak te ce Gh te chen 188 De GO edly Me ngs cae a a Dee Sete a ds ante ag a an a es PA tence tech alee au 190 Understandings the System Features 4 0 04 usc tgeee dad oad oes E 191 Camera Line Number Featur o oo occae eae ei ee SV E a 191 Camera Finer Peattre da ia seeks aair a E S 191 Fow Speed Ferie eaei aea r a Vewat 191 Object Number Feature 24 ve 2 54 Ye bs Re Rare REG A 191 ODjects ml Pen ure 655 5 Sot k Sane Sees E A A S Se 191 Objecte C Tee e ea a sra ar Ma ees ME es RE eS eh os 192 Emmet aUe cS olay tt aa ea a ae ie Ss an GE a a a eae eae A 192 ADOTE IVS keea 3 ee Sree Soe we ties So Wiech ee ee n 193 CHAPTER 8 CHAPTER 9 Etol Funcion Maks 6 3 iy bed doe ew dao the ak bak eos ewok wae 195 Mate MASE wae iat es ak ees Bae eos ain ee Ay See Ses 195 Prode NIE saye ae ra tes ee area toate sat teh ula ere nd eta tgs Ae ted tec Be eee 195 E E EE canes E E E E E E E E EEE 195 Mspr Mikese rekaan eka eae eee N A EAR EEA 196 eME 15a Rots Seer ac Shes a eas oes oe ene a 196 Mea ACe MIE aaor cara eon oc E eee a aes totale Oe 197 Morpnoloey Mask zerer nita isis NN ee WE EEE oe 198 CGI ete oe cork a See ts See oe he en eae ee Dean ae eee SUE 198 NG teehee aes ata ar ee ae Ra a ata honey isn alata ah tae tna eh Acetic 199 Rance Ma Kn eld cates cate ae ue test ca es acetic a beatae ee ee gas 199 Skelm Maik J dec
111. ee hard A 167 H Texture Fearne 2 6 8 ot ee i he os Bo ek a a a 168 Modulation Feature 2 0 0 169 Spot COMME TEIE ns tae ars wee wa ee a ee A A ae 170 oA DPev Ferer of van ese ya eb ad Ae od aed eS re ea a 171 Understanding the Signal Strength Features 000000005 172 Bhed Mein Feat cc cee pd Sau ele su ota E ooo a 172 kod Stdlev Peas vrsi enerne ely pais in ten eee ee a 172 TAGES ICY FOUTE os ine one on E gc re aan Aine ln Bove ass ames esas ee 2G SS 173 Max Pixel PSat aire ieena idana Sent Setanta te de a de a Stace 174 Mear Piz Feno 0 2 ob ut th Yee OMe te SM eM aM be 175 Medon Pixel Features cccce 5 ter aee tene ie See os ie eee ad eke S Sone 176 Win Pixel Peat 2 o 5 0 5 oso bh wed so S Se oe ee ee ee HOSES 176 Raw Intensity Ferie cear eare kaa Aces ao Slee we dee ae Re ae ae 177 Raw Max Pixel Featite sence renia enra SES Pw SEY Go eS Go Bee 177 Raw Mean Pixel Perre o eate a eat eee eee So 179 Raw Median Pixel Feature wpa toe unsa e v6 CSS GER OETA 179 Raw Min Pixel Feature 65 644 44664 40654 4466466624 aL 180 Saturation Count Feat re rsierryprdirpori t eria pania Eaa 181 Saturation Percent Features e aa Ste i ei ee i ee er 182 Spot Intensity Min and Spot Intensity Max Features 183 Understanding the Comparison Features 26 evo 24 Seder OES SS St Shea 184 Bright Detail Similarity R3 Feature 0 002 0000000005 184 Intensity Concentration Ratio Feature senec 21h digas I E cas
112. eleton Mask on page 200 e Spot See Spot Mask on page 201 e System See System Mask on page 203 e Threshold See Threshold Mask on page 204 e Walley See Valley Mask on page 205 USING THE DATA ANALYSIS TOOLS 95 se Define Mask Function Function Select the object number and channel image to Dilate use as a background for digplaying the mask Mask Object 0 Ch 1 Number of Fisels 1 l l l l 0 5 10 15 OF Cancel 4 Select a mask and change the scalar parameters as needed The right side of the window adjusts the display and view of the channel image e To view a different object in the file select it in the Object list e To view a different channel image for the object select it in the Ch list Click OK The new function is added to the mask definition Click OK Review your information and click OK COON DBD UI The new mask name will appear in the list of Masks on the left side TO CREATE A NEW COMBINED MASK 1 Select Analysis gt Masks 2 Click New The Name box and Definition toolbar become enabled 96 USING THE DATA ANALYSIS TOOLS 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 3 MASK TASKS AND TOOLBAR TASK TOOLBAR Add a mask tothe Double click the feature in the Masks list definition Or single click the featur
113. es other types of files that are used for data correction and presentation 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 on page 8 for more information SAVING A DATA ANALYSIS FILE DAF A daf file contains a snapshot of an analysis as described in Data Analysis File daf on page 14 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 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 On the File menu click Save as Data Analysis File daf 2 Enter a 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
114. essage appears 7 Click OK MERGING COMPENSATED IMAGE FILES You can merge cif files together for analysis TO MERGE CIF FILES 1 On the Tools menu click Merge rif Files The Merge Raw Image Files window appears E Load Multiple cif Files Files to Load Select cif files to load Enter the number of objects to load from each file A population will be created for each file Specify the population name File eects Population Name the output files to be created To use a custom template for analysis Compensated image file cif Select a template or data analysis file ast daf fae J Data analysis file dat 2 To select the cif files to merge click Add Files 50 GETTING STARTED WITH THE IDEAS APPLICATION The cif file names appear in the list If you want to remove a file from the list select it and then click Remove File Type a unique name for the output files Select a template Click OK The merged files are created and the new daf file is loaded with a population cre ND OF A OW ated from each file 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 populations Compensated image files and Data analysis files are saved when opening rif files opening multiple cif files using the file menu or when running a batch analysis The IDEAS application also sav
115. etaphase plates in the bright image are masked Threshold 50 Intensity 250 1023 APPLICATION EXAMPLE Used with the Area feature to define apoptotic cells 204 UNDERSTANDING THE IDEAS FEATURES AND MASKS VALLEY MASK The Valley mask is a rectangular mask that sits between two bright regions in a starting 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 on page 200 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 1s defined by the user in pixels APPLICATION EXAMPLE Quantify the intensity of a probe in an immune synapse 205 UNDERSTANDING THE IDEAS FEATURES AND MASKS 206 UNDERSTANDING THE IDEAS FEATURES AND MASKS CHAPTER 8 Troubleshooting This chapter covers common issues and provides solutions Application Hanging on page 207 Compensation on page 207 Object Number set to Zero on page 209 Object Number set to Zero on page 209 Delay in Copy Paste on page 209 Images and brightfield channel appear uniformly bright on page 210 APPLICATION HANGING If the IDEAS applicatio
116. event in that population The exported TIF files can be opened in image viewing applications that support 16 bit Tif format TO CREATE TIFS FROM POPULATION FOR EXPORT 1 On the Tools menu click Export tif Images CREATING REPORTS AND EXPORTING DATA 121 The Create TIFs From Population window appears Ee Create TIFs From Population Select population Select Channels CHO Choe CHO 2 Select the population and channels 3 Type a prefix for the TIF file name 4 Select the bit depth 5 Select padded or raw 6 Click OK TIF Settings File name pretis EEE Bit Depth bit for display 16 bit for analysis Pixel D ata padded for display O raw for analysis A TIF file is created for every selected channel within the selected population 122 CREATING REPORTS AND EXPORTING DATA CHAPTER 7 Understanding the IDEAS Features and Masks This section contains the following subsections which describe the features that the IDEAS application uses for data analysis Overview of the IDEAS Features and Masks on page 124 The Base Features at a Glance by Category on page 128 Understanding the Size Features on page 134 Understanding the Location Features on page 143 Understanding the Shape Features on page 154 Understanding the Texture Features on page 163 Understanding the Signal Strength Features on page 172 Understanding the System Features o
117. ey while selecting the files e To remove 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 ND OF A If the template contains a Statistics Report template click on the Preview Statis tics Report button Order the files as you wish them to be reported by selecting a file with a left click then right click the desired position and select move here See Creating a Statistics Report Template on page 116 for more information 8 Click OK 55 GETTING STARTED WITH THE IDEAS APPLICATION The Define a Batch window closes The batch appears in the Batches window Batches Batches ta Run Add Batch Processed Batches HE Batch 12 21 2010 10 10 02 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 o Unprocessed o Proces
118. f w IDEAS files nif cit daf Faw image files rif Compensated image files cif Data analysis files daf Files of type 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 30 GETTING STARTED WITH THE IDEAS APPLICATION 3 6 ree Opening C Training Data Files 3 0 NFKB Translocation Dose an Sele To perform fluorescence compensation Select a compensation matris compensated image file or data analysis file ctm cif dat O E Or Create a compensation matris from control files New Wlatris To uze a custom template for analysis Select a template or data analysis file ast dat Oo D Name the output files to be created Compensated image file cif Data analysis file dat Enter the number of objects to process 1000 of 1000 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 generated from the controls used for the experiment If you leave it blank the default compensation matrix will be used but this is not recommended unless you do not want to compensate your data e Ifa compensation matrix for the experiment has not been made click New Matrix For more information on creating a compensation matrix see Creat ing a New Compensation Matrix File
119. f interest of the image A few system features such as Object Number Camera Background and Flow Speed do not require calculations masks or image intensity information 124 UNDERSTANDING THE IDEAS FEATURES AND MASKS ABOUT FEATURES The IDEAS application provides a large selection of criteria or features for analyzing images A feature is described by a mathematical expression that contains quantitative 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 Background and Flow Speed do not require calculations masks or image intensity information Features are created in IDEAS using base feature algorithms such as Area or Intensity along with a mask and or a channel image New masks and features can be created by the user using the Mask Manager and Feature Manager tools New features can be created by combining existing features in mathematical expressions using the Feature Manager For more information see Using the Mask Manager on page 94 and Using the Feature Manager on page 101 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 diagram represents a pixel that equals approximately 0 5 um X 0 5 um Each channel is 88 pixels
120. features and the size of the image file USING THE DATA ANALYSIS TOOLS 103 TO CREATE MULTIPLE FEATURES 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 gt oO N e Select one image or check the box to create for all channels using default masks and images FS Add Features Select base features Select feature inputs B f Texture C Create for all channels uzing default masks and images Bright Detail Intensity R3 Bright Detail Intensity A7 Contrast Gradient Max 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 H Variance Mean Fal U tienswan Cr ill Select masks Sort Order Alphabetical Category Clear Selected Clear Selected Add Features 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 104 USING THE DATA ANALYSIS TOOLS a Confirm Feature Creation The following features will be created if they do not already exist Do vou want to continue Bright Detail Intensity A3_ Mo4_C h4 Bright Detail Intensity AY_MO4_ Ch Contrast_MO4_ Ch
121. file O 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 You may repeat editing the matrix and previewing until satisified When done click OK and save the matrix MERGING RAW IMAGE FILES You can merge rif files together for analysis TO MERGE RIF FILES 1 On the Tools menu click Merge rif Files The Merge Raw Image Files window appears Merge Raw Image Files Raw Image Files to Merge Select raw image files to combine rif file will be created containing the objects in each file Add Files Remove File 2 To select the rif files to merge click Add Files The rif file names appear in the list 49 GETTING STARTED WITH THE IDEAS APPLICATION If you want to remove a file from the list select it and then click Remove File When the merge list is complete click OK The Save Merged Raw Image rif File dialog box appears Type a unique file name Click Save The Creating merged rif file window appears Creating merged rif file C Documents and Settings Marita My Docum Sele Hf files to merge i C Documents and Settings MartayhMy Documents 4nmnisBookFile unezO Demo set for Marta merg O CADocuments and patings M antaMy Documents4nmnis BookFile unezO Demo set for Martas 0606 i gt O Unprocessed In process Processed Cancel When the merge is complete the Merged rif Created m
122. file rifj compensated image file cif and data analysis file daf This section describes each file type and the table summarizes the features of each file OVERVIEW OF THE IDEAS APPLICATION 13 RAW IMAGE FILE RIF The INSPIRE application saves the image data that were acquired by the ImageStream cell analysis system 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 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 boundaries 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 corrections 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 multi composite imagery and the calculation of multi feature values such as the Similarity value e Coincident objects are cut apart to place into individual image frames Note that this will increase the number of objects in the
123. g image in the Image Gallery if the popu lation that is currently displayed in the Image Gallery contains that point Click an image to display the corresponding statis tics in the Statistics Area Click the top of a bin in a histogram to select the bin In the Image Gallery you can view images of cells in the bin by clicking the Selected Bin popu lation Click Pointer Tool while drawing a region on a graph to cancel the creation of a region Allows you to create a population of hand picked objects For more information see Creating Tagged Populations on page 71 Creates a new histogram Refer to To create a graph on page 75 Creates a new scatter plot Refer to To create a graph on page 75 Allows user to add text notes to the Analysis Area Refer to Adding Text to the Analysis Area on page 88 Draws a horizontal line on a histogram to define a region Draws a rectangular region on a scatter plot 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 TABLE 2 ANALYSIS AREA TOOLS TOOL DESCRIPTION Changes the background of the graphs to black or white Graph Bked Tool Tiles graphs in the analysis area after changing the size of the analysis area to fit all graphs to the new Tile Graphs Tool space Chooses a layout for
124. ge 191 Camera Timer Feature on System Intensity Concentration Ratio Comparison page 191 Feature on page 186 Centroid X and Centroid Y Location Intensity Feature on page 173 Signal Strength Features on page 144 Centroid X Intensity and Location Internalization Feature on Comparison Centroid Y Intensity Features page 187 on page 145 Circularity Feature on Shape Length Feature on page 136 Size page 156 Compactness Feature on Shape Lobe Count Feature on page 160 Shape page 158 Major Axis and Minor Axis Size Raw Median Pixel Feature on Signal Strength Features on page 137 page 179 126 UNDERSTANDING THE IDEAS FEATURES AND MASKS TABLE 1 FEATURES LISTED ALPHABETICALLY FEATURE NAME CATEGORY FEATURE NAME CATEGORY Major Axis Intensity and Saturation Count Feature on Signal Strength Minor Axis Intensity Features page 181 on page 138 Max Contour Position Fea Location Saturation Percent Features on Signal Strength ture on page 149 page 162 Max Pixel Feature on Signal Strength Shape Ratio Feature on page 161 Shape page 174 Mean Pixel Feature on Signal Strength Similarity Feature on page 188 Comparison page 175 Median Pixel Feature on Signal Strength Spot Area Min Feature on Size page 176 page 140 Min Pixel Feature on Signal Strength Spot Count Feature on page 170 Texture page 176
125. hannel 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 1s 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 208 TROUBLESHOOTING 5 Click Preview and choose the tagged population to view the results of the changed coefficient Repeat steps 4 and 5 until the matrix is corrected Click Save append manual to the matrix name then click OK 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 DELETING A POPULATION AND REGION Often a user deletes a population but forgets to delete the region Deleting a population does not delete the region You must delete the region itself DELAY IN Copy PASTE When copying and pasting histogram information to a clipboard you may experience a delay In this case there may be t
126. her 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 communications 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 contact 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 SOFTWARE PLEASE CLICK THE I ACCEPT BUTTON OTHERWISE PLEASE CLICK THE I DO NOT ACCEPT BUTTON AND THE INSTALLATION PROCESS WILL STOP DISCLAIMERS The screen shots presented in this manual were created using the Microsoft Windows XP operating system and may vary sligh
127. hoose a compensation matrix at this time by using the control files that were collected for a particular experiment For more information refer to Creating a New Compensation Matrix File on page 40 The application performs the corrections by using calibration information that was saved to the rif file during acquisition GETTING STARTED WITH THE IDEAS APPLICATION 29 TO OPEN A RIF FILE To use a wizard to do this see Open File on page 19 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 window to rif files Select File To Load Lookin Of gt m 0 0ng_2_9 rif 121906 C16 72 06 DRAQS _ noBF4 rif I 2 O 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 if Documents o 1ng 45_11_18 riF O 1ng 60_16_23 rif O 1ng 75_21_4 rif 0 1ng 90_26_9 tiF 10mg 15_3_10 rif 10ng 30 _8_15 rif 10ng 45_13_20 rif 10ng 60_18_1 rif 10ng 75_23_6 rif 10ng 90 _28_11 rif 1000ng 15_5_12 rif 1000mg 30_10_17 rif 1000ng 45_15_22 rif 1000ng 60_20_3 rif 1000ng 75_25_8 rif 1000ng 90_30_13 rif My Documents hy Network Places File name Ding 15_1_8 rif v Raw image files ri
128. ial Image Display Mapping Aas Scale Set Range to Fisel Data Full Scale Set Linear Curve Autoscale 4 Ifyou want to preview the changes in the Image Gallery click Preview Changes in Gallery 5 Continue customizing the Image Gallery display properties with another procedure in this section or click OK to finish and save changes or Cancel to finish and dis card changes TO CUSTOMIZE THE IMAGE GALLERY VIEWS IMAGES AND MASKS 1 Within the Image Gallery Properties window click the Views tab Note The Image Gallery view can be customized to view any combination of channel images or composites The default view All Channels is a view that displays all image channels that were included during acquisition of the file with their associated default masks This mask may be changed for the default view however the images in this view cannot be changed USING THE DATA ANALYSIS TOOLS 67 vine Image Gallery Properties Display Properties Views Composites Views View Definition BF Name All Channels Ch2 Ch4 DRAQS cH NFkB H SSC Column S All Channels i SSC mask M01 Ch2 mask M02 i NFkB mask M03 i Ch4 mask M04 BF mask M05 i DRAGS mask MOB Preview Changes in Gallery To create a new view Click New 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 Oo A WwW N dropdown menu
129. ick Export Statistics To Clipboard TO EXPORT POPULATION STATISTICS OR OBJECT FEATURE VALUES FROM THE STATIS TICS AREA e Right click the table and then click Copy data to clipboard TO COPY THE ENTIRE SCREEN TO THE CLIPBOARD e Press CTRL PRINT SCREEN TO COPY A WINDOW TO THE CLIPBOARD e Select the window and then press ALT PRINT SCREEN EXPORTING FEATURE DATA You can export feature values for a population to the Clipboard a text file or a Flow Cytometry Standard FCS file You can export pixel intensity values for an object to the Clipboard or a text file Later you can open or paste the FCS file into a spreadsheet or other programs that uses the FCS file format Keep in mind however that limitations might exist on the number of values that these programs can import TO EXPORT FEATURE DATA 1 On the Tools menu click Export Feature Values The Export Feature Data window appears Fs Export Feature Data EBR Select daf files to process Select features to export Area _ MOT Area_MO Area_MO6 Area_MC Aspect Ratio Intensity MOT Cho Aspect Ratio Intensity _MO2 Choe Aspect Ratio Intensity MOB _CHOE Aspect Ratio M1 Aspect Ratio MO Aspect Ratio M06 Bkod Mean _Cholz Bkod Mean _ChOE Bkgd StdDey_ChO1 Select a population All Sort features by Export to Order by Clipboard Object C Export all used features Text File Feature Export all features 120 CREATING REPORTS AND EXPORTING
130. ied in the final graph presented by the wizard An example 1s shown below Apoptotic cells have low nuclear area and high brightfield contrast ADE Ce ON i ap Dd e w P 1 GETTING STARTED WITH THE IDEAS APPLICATION 21 CELL CYCLE MITOSIS The cell cycle mitosis wizard will guide you through the process of creating the features and graphs to analyze the cell cycle and identify mitotic events using the images of a nuclear dye TO BEGIN DOUBLE CLICK ON CELL CYCLE MITOSIS Follow the instructions to open and analyze your file The analysis includes e Opening the data file e Setting the display properties e Gating on single cells e Gating on focused cells e Gating on fluorescent positive cells e Creating and graphing the features that measure cell cycle and mitosis e Creating a statistics report Normalized Frequency m tes 1 Set 2e4 Bright Detail intensity R3_MC_Channel 6 Rago sista x 2504 aes E EEEIEI Area_Threshold M06 Channel 6 50 Channel 6 i y i y J J 7 i r J f r 0 10 20 3 40 60 70 Contrast_M04_ Channel 4 22 GETTING STARTED WITH THE IDEAS APPLICATION CO LOCALIZATION The co localization wizard will guide you through the process of creating the features and graphs to measure the co localization of two probes in any population of cells you identify TO BEGIN DOUBLE CLICK ON CO LOCALIZATION Follow the instructions to open
131. ile Click Add The information pairs in the Statistic Columns area Add Statistics Columns as necessary Adjust the Statistic Columns as necessary e Edit or double clicking allows changes to a previously created statistic e Delete removes a selected statistic e Right clicking a statistic and drag and dropping it to a new location changes the order of the columns as they will appear in the report table M Statistic Columns RB A 7 Cancel GeoMean 44ngle gdouble click here fo add e To reorder longer lists hold the Ctrl key for an individual item or the shift key for multiple items and click each individual statistic in the desired order Then right click and select Move Here 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 If you do not want to generate a report click OK to save your changes and exit the window The saved template can generate statistics for multiple data files during batch pro cessing See Batch Processing on page 54 for more information CREATING REPORTS AND EXPORTING DATA 117 GENERATING A STATISTICS REPORT USING DAF FILES Once a Statistics Template has been created the user can generate a statistics report from multiple daf files However these files must use the same template The Batch Processing feature can also generate a statistics report wher
132. in the X direction and varies in the Y direction depending on the size of the imaged object IDEAS groups the features into eight categories size location shape texture signal strength comparison system and combined UNDERSTANDING THE IDEAS FEATURES AND MASKS 125 THE BASE FEATURES AT A GLANCE SORTED ALPHABETICALLY TABLE 1 FEATURES LISTED ALPHABETICALLY FEATURE NAME CATEGORY FEATURE NAME CATEGORY Angle Feature on page 143 Location Contrast Feature on page 165 Texture Angle Intensity Feature on Location Delta Centroid X and Delta Cen Location page 143 troid Y Features on page 146 Area Feature on page 134 Size Delta Centroid XY Feature on Location page 147 Aspect Ratio Feature on Shape Diameter Feature on page 135 Size page 154 Aspect Ratio Intensity Fea Shape Elongatedness Feature on Shape ture on page 156 page 159 Bked Mean Feature on Signal Strength Flow Speed Feature on page 191 System page 172 Bked StdDev Feature on Signal Strength Gradient Max Feature on Texture page 172 page 166 Bright Detail Intensity R3 and Signal Strength Gradient RMS Feature on Texture Bright detail Intensity R7 Fea page 167 tures on page 163 Bright Detail Similarity R3 Comparison Height Feature on page 136 Size Feature on page 184 Camera Line Number Fea System H Texture Features on page 168 Texture ture on pa
133. ing a cif file an analysis template is selected The template provides the initial characteristics of the analysis Opening the cif file causes the IDEAS 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 on page 19 otherwise 1 From the File menu choose Open or drag the file into the IDEAS window GETTING STARTED WITH THE IDEAS APPLICATION 33 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 window to cif Select File To Load 7 0 0ng_2_9 cif B o 1ng 15_1_8 cif My Recent 0 1ng 30_6_13 ciF Documents 0 1ng45_11_18 cif O 1ng 60_16_23 cif O 1ng 75_21_4 cif o 1ng 90_26_9 cif 10ng 15_3_10 cif 10ng 30_8_15 cif 10ng 45_13_20 cif 10ng 60_18_1 cif 10ng 75_23_6 cif 10ng 90_28_11 cif 1000ng 15_5_12 cif 1000nq 30_10_17 cif 1000ng 45_15_22 cif 1000ng 60_20_3 cif 1000ng 75_25_8 cif i 1000ng 90_30_13 cif hy Network Places File name Files of type In the next window you will e Choose a template e Name the output file Opening 10ng 60_16_1 cif To use a custom template for analysis Select a template or data analysis file ast daf Name the analvsis file to b
134. installing 6 interface 13 upgrading 6 Image Gallery channel view 62 composites 68 overview 61 population 62 printing 113 properties 65 resize 63 show hide color 63 show hide masks 62 tools 6l using 60 individual image display properties 86 manipulating 70 measurement tool 85 pixel intensities 85 show hide mask 88 M Mask Manager overview 94 tools 97 masks about 193 creating new 94 dilate 195 erode 195 examples 98 fill 195 intensity 196 morphology 198 object 198 peak 199 range 199 skeleton 200 spot 201 216 INDEX system 203 threshold 204 valley 205 viewing definitions 100 merging raw images files 49 50 O Object Data 92 P Population Manager 108 111 tools 110 Population Statistics 89 populations creating 71 creating combined 109 deleting 109 display properties 108 111 viewing 108 111 R reports creating 116 printing 113 rif about 14 opening 29 S saving data files 51 scatter plots show hide populations 84 screen resolution 6 software requirements 5 Statistics Area overview 89 T template file about 15 saving 52 TIFs creating 121
135. is Scale Display Width Display Height Set Range to Pixel Data Full Scale Channel Width Auto Fit Auto Fit O 88 o 2s Preview Changes in Gallery TO CHANGE THE SIZE OF THE PANELS IN THE IMAGE GALLERY 1 Display Width and Display Height can be specified or changed to Auto Fit in the lower left section of this window TO CHANGE THE NAME OR COLOR FOR EACH IMAGE 1 Select an image in the list of images on the Display Properties tab of the Image Gallery Properties window 2 On the right side of the window you can type a new unique name for the selected image Note that each image 1s 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 USING THE DATA ANALYSIS TOOLS 65 Tip The grayscale image in each channel is assigned a default color for image dis play 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
136. istics are presented in tabular form rather than graphical form You can copy data from the Statistics Area to the Clipboard as well as export the data to applications such as Microsoft Excel and Microsoft Word VIEWING THE POPULATION STATISTICS The Population Statistics tab displays selected feature values and statistics for chosen populations The statistics that are supported are the Count Percent Total Percent Gated Percent Concentration count sample volume Mean Median Standard Deviation MAD Median Average Deviation RD Mean RD Median CV Minimum Maximum Geometiric Mean Mode variance and NaN not a number TO VIEW AND CUSTOMIZE THE POPULATION STATISTICS 1 Click the Populations Statistics tab in the Statistics Area 2 Right click anywhere in the tab and the menu opens Population Statistics Object Feature Values Add Statistics Order Statistics Edit Statistic Colurnn Delete Statistic Column Clone Statistic Colurnn Clear All Use Short Population Names Copy Statistics to Clipboard Use Short Population Names is enabled by default This will display only the last part of the population name and not the entire tree For example the short name for Population R1 amp R2 amp R3 amp R4 is R4 3 Select Add Statistics The Add Statistics Rows and Columns window appears USING THE DATA ANALYSIS TOOLS 89 Gi 7 Z Add Statistics Rows and Colum _ El pt Rows Columns Statistics
137. ith a specified set of features available depending on the purpose of the graph 1 Choose the specific Building Block from the drop down menu Building Blocks Select Predefined Building Block Fluorescence Positives One Color Fluorescence Positives Two Color Focus Single Cell Single Cell Default 2 Choose the population s to graph Building Blocks Select Predefined Building Block Fluorescence Positrves One Color we Use the control key to select multiple populations Spo All 5 0 Single cells B m Re J apoptotic GETTING STARTED WITH THE IDEAS APPLICATION 27 3 Choose the X Axis Feature and the Y Axis feature if applicable Tithe and Axes ie g _ Axis Feature a Osis Label Y Axis Feature Y Asis Label 4 Click OK Intensity MEC Cho Intensity MC CAO Intensity MC CHU Intensity MC CAUS Intensity MC Cho Intensity MEC CHOB NomalzedFrequency i 5 The graph is added to the analysis area TABLE 1 BUILDING BLOCKS OVERVIEW BUILDING BLOCK Flourescence Pos itives one color Flourescence Pos itives two color Focus Single Cell Single Cell Default X AXISFEATURES Intensity_MC_ChX for all channels Intensity_MC_ChX for all channels Gradient RMS_MX_ChX for all channels Note Gradient RMS of brightfield is default Area_brightfield default Area_scatter Intensity_MC_ChX for all channels Area_brightfield 28 GETTING STA
138. jects sec Feature on page 192 No Yes A local concentration of number of objects per second Note to get objects per ml of a population use the statistic Concentration Time Feature on page 192 Yes Yes The camera timer feature converted to seconds COMBINED Any combined feature will be listed under Combined No No UNDERSTANDING THE IDEAS FEATURES AND MASKS 133 UNDERSTANDING THE DETAILED FEATURE DESCRIPTIONS UNDERSTANDING THE SIZE FEATURES Size features are in microns and include Area Diameter Length Major Axis Minor Axis Major Axis Intensity Minor Axis Intensity Perimeter Thickness Max and Min Spot Area Min and Width and Height AREA FEATURE The number of microns squared in a mask 1s equal to the Area In the following figure a 1 symbolizes whether the area is included in the mask The number of pixels is converted to um Note that 1 pixel 0 25 um As an example a cell with a mask that includes 2000 pixels is therefore equal to 500 uum Brightfield Sie ee lM Cut nhere ALE eee 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 134 UNDERSTANDING THE IDEAS FEATURES AND MASKS DIAMETER FEATURE Homadzed Frequency
139. k In the Mask Manager window click New Double click Morphology Intracellular in the Masks list Click the AND button on the toolbar m Click the NOT button on the toolbar o Double click Morphology Nuclear in the Masks list Enter a new mask name Click OK to add this mask to the list Click Close 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 1 Intercellular 9g 1 You can further refine this mask by eroding the Morphology Nuclear mask such that it allows the Cytoplasm mask to better capture the cytoplasm close to the nuclear boundary To do so open the Mask Manager window Click Cytoplasm in the Masks list and click Edit Select the Morphology Nuclear mask in the Cytoplasm mask definition Click the Function toolbar button The Define Mask Function window appears Select Erode in the Function list The mask will already be selected Set the num ber of pixels to 1 Click OK to complete the 1 pixel erosion of the Morphology Nuclear mask The eroded mask appears in the definition Click OK to complete the edit of the Cytoplasm mask Click Close in the Mask Manager window To view the resulting mask on a Channel 3 image open the Image Display Prop erties window and if necessary select the new mask for the channel USING THE DATA ANALYSIS TOOLS 99 Intercellular VIEWING AND EDITING A MASK TO VIEW A MASK DE
140. k Tool show or hide masks on page 62 Sets the Image Gallery color See To show Show Color Tool or hide color on page 63 USING THE DATA ANALYSIS TOOLS 61 TABLE 1 IMAGE GALLERY TOOLS TOOL DESCRIPTION A Click on the tool and it will show any satu Show Saturation Color Tool rated pixels will turn red See To show sat uration on page 63 Zoom Tools Zoom in or out and reset zoom on the image gallery See To zoom on the image gallery S on page 63 Q Q 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 Popula tions on page 71 2 To select an individual image click it A thin green frame indicates the selected object e The Statistics Area displays the object s feature values if an object is selected See Overview of the Statistics Area on page 89 e The Analysis Area identifies the object in each scatter plot graph with a green cross See Overview of the Analysis Area on page 73 e The image can be placed in the Analysis area by right click gt Display Single Image Tip Conversely in the analysis area clicking a graphical point causes the Image Gallery to highlight and display the corresponding object TO CHANGE THE VIEWING MODE e Inthe View drop down
141. k consists of the union of the masks of all the channels of the object A Not Combined Mask is all of the pixels with no intensities above background You might need to adjust the masks or create new ones that include only a specific area of a cell such as the nucleus You can combine masks by using Boolean logic or you can adjust them by applying functions CREATING NEW MASKS WITH THE MASK MANAGER There are two ways to work with new masks in the Mask Manager First masks can be created by using functions which allows you to choose an input mask and if needed adjust the channel and scalar input Alternatively masks can be created by combining masks through Boolean logic TO CREATE A NEW MASK USING FUNCTIONS 1 Select Analysis gt Masks 2 Click New The Name box and Definition toolbar become enabled 94 USING THE DATA ANALYSIS TOOLS Name Definition Function 4 m ol e OK Cancel Close Click Function The Define Mask Function window appears with 13 available masks to use e Dilate See Dilate Mask on page 195 e Erode See Erode Mask on page 195 e Inspire See Inspire Mask on page 196 e Intensity See Intensity Mask on page 196 e Interface See Interface Mask on page 197 e Morphology See Morphology Mask on page 198 e Object See Object Mask on page 198 e Peak See Peak Mask on page 199 e Range See Range Mask on page 199 e Skeleton See Sk
142. lication uses the rif file to create a com pensated image file cif file cif User creates a cif Contains imagery that has been corrected for variations in Compensated from the rif and ctm the camera background camera gains flow speed and Image File vertical and horizontal positioning between channels Serves as a database of images used for feature value cal culations 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 daf References the cif The main working data file that contains the calculated Data Analysis feature values the graphs and the statistics used for analy File sis The daf file references the cif cast Created from the daf This file contains no data it contains the structure for the Template File analysis such as definitions for features graphs regions and populations image viewing settings image names and statistics settings ctm User creates new Contains compensation values that are created and saved Compensation ctm when openinga during the spectral compensation of control rif files This Matrix File rif file has no associated object data it is created and saved to be applied to experimental rif files Note about Case Sensitivity Even though Windows does not treat file names as case sensitive the IDEAS ap
143. ls in order to observe the over or under compensation 3 Identify the matrix values that need adjusting by inspecting the scatter plots and images Each column contains the coefficients for the peak channel into the corre sponding crosstalk channels rows For example the crosstalk of channel 2 green into channel 3 is highlighted in the matrix below Compensation Matrix Ses Select a compensation matris 081109 G241 shape change MCP1_2 cif Ch ChO2 ChO3 ChO4 ChO5 ChOB ChO CHOS CROS ChiO Chil Chi2 1 0 048 0 003 1 o ao2 DEA o O05 o 0o om7 0 07 0 044 o oo 0 002 0 001 mm mm mm mm mm mm mm mm mm mmn S GE mm A G mn S GE m A A ma G GE m GA A mn S GE m S O E a B G m DOJAJA lalalala eala aa a Aala Aaja Aje A A m B GE mn A E n S GE m A A man G a A n S GE aa B GA oan S GE m B GE m a Aala Aafje a AA A mn B GE mn A E an S a A a S GE m G G man S GE ma B GA oan G GE m B GE m 2 Aale ala Ajla A AA A n S a O S G m A A an S GE m G GA mn S GE ma G G aan G GOE m B G m jo oaloao oy ljo o y oa o oay a 0 0 0 0 0 0 0 0 Preview a file with this matris applied Select an existing rif file Select a population from the current file eooo 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 c
144. mage File rif check box the popu lation name is used as a default You may enter a new name 4 To create a cif file select the New Compensated Image File cif check box the population name is used as a default You may enter a new name 5 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 53 GETTING STARTED WITH THE IDEAS APPLICATION BATCH PROCESSING Batch processing allows you to automatically analyze a group of files with one template when a compensation matrix has already been generated for the experiment TO PERFORM BATCH PROCESSING 1 On the Tools menu select Batch Data Files The Batches window appears It lists a record of all batches you have processed Batches to Run Add Batch Remove Batch Processed Batches HE Batch 12 21 2010 10 10 02 AM 2 Click Add Batch The Define a Batch window appears 54 GETTING STARTED WITH THE IDEAS APPLICATION ge Define a Batch Ses Input Files Select cof cif or daf files to process Add Files Select a compensation matrix compensated image file or data analysis file ictm cif daf Select a template or data analysis file ast daf Do not overwrite files Batch Name Batch File sufix 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 k
145. menu of the Image Gallery select a specific view The imagery display changes according to the new view See To customize the Image Gallery views images and masks on page 67 for more information TO SHOW OR HIDE MASKS e Click the Show Segmentation Masks toolbar button to toggle between showing and hiding the selected masks for all images in the Image Gallery See To change the name or color for each image on page 65 for more informa tion The mask is shown as a transparent cyan layer over each image 62 USING THE DATA ANALYSIS TOOLS Channel amp Tip To hide the mask for a specific channel only set the individual channel mask to None For more information see Setting the Image Gallery Proper ties on page 64 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 To change the name or color for each image on page 65 for more information TO ZOOM ON THE IMAGE GALLERY e Click the Zoom In toolbar button to view the images in the gallery closer and the Zoom Out or Reset Zoom to reverse the zoom amp Zoom In amp Zoom Out a Reset zoom TO SHOW SATURATION e Click the Show Saturation Color toolbar button m Saturated pixels in images if any appear in red a USING THE DATA ANALYSIS TOOLS 63 SETTING THE IMAGE GALLERY PROPERTIES When a new data file opens in the default template yo
146. n is hanging there may be a memory issues especially with large file processing You must use the Task Manager to force quit the application Press and hold Ctrl Alt Delete The Window Task Manager appears Under the Applications tab select IDEAS Application If the status is Not Responding select End Task Jv A O N e 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 samples different cell type different probe or 3 cells exhibit substantial autofluorescence This protocol describes a method for manually adjusting and validating a compensation matrix for difficult samples TO TROUBLESHOOT AND REPAIR A COMPENSATION MATRIX 1Create a population of cells that are miscompensated using the tagging tool See Creating Tagged Populations on page 71 Choose single cells that are exhibiting crosstalk Choose a range of intensities from negative to bright but not saturated TROUBLESHOOTING 207 preferably single color If single color cells are not available choose cells with a dis tinct staining pattern in the peak channel 2 Create Intensity scatter plots of adjacent channe
147. n of 2 probes e Internalization on page 24 Guides you through the process of creating the features and graphs for ana lyzing the internalization of a probe e Nuclear Localization on page 25 Guides you through the process of creating the features and graphs for ana lyzing the nuclear localization of a probe e Shape Change on page 26 Guides you through the process of creating the features and graphs for ana lyzing the circular shape of a cell using a surface stain or brightfield image 18 GETTING STARTED WITH THE IDEAS APPLICATION OPEN FILE This wizard will guide you through the opening of a data file Use this wizard to open a file if you are not using one of the application specific wizards The application specific wizards incorporate opening a file 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 compensation matrix boxes are left blank the default template and or matrix will be applied For more information on opening data files see The File Menu on page 29 EJ Open File Wizard Step 1 Select the data file you wish to open
148. n page 191 Understanding the Comparison Features on page 184 About Masks on page 193 List of Function Masks on page 195 UNDERSTANDING THE IDEAS FEATURES AND MASKS 123 OVERVIEW OF THE IDEAS FEATURES AND MASKS Objects passing through the ImageStream 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 coupled detector CCD as shown in the following diagram Therefore each object has six images that can be individually analyzed or because they are in spatial register with respect to one another reconstructed Each of the separate bands is called a channel Below is an example of collecting 6 images The ImageStreamx system has a second camera option which enables collection of up to 12 images per object detector Spectral decomposition element cells in flow ye autofocus velocity j ry detector i y N brightfield illuminator The IDEAS application provides a large selection of criteria or features for analyzing images A feature is described by a mathematical expression that contains quantitative 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 o
149. nd Shape Ratio Feature on page 161 for more information Shape ratio Thickness min Length Thickness min UNDERSTANDING THE IDEAS FEATURES AND MASKS 141 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 on page 159 APPLICATION EXAMPLE These features can be used to separate rectangular shaped objects For curved objects measurement 1s more accurately obtained using the thick ness features 142 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 radians Brightfield 7AAD Composite Major Axis s _ Major Axis Angle 7AAD 1 2 Angle 7AAD 0 83 Angle Intensity 7AAD 1 2 Angle Intensity 7AAD 0 81 APPLICATION EXAMPLE Identify the orientation of an image relative to the image frame UNDERSTANDING THE IDEAS FEATURES AND M
150. nd Desktop IS100 NFkB Translocation Dose and Time 4 0 analyzed cif and daf files FITC DRAG ctm Corrections Alignment Camera Background Brightfield Gain EDF Flow Speed Output Options Cell classifiers applied Single objects separated Clipped objects removed Non4ramed objects erased C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 0ng_2_9 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 15_1_8 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 1ng 30_6_13 0f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 1ng 45_11_18 0f C Users sfriend Desktop IS 100 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 rfs 0 1ng 75 21 Arf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_26_9 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 15_ EE 1O 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 vifs 10ng 45_13_20 nf 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 rfs 10ng 75 23 Grif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 90_ 28_11 sf C Users sfrie
151. nd Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 15_5_12 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 1000ng 30_10_17 1f C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 nfs 1000ng 45_ 15 22 rif l C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 60_20_3 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 vifs 1000ng 75_25_8 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 1000ng 90_30_13 nf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 0ng_2_9 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 1ng 15_1_8 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 30_6_13 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 1ng 45_11_18 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 60_16_23 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 0 1ng 75_21_4 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 0 1ng 90_ 26 9 cf C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 15_3_10 cif C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rifs 10ng 30_8_ bot C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 rfs 10ng 45 _13_20 cif C Users sfriend Desktop IS100 NFkB
152. nd 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 population and click OK Note When you copy a region the scale is copied and is no longer associated with the feature from which it was originally drawn Therefore the region might not fit on the new graph USING THE DATA ANALYSIS TOOLS 83 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 fet Apply Graph Regions BHA Check the regions to be added to the graph Uncheck the regions to be removed yi Note Only regions that have the same scaling types ie linear log as the graph may be added OF 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 fet Show Hide Populations BHA Select the populations to view 4 Click OK Ti
153. nnels on the detector With this technique an image is optically decomposed into a set of six sub images each corresponding to a different color component and spatially isolated from the remaining sub images spatial offset The registration error of the six channel images for a single cell The spatial offset is measured during calibration and the values are saved to the image database Table of Coefficients 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 template A file that saves the set of instructions for an analysis session Note that a template contains no data it simply contains the structure for the analy sis This structure includes definitions of features graphs regions and populations image viewing settings channel names and statistics set tings GLOSSARY 213 214 GLOSSARY Index A Analysis Area adding an image panel 85 adding text 88 overview 73 printing 113 tools 74 application defaults 7 ast about 15 B batch processing 54 C cif about 14 opening 33 saving 52 compensation creating 40 overview 38 compensation matrix file about 15 creating 40 composites 68 copying example data files 7 copying images 70 ctm about 15 creating 40 D daf about 14 opening 35 saving 51 data analysis tools about 59 data analysis workflow 10 E exporting data 119 F Feature Manager
154. nt Max feature measures the sharpness quality of an image by detecting largest change of pixel values in the image and is useful for the selection of focused objects This figure shows the change in intensity across the red line The top image has a larger slope change than the lower image APPLICATION EXAMPLE Determine peak focus quality of images Also used to characterize texture However the Gradient RMS and Con trast feature are more robust for these applications See also Gradient RMS Feature on page 167 and Contrast Feature on page 165 166 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 calculation with different weighted assignments to the pixel arrays and with background subtracted Example images are shown in the figure below re E single cells n gt So S i or 90 y iy wal ae 32 i Le co Ta m Z a uo 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 on
155. ntensity of the red is high and the Similarity value is a high positive number Untranslocated Translocated NF kB image gt gt z z m m Cc Cc LB g e a L E L oF A m m x Ee x E ST I LL LL Z Z L T 80 100 120 7 AAD Pixel Intensity 7 AAD Pixel Intensity 7 AAD image 7 AAD image Below are examples of cells with varying amounts of similarity between the NFkB image in green and 7 AAD image in red shown here as a composite image The most dissimilar image pairs in the upper left to the most similar image pairs in the upper night Frequency l l 0 3 Similarity_NFkB 7 AAD 0 1 40 5 09 14 18 188 UNDERSTANDING THE IDEAS FEATURES AND MASKS APPLICATION EXAMPLES Quantify translocation Identify copolarization of two probes UNDERSTANDING THE IDEAS FEATURES AND MASKS 189 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 1s 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 190 UNDERSTANDING THE IDEAS FEATURES AND MASKS UNDERSTANDING THE SYSTEM FEATURES The system features do not require a mask CAMERA LINE NUMBER FEATURE
156. o have a sin gle axis of elongation a three fold and a four fold variation of the shapes See also Lobe Count Feature on page 160 Texture features measure pixel or regional variation and indi cate the granularity or complexity of the image Bright Detail Intensity R3 and Bright detail Intensity R7 Features on page 163 The Intensity of the pixels in the bright detail image using a 3 or 7 pixel structuring element Also see Spot Mask on page 201 for a description of the bright detail image Contrast Feature on page 165 Enumerates changes of pixel values in the image to measure the focus quality of an image Gradient Max Feature on page 166 The maximum slope of the pixel value changes in the image to mea sure focus quality of an image Gradient RMS Feature on page 167 Enumerates changes of pixel values in the image to measure the focus quality of an image H Texture Features on page 168 Measures Haralick texture features 130 UNDERSTANDING THE IDEAS FEATURES AND MASKS IN DEFAULT TEMPLATE No No No Yes R3 R7 IN EXPANDED DEFAULT TEMPLATE Yes No No Yes Yes Yes Yes Yes Yes Yes MASK_IMAGE USED IN DEFAULT TEMPLATE M1_Ch1 Mo6_Ch6 M1 Mo6 M1 Mo6 MC_Chi1 MC_Ch6 M1_Ch1 Mo6_Ch6 M1_Ch1 Mo6_Ch6 M1_Ch1 Mo6_Ch6 M1_Ch1_5 Mo6_Ch6_5 TABLE 2 LIST OF FEATURES BY CATEGORY FEATURE CATEGORY SIGNAL STRENG
157. oard Copy Displayed Images to Clipboard e To place the image in the Analysis Area click Display Single Image For more information see Analyzing Images on page 85 e 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 e To turn the colors on or off for the object image click Color On or Color Off respectively One or the other will appear depending on the current state e To show or hide the saturation color for the object image click Show or Hide Saturation Color respectively One or the other will appear depending on the current state TO COPY IMAGES FOR USE IN REPORTS 1 Inthe Image Gallery right click an image that you are interested in A menu appears Display Single Image Show Masks Color On Hide Saturation Color Copy Image to Clipboard Copy Column to Clipboard Copy Column Image to Clipboard Copy Displayed Images to Clipboard e To copy the image to the Clipboard click Copy Image to Clipboard e To copy the single channel image to the Clipboard click Copy Column Image to Clipboard 70 USING THE DATA ANALYSIS TOOLS e To copy the single channel image for all of the displayed images to the Clip board click Copy Column to Clipboard e To copy all the visible images in the Image Gallery to the Clipboard click Copy Displayed Images to Clipboard CREATING TAGGED POPULATIONS
158. on window appears 80 USING THE DATA ANALYSIS TOOLS Name the region Click the colored box to select an alternate color Select Use for statistics only if you do not want to create a population from this region Click OK The region appears on the graph with the name and color that you selected POLYGON TOOL OPTION The Polygon tool works by clicking the scatter plot at the point where you would like to start the polygon Click once for each vertex of the polygon Double click to complete the drawing of the region A window appears that allows you to name the population created by the polygon region and to assign the region s display properties Click OK The region appears on the graph with the name and color 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 L3 USING THE DATA ANALYSIS TOOLS 81 TO DRAW A REGION ON A HISTOGRAM 1 On the Analysis Area toolbar click the Line Region tool i 2 Drag the line across the histogram TO MOVE OR RESIZE A REGION ON A GRAPH 1 Click the Move Resize Region toolbar button on the graph panel toolbar 2 Click the region that you would like to move or resize When the region is selected squares that can be moved appear at the vertices and the label The first time that you drag the region the entire region and label move Dragging a specifi
159. on appears on the desktop and IDEAS Application appears on the All Pro erams menu SETTING YOUR COMPUTER TO RUN THE IDEAS APPLICATION Setting the Screen Resolution on page 6 Viewing File Name Extensions on page 6 Copying the Example Data Files on page 7 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 resolution 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 determine the file type It will be easier for you to distinguish raw image files compensated image files and data analysis files from each other if Windows Explorer does not hide the extensions TO VIEW FILE NAME EXTENSIONS In Windows Explorer go to Tools gt Folder Options 2 Click the View tab and make sure that the Hide extensions for known file types check box is not selected 3 Click OK 6 SETTING UP THE IDEAS APPLICATION COPYING THE EXAMPLE DATA FILES If 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 ac
160. on page 150 Spot Count Feature on page 170 Spot Intensity Min and Spot Intensity Max Features on page 183 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 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 140 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 Selecting an input mask that can accurately capture the object shape is important See also Thickness Min Feature on page 141 Length Feature on page 136 and Shape Ratio Feature on page 161 for more information Thickness max Le n gth a i Shape ratio Thickness min Length Thickness min THICKNESS MIN FEATURE Thickness Min measures the smallest width of an object This feature is based on an input mask and therefore sensitive to the variation of the input mask shape Selecting an input mask that can accurately capture the object shape is important See also Thickness Max Feature on page 141 Length Feature on page 136 a
161. oo many bins displaying Adjust the number of bins through the following steps 1 Right click on the histogram and select Graph Properties 2 Inthe Graph Properties window click Display Properties 3 In the Bin count drop down menu decrease the bin count as needed 4 Click OK in both windows to return to the histogram 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 Select Tools gt Merge rif Files Click Add Files to select the single rif file Click OK Enter a new name if desired The single rif file will merge with itself and rewrite the file with the proper object count BUTTONS OR OPTIONS IN WINDOWS ARE NOT APPEARING When the font size setting is set to large some windows will not size properly causing 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 209 TROUBLESHOOTING IMAGES AND BRIGHTFIELD CHANNEL APPEAR UNIFORMLY BRIGHT Image files collected on early ImageStream instruments may have incorrect flowspeed information IDEAS versions 2 2 and later automatically perform flow speed normalization and will attempt to use the incorrect information causing the imagery to appear uniformly bright Here is an example of imagery t
162. ore the Circularity value will be low See also Compactness Feature on page 158 156 UNDERSTANDING THE IDEAS FEATURES AND MASKS Below is an example using Circularity and Compactness to characterize the shape of peripheral blood mononuclear cells stained with the DNA dye Draq 5 Comp actne ss_D raq 5 W e G Circu larity Drag 5 APPLICATION EXAMPLES Distinguish singlets and doublets Separate circular and non circular shapes UNDERSTANDING THE IDEAS FEATURES AND MASKS 157 COMPACTNESS FEATURE Compactness measures the degree of how well the object is packed together This feature is similar to the Circularity feature but unlike Circularity this feature includes all of the pixels within the mask and 1s intensity weighted The higher the value the more condensed the object See also Circularity Feature on page 156 Below is an example using Circularity and Compactness to characterize the shape of peripheral blood mononuclear cells stained with the DNA dye Draq 5 Comp actne ss_D raq 5 el 2h 10 a le Circu larity Drag 5 Muclear Brightfield Drag 5 Circularty Compactness 0 942 J cy a t gt APPLICATION EXAMPLE Differentiate between rounded objects with smooth boundary to less regu lar objects 158 UNDERSTANDING THE IDEAS FEATURES AND MASKS ELONGATEDNESS FEATURE Elongatedness is the ratio of the Height over Width of the object s
163. ors greater than 1 A graph representing the coefficient appears The population potted in the graph is the positive control population of the column of the coefficient The X Axis represents the intensity in the assigned channel of the fluorochrome The Y Axis represents the intensity in the channel of leakage The coefficent value and error are also displayed 3 Positive aa ae 20000 40000 60000 80000 Intensity MC _ChOS Coefficient value 019 Coefficient emor 0 00036 Add Graph to Analysis Area You can use the automatically generated control populations as they are or you can refine them and create different populations by using the region tools See 44 GETTING STARTED WITH THE IDEAS APPLICATION the option below to remove objects from the population By default the popu lations are named 3_ Positive 5_Positive and so on You can view the popula tions in the Image Gallery Some populations may be empty e To choose a different population use the arrow and select the population from the list The hierarchical relationship is shown in the population list Assign populations only to the channels that correspond to the fluorochromes used in the experiment e Ifyou want to clear a column double click on the channel heading e Ifneeded you can create new scatter plots by using the Analysis Area toolbar For example a 4_Intensity versus 5_Intensity plot may be useful See Creating Graphs on page 75 for more inform
164. ot fill the line for a population select or clear the Fill checkbox e Ifyou 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 USING THE DATA ANALYSIS TOOLS 77 to create the graph will reappear and you can then make any changes that you want Graph Properties Statistics Show Hide Legend Order Plots 4pol Remove Regions Clear Selected Bin Similarity Dilate Object MOe 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 1s by clicking the Statistics toolbar button in the panel that contains the graph Fil r 60 mn ag a Frequency i F ae o 10 2 Or right click anywhere on the graph and click Statistics on the graph context menu that appears The Statistics window appears 78 USING THE DATA ANALYSIS TOOLS 3 To Statistics C Show statistics Hide statistics Ais Statistics Y Ass Statistics w Count 7 otal w
165. p On a scatter plot you may show or hide any population on the graph regardless of the features on the axes Each scatter plot has an original or base pop 84 USING THE DATA ANALYSIS TOOLS ulation When you show a population on a scatter plot only those objects that are also in the base population will be shown ANALYZING 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 context menu that appears when you right click an image An image in the Analysis Area is three times the size of an image in the Image Gallery TO ADD AN IMAGE PANEL TO THE ANALYSIS AREA e Right click an image in the Image Gallery or Analysis Area and click Display Single Image on the context menu that appears The image panel appears in the Analysis Area 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 1s positioned at the upper left of the image Pixel 43 34 Intensity 264 TO DISPLAY THE MEASUREMENT TOOL IN AN IMAGE PANEL e Right click the image panel and click Show Measurement Tool on the context menu that appear
166. page 166 and Contrast Feature on page 165 UNDERSTANDING THE IDEAS FEATURES AND MASKS 167 H TEXTURE FEATURES H Texture features include the following H Energy Mean and Std H Entropy Mean and Std H Contrast Mean and Std H Homogeneity Mean and Std H Correlation Mean and Std H Variance Mean and Std Features R M Haralick H defined a set of texture features that characterize the spatial relationships amongst the pixel values in an image IDEAS uses a common normalization 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 textures 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 individually images often contain a mixture of different textures at different grains Therefore these features are most powerful when combined APPLICATION EXAMPLE Quantify texture in cells Haralick R M K Shanmugan and I Dinstein Textural Features for Image Classification IEEE Transactions on Systems Man and Cybernetics Vol SMC 3 1973 pp 610 621 168 UNDERSTANDING THE IDEAS FEATURES AND MASKS MOD
167. play and hand select objects for a population Overview of the Image Gallery on page 61 Setting the Image Gallery Properties on page 64 60 USING THE DATA ANALYSIS TOOLS Working with Individual Images on page 70 Creating Tagged Populations on page 71 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 information see Setting the Image Gallery Properties on page 64 IDEAS Application Z DataForShows_051507 Immune Synapse h Population A5 amp RARASEAZ EAT o WEN comp H EF Actin ADAF FE 74 40 40477 4 22 Actin i ADAF eae T g ei TABLE 1 IMAGE GALLERY TOOLS TOOL DESCRIPTION Allows you to create a population of hand Tagging Mode Tool picked objects See To create a hand selected population on page 71 ol Provides custom display features See To Image Gallery Properties Tool customize the Image Gallery display proper ties on page 65 Displays masks on the imagery See To Show Segmentation Mas
168. plication 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 2 lt gt 16 OVERVIEW OF THE IDEAS APPLICATION CHAPTER 4 Getting Started with the IDEAS Application 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 E IDEAS 4 0 jae h o Pe i Welcome to IDEAS Yersion 4 0 274 0 To begin using IDEAS double click on an application below or choose the Open File option to perform manual analysis or view previously processed ff Open File Open ImageStream data files Apoptosis Identify apoptotic events based on brightfield and nuclear morphology Cell Cycle Mitosis Distinguish mitotic and apoptotic events Co localization Measure the co localization of two probes on in or between cells Internalization Measure the internalization of a probe Nuclear Localization Measure the nuclear localization of a probe Shape Change Measure circular morphology This chapter is divided into two sections First guided analysis is described using the analysis wizards and second advanced analysis with more detailed instructions that describe how to open compensate merge save and create data files without using the wizards Building blocks are also discussed whi
169. port Template Statistic Columns Column Details doube otek Dere fo adoh Column Heading pete ooo Statistic Population E New Edit Delete Generate Report Cancel Report title Enter a Report title In the Statistic Columns area double click the blue bar or select New Select a statistic in the Statistic drop down menu e Gated the percent of one population as a percentage of another but not used for tagged populations e Total percentage of a population as a percentage of All e the percentage of one population as a percentage of another also is used for tagged populations e Count the absolute count of the population e CV the coefficient variable e Geometric Mean standard statistical definition e Maximum standard statistical definition e Mean standard statistical definition 116 CREATING REPORTS AND EXPORTING DATA O N DW UI 10 11 12 13 e Medium standard statistical definition e Minimum standard statistical definition e Mode standard statistical definition 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 Based on the selected statistic select a population Select a Feature This is not available for the related statistics or the Count Enter an appropriate name This will be name of the column in the Excel f
170. rface bound antibodies UNDERSTANDING THE IDEAS FEATURES AND MASKS 175 MEDIAN PIXEL FEATURE The Median Pixel feature is the median of the background subtracted pixels contained in the input mask It is more robust than the mean as an estimate of the average fluorescence since it is less influenced by outliers The relationship of Max Mean Median and Min Pixel is shown in the figure below MaxPet ss MinPixel os ot Intensity 230 000 230 000 APPLICATION EXAMPLE Estimate the 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 contained in the input mask There will be some negative numbers due to the background subtraction therefore the Raw Min Pixel feature is preferred APPLICATION EXAMPLES 176 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 PIXE
171. run them together but you must be careful that the fluorochromes do not bleed onto other singly stained cells Because it is critical that matrix values be calculated from intensities derived from a sole source of light control files are collected without brightfield illumination 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 GETTING STARTED WITH THE IDEAS APPLICATION 39 CREATING A NEW COMPENSATION MATRIX FILE The compensation matrix is a table of coefficients The IDEAS application uses this table to place the detected light that is displayed in each image into the proper channels on a pixel by pixel basis The coefficients are normalized to 1 Each coefficient represents the normalized amount of the leakage of the fluorochrome into the other channels The default matrix which is used if no compensation matrix is chosen 1s the identity matrix TO GENERATE A NEW COMPENSATION MATRIX FILE 1 Start the Compensation Wizard in one of two ways Click the New Matrix button when opening a rif file OR select Compensation gt Create New Matrix The compensation wizard opens to Step 1 Z Create Compensation Matrix ole Step 1 Select the control files for compensation The control files must be all 6 channel files or all 12 channel files Control Files ha Fes 2 Add compensation control files b
172. s The 10 micron bar appears USING THE DATA ANALYSIS TOOLS 85 O Microns 10 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 window appears next to the image panel The statistics are calculated 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 fee Image Statistics aj f Object 74 Ch Che Selected Region Upper Left Corner 38 17 Lower Right Comer 74 35 Area pixels pie Raw Fisel Intensities Minimum 20 Maximum 175 Mean 64 2376 Standard Deviation 39 2358 Close TO CHANGE THE DISPLAY PROPERTIES OF AN IMAGE 1 Click the Channel Display Properties button on the image panel toolbar The Display Properties window appears 86 USING THE DATA ANALYSIS TOOLS e For single channel image you can change the displayed mask and adjust the display intensity mapping For more information see Setting the Image Gal lery Properties on page 64 roe Display Properties Object 74 Image Ch Select a different mask to display MOB Minimum Fisel Intensity 26 Maximum Fisel Intensity 178 l l l l a0 100 120 140 Fisel Value Automatic Settings Manual Settings Pixel Intensity Asie Scale e Fora composite image you can change the images in the composite and adjus
173. s a different spectral band of imagery which allows for the collec tion of brightfield darkfield and up to four fluorescence images per object A sensor for recording images that consists of a particular type of inte grated circuit one that contains an array of linked or coupled capaci tors Under the control of an external circuit each capacitor can transfer its electric charge to either of its neighbors The mean normalized standard deviation expressed as a percentage The CV measures the variation of a feature value independent of the population mean value The formula is CV 100 x standard deviation mean See coefficient of variation CV GLOSSARY 211 TABLE 1 GLOSSARY OF TERMS TERM compensation compensation matrix crosstalk darkfield FISH fluorochrome fluorescence fluorescence compen sation fluorescent in situ hybridization FISH gain grayscale pixel saturation 212 GLOSSARY DEFINITION The process of removing intensity specifically intensity that was derived from fluorescence crosstalk that originated from dyes centered in other channels The IDEAS application performs compensation on a pixel by pixel basis The set of values that report the relative amount of fluorescence of each probe in each channel The compensation matrix is used to subtract intensity originating from dyes centered in other channels Leakage of fluorescence signal from a fluorochrome into adj
174. saturated and cannot be quantified after 1023 See also Saturation Count Feature on page 181 An object with saturated pixels shown in red APPLICATION EXAMPLE Measure the validity of the experiment setup Saturated data may not pro duce useful information 182 UNDERSTANDING THE IDEAS FEATURES AND MASKS SPOT INTENSITY MIN AND SPOT INTENSITY MAX FEATURES Spot Intensity Min provides the smallest Raw Mean Pixel value not background subtracted of the dimmest spot connected component The Raw Mean Pixel values for each spot is computed and the smallest value is reported Spot Intensity Max provides the largest Raw Mean Pixel value not background subtracted 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 properties 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 on page 140 Raw Centroid X and R
175. scale to increase the dynamic range to values between inf inf Internalized cells typically have positive scores while cells with little internalization have negative scores Cells with scores around 0 have a mix of internalization and membrane intensity Composite Images of brightfield and channel 6 are shown for High Medium and Low Internalization values Mid Internalization Single Focused Cells jy x 25 Low Internalization uo Normalized Frequency High Internalization APPLICATION EXAMPLES Quantify internalization when supplied with the internal mask Quantify the intensity ratio of a region of interest to the whole cell UNDERSTANDING THE IDEAS FEATURES AND MASKS 187 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 1n 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 intensity of the green is high the intensity of the red is low and vice versa The Similarity value for this cell 1s 2 067 indicating that the image pair has a high degree of dissimilarity Analysis of the image pair on the right shows that when the intensity of the green is high the i
176. signed to a column of a viewing mode regardless of the image in that column Compensation Select any cif daf or ctm file to obtain a compensation matrix New compensation menu for viewing and editing compensation matrices and preview process for testing a matrix Improved algorithm for selection of positive populations that eliminates satu rated events Feature and Mask Improvements Features Improved the Object per second and Objects ml calculations New Spot Intensity Max feature New features Ensquared energy Raw Centroid X Raw Centroid Y Shift X Shift Y XCorr Masks The Spot mask s spot to cell background ratio has improved Inspire mask is new PREFACE 3 4 PREFACE CHAPTER 2 Setting Up the IDEAS Application This chapter describes the hardware and software requirements for the application which includes the procedures for installing removing and upgrading the application The following subsections cover this information Hardware and Software Requirements on page 5 Installing the IDEAS Application on page 6 Setting Your Computer to Run the IDEAS Application on page 6 Viewing and Changing the Application Defaults on page 8 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 req
177. sing Processed rf File S 0 0ng_2_9 rif O O 1ng 15_1_8 f 0 1ng 30_6_13 nf 0 1ng 45_11_18 nf O 0 0ng_2_S cf O 0 1ng 15_1_8 cif O 0 1ng 30_6_13 cf O 0 1ng 45_11_18 cif O Nina 6M 16 23 cif 4 daf File Total elapsed time 0 minutes 56 GETTING STARTED WITH THE IDEAS APPLICATION 11 IZ 13 14 Z Batch Results f Se Tip To cancel the batch processing at any time click Cancel Batch The IDEAS application will confirm cancellation and complete the file it is working on When the batch processing is complete the IDEAS application saves the rif cif and daf files in the batch results directory In the Batches window a list of pro cessed batches appears in the Processed Batches list If a batch did not successfully complete it will appear in red Tip To display the error that occurred during processing double click the batch If you want a batch report double click the batch in the Processed Batches list of the Batches window The Batch Results window appears In the Batch Results window click Print In the Batch Results window click Close In the Batches window click Close Batch Report C Users sfriend App Data Roaming Amnis Corporation batches Batch 2 1 2011 11 53 28 AM Batch Batch1 Template C Users sfriend Desktop IS100 NFkB Translocation Dose and Time 4 0 analyzed cif and daf files 0 0ng_2_9 _4 Analyzed daf Compensation C Users sfrie
178. 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 Brignttield YoYol ow ho Normalized Frequency i M lial ML 120 150 180 Raw Min Pixel APPLICATION EXAMPLE Quantify spectral absorbance using the brightfield image Identify over compensated images Measure the level of malaria infection in RBCs 180 UNDERSTANDING THE IDEAS FEATURES AND MASKS SATURATION COUNT FEATURE The Saturation Count feature reports the number of saturated pixels in an object See also Saturation Percent Features on page 182 In the figure below objects with saturated pixels are lined up at the Raw Max Pixel value of 1023 and a selected image 1s shown with saturated pixels in red D l Saturation Count Channel 3 i i l i Feature Value 0 300 Bod gpg 12e3 Saturation Count_Channel 3 3 Raw blas Pixel Channel 3 Saturation Percent Channel 3 4 2062 APPLICATION EXAMPLE Measure the validity of the experiment setup Saturated data may not pro duce useful information UNDERSTANDING THE IDEAS FEATURES AND MASKS 181 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 O to 1023 10 bit and therefore become
179. such activities are expressly permitted by law notwithstanding this prohibition 3 Ownership The copy of the Software is licensed not sold You own the media on which the Software is recorded but Amnis retains ownership of the copy of the Software itself including all intellectual property rights therein The Software is protected by United States copyright law and international treaties You will not delete or in any manner alter the copyright trademark and other proprietary rights notices or markings appearing on the Software as delivered to you 4 Term The license granted under this Agreement remains in effect for a period of 75 years unless earlier terminated in accordance with this Agreement You may terminate the license at any time by destroying all copies of the Software in your possession or control The license granted under this Agreement will automatically terminate with or without notice from Amnis if you breach any term of this Agreement Upon termination you must at Amnis option either promptly destroy or return to Amnis all copies of the Software in your possession or control 5 Limited Warranty Amnis warrants that for thirty 30 days following the date of purchase or delivery if Amnis has made the Software available to you without charge the Software will perform in all material respects in accordance with the Documentation As your sole and exclusive remedy and Amnis entire liability for any breach of this limi
180. t the percent contribution of each image see Setting the Image Gallery Proper ties on page 64 2 Click OK Display Properties Object 61 Composite Ch6 Ch3 Object E Che Chs Che 10072 Cha 10072 Percent USING THE DATA ANALYSIS TOOLS 87 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 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 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 appears Enter title Enter text here 2 Type a title and text 88 USING THE DATA ANALYSIS TOOLS USING THE STATISTICS AREA This section contains the following subsections which describe how to view the population statistics the object feature values and the compensation matrix Overview of the Statistics Area on page 89 Viewing the Population Statistics on page 89 Viewing the Object Feature Values on page 92 OVERVIEW OF THE STATISTICS AREA The Statistics Area allows you to view both multiple feature values for an object and population statistics Feature values and population stat
181. ted 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 Software 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 DEALING OR USAGE OF TRADE NO ADVICE OR INFORMATION WHETHER ORAL OR WRITTEN OBTAINED FROM AMNIS OR ELSEWHERE WILL CREATE ANY WARRANTY OR CONDITION NOT EXPRESSLY STATED IN THIS AGREEMENT 7 Limitation of Liability AMNIS TOTAL LIABILITY TO YOU FROM ALL CAUSES OF ACTION AND UNDER ALL THEORIES OF LIABILITY WILL BE LIMITED TO THE AMOUNTS PAID TO AMNIS BY YOU FOR THE SOFTWARE OR IN THE EVENT THAT AMNIS HAS MADE THE SOFTWARE AVAILABLE TO YOU WITHOUT CHARGE AMNIS TOTAL LIABILITY WILL BE LIMI
182. text Each panel will contain its own toolbar and context menu To move a panel click on the bar at the top and drag to another location R3 8hr DNA FEREN RAE lebS NFKb Nuclear Translocation DNR 8 hour treatment x R4 Shr DNA CERENE AE Text Area Frequency l O iia imal 1 10 1e2 le e 0 3 Spot Small Total DRAGS Gradient RMS_DRAQ5 Similarity_NFkB DRAGS Spot Small Total DRAGS aeleze x 23 NFkB y pRags 10 x 7765 FITC NFkB maa x Population Count Gated Mean Composite Channel Image Image gt Ri 4148 100 2200 R3 amp R1 R24 Statistics Area_DRAQ5S_T30 Histogram Overlay Population Mean gt Ri 170 81 R3 amp A1 224 84 R2 amp R1 47 92 Normalized Frequency o 3 0 3 6 Similarity_C3 amp C6_Morphology Channel 6 USING THE DATA ANALYSIS TOOLS 73 A toolbar is visible on the left side of the Analysis Area The following table describes the function for each tool TABLE 2 ANALYSIS AREA TOOLS TOOL La Pointer Tool ko Tagging Tool New Histogram Tool New Scatter Plot Tool Text Tool Line Region Tool Rectangle Region Tool Oval Region Tool ine Polygon Region Tool 74 USING THE DATA ANALYSIS TOOLS DESCRIPTION Provides the normal mode of interaction with the graphs Clicking a point on a scatter plot graph causes the IDEAS application to display the corre spondin
183. the IDEAS window _ Ed HI H Layout Tools Short cut to using Building Blocks for guided e lysis Building Blocks Tool ia a Short cut to using Wizards for guided analysis T Wizards Tool 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 TO CREATE A GRAPH 1 Click the New Histogram or New Scatter Plot toolbar button u The New Histogram or New Scatterplot window appears respectively USING THE DATA ANALYSIS TOOLS 75 me New Histogram Use the control key to select multiple populations Scaling Auto O Manual X Axis 3 0 0ng_2_3 cif Minimum Maximum Linear Title and Axes O log x Title Y Axis X Axis Feature Choose X Axis Feature x Minimum x Axis Label Maximum Y Axis Feature Linear OAS 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 Ifyou want to change the label for the X axis edit the text in the X Axis Label field The label defaults to the name of the selected feature If you are creating a scatter plot select a feature and a label for the Y
184. tion compensation E Update automatically when file is selected E Use default data directory 8 SETTING Up THE IDEAS APPLICATION CHAPTER 3 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 workflow Understanding the Data Analysis Workflow on page 10 Overview of compensation analysis tools and file structure on page 12 The ImageStream cell analysis system possesses unique capabilities that neither flow cytometry nor microscopy alone can deliver Examples include the analysis of molecule co localization and translocation 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 compensates the images and allows the user to evaluate the images with data analysis tools OVERVIEW OF THE IDEAS APPLICATION 9 UNDERSTANDING THE DATA ANALYSIS WORKFLOW Data analysis in IDEAS begins with opening a raw image file rif that was collected and saved using INSPIRE on the ImageStream Then an existing compensation matrix or a new compensation matrix is applied to the rif file and two additional files are created the cif compensated image file and daf data analysis
185. tion can quantify cellular activity by performing statistical analyses on thousands of events and at the same time permit visual confirmation of any individual event Furthermore you can operate the application in a batch processing mode and store specific analysis templates for either repeated use or sharing with colleagues The fastest way to put the IDEAS application to work 1s 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 experimental 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 PREFACE 1 WHAT S NEw IN IDEAS 4 0 2 PREFACE IDEAS 4 0 is required to analyze data from the ImageStreamX and offers numerous improvements for analyzing data from any ImageStream instrument Please refer to the our web site for the latest improvements and updates to this manual 1 ImageStreamX Data files collected on the _ImageStreamX have new requirements that are built in to IDEAS 4 0 software 2 General Multiple files can be opened in a single instance of IDEAS Multiple window layouts can be displayed with resizable panels Open large data files that would not previously load due to memory con straints Drag and drop
186. tions for e Features e Graphs e Regions e 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 subsequent 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 template is also available that includes over 600 calculated features per object 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 OVERVIEW OF THE IDEAS APPLICATION 15 REVIEW OF DATA FILE TYPES TABLE 1 REVIEW OF DATA FILE TYPES FILE ACRONYM FILE CREATION DESCRIPTION AND NAME rif Created in INSPIRE Contains instrument setup data pixel intensity data and Raw Image File uncorrected image data from the INSPIRE application The IDEAS app
187. tliers Click the Resize S and Zoom buttons on the graph toolbar to more clearly see the population of interest Using one of the region buttons on the tool bar draw a region that contains only the cells you want to use for determining compensation You can click a point on the graph and view the image to help you decide where the region boundaries should be In the example below the Polygon Region tool was selected to draw a border around a selection of cells Clicking within the graph anchors the line and double clicking completes the region 12 Positive eir A E 5 Create a Region Name R1 MC_Ch07 Color A C Use for statistics only Intensity I 1e5 ies 2e5 2 5 Intensity_MC_Ch12 For more information refer to Creating Regions on Graphs on page 80 4 Assign the new population to the appropriate channel by using the Positive Con trol Populations list for that channel 46 GETTING STARTED WITH THE IDEAS APPLICATION Create Compensation Matrix Step 2 Validate the compensation matrix refine the postive control populations Double click each matris coefficient to validate the fit of the positive control population The resulting graphe can be added to the analysis area to Cho Choe Chos Cho4 ChoOS Choe Cho Choe Chog 1 oie pors ose a am omer Best Fit Means o C DE oo om o fo D ee E oia o h Foszitive Control Populations 3
188. tly from those created using other operating systems The Amnis ImageStream cell analysis system is for research use only and not for use in diagnostic procedures TECHNICAL ASSISTANCE Amnis Corporation 2505 Third Avenue Suite 210 Seattle WA 98121 Phone 206 374 7000 Toll free 800 730 7147 Wwww amnis com CHAPTER 1 CHAPTER 2 CHAPTER 3 CHAPTER 4 PREFACE How to use this manual 0 0 0 ce e ne ne nen What s New IDEAS 4 0 2 42 yalan Goh ay Serko Sorhae eat Sees Mess SETTING UP THE IDEAS APPLICATION Hardware and Software Requirements 0 00000 cee eee eee Hardware Requirements 1 0 5 0 e050 pao Ae acne h Erne h One 6 8GRG HS SOlewares Requirements yieee 44 ue Cae SAR Rae eee Installing the IDEAS PIG ALOU eet sand of GA GANS el antes oo A Gino Gio Setting Your Computer to Run the IDEAS Application co Baek ie B naaa Setting the Screen Resolution 2 26 2s eee yas ee he et Ge es Viewing File Name Extensions 23 44 i 446044266 42800006 donk 2 Copying the Example Data Files x 2 65 ia 6 05 36h4 e abe beoe G we ee 3 Viewing and Changing the Application Defaults OVERVIEW OF THE IDEAS APPLICATION Understanding the Data Analysis Workflow 0 0 00000002 ae Overview of compensation analysis tools and file structure Data Acquisition and Compensation 352404 it0simeivb tence ieness Daw Analysis Tolis etinren le dug ext T
189. to 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 window to daf Select File To Load Look in 3 analyzed cif and daf files wt J a pr E A 0 0ng_2_9 Default daf 1000ng 90_30_13 daf E 0 0ng_2_9 daf My Recent 0 1ng 15_1_8 daf Documents 0 1ng 30_6_13 daf O 1ng 45_11_18 daf O 1ng 60_16_23 daf Desktop E 0 1ng 75_21_4 daf 0 1ng 90_26_9 daf e 10ng 15_3_10 daf i 10ng 30_8_15 daf 10ng 45_13_20 daf My Documents a ng Taid a 10ng 90_28_11 daf 1000ng 15_5_12 daf 1000ng 30_10_17 daf 1000ng 45_15_22 dar e 1000ng 60_20_3 daf D 1000ng 75_25_8 daf hy Network Places 10nq 60_18_1 daf 10ng 5_23_6 dar My Computer File name Files of type IDEAS files rif cif dat Raw image files rif Compensated image files ci Data analysis files dar GETTING STARTED WITH THE IDEAS APPLICATION 35 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 MERGING CIF FILES You can open multiple cif files to combine their data and create a single daf file This is useful when you would like to create one graph with multiple data files TO OPEN MULTIPLE CIF FILES COMBINE THEIR DATA AND CREATE A SINGLE DAF FILE 1 From the Tools menu select
190. ty Raw does not have background sub tracted IN DEFAULT TEMPLATE No No No No IN EXPANDED DEFAULT TEMPLATE Yes No Yes Yes Yes Yes Yes Yes Yes Yes No UNDERSTANDING THE IDEAS FEATURES AND MASKS 131 MASK_IMAGE USED IN DEFAULT TEMPLATE M1_Ch1 Mo6_Ch6 M1_Ch1 Mo6_Ch6 Ch1 Ch6 Ch1 Ch6 MC_Ch1 MC_Ch6 MC_Ch1 MC_Ch6 M1_Ch1 M6_Ch6 M1_Ch1 M6_Ch6 MC_Ch1 MC_Ch6 TABLE 2 LIST OF FEATURES BY CATEGORY IN MASK_IMAGE FEATURE k EXPANDED USED IN FEATURE NAME DEFAULT DEFAULT CATEGORY TEMPLATE TEMPLATE DEFAULT TEMPLATE Raw Median Pixel Feature on page 179 No No The median pixel intensity Raw Min Pixel Feature on page 180 Yes Yes MC_Ch1 The lowest pixel value within the mask MC_Ch6 Saturation Count Feature on page 181 Yes Yes M1_Ch1 The number of pixels in the mask that are saturated Mo6_Ch6 Saturation Percent Features on page 182 Yes Yes The Percentage of pixels in the mask that are saturated Spot Intensity Min and Spot Intensity Max Features on page 183 No No The raw intensity not background subtracted of the dimmest com ponent spot See also Spot Count Feature on page 170 Raw Centroid X and Raw Centroid Y Features on page 150 and Spot Area Min Fea ture on page 140 Difference of intensity measurements between masks or COMPARISON pixels Bright Detail Similarity R3 Feature on page 184 No No
191. u 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 on page 20 to set the pixel intensity mapping to the display range Manual adjustment is described below Clicking the Image Gallery Properties toolbar button opens the Image Gallery Properties 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 1s included in a composite image 64 USING THE DATA ANALYSIS TOOLS TO CUSTOMIZE THE IMAGE GALLERY DISPLAY PROPERTIES 1 Click the Image Gallery Properties toolbar button to begin The Image Gallery Properties window appears with the Display Properties tab displayed Ta Image Gallery Properties Display Properties Views Composites Images Object 0 Name SSC Color w Minimum Pixel Intensity 26 Maximum Pixel Intensity 545 250 4 on i imi o Co pea 22 S y D 5l 2 a ao O rel 50 150 200 250 300 350 Pixel Value Automatic Manual Image Display Mapping x As
192. uirements are 512 MB of RAM and a 1 GHz processor However due to the large size of the image files that the ImageStream cell analysis system creates a larger amount of RAM will prevent paging and thus increase performance SOFTWARE REQUIREMENTS IDEAS 64 bit version requires that the Windows 7 operating system be installed on your computer IDEAS 32 bit version requires Windows SP 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 Microsoft 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 Security Web Site SETTING UP THE IDEAS APPLICATION 5 INSTALLING THE IDEAS APPLICATION If the IDEAS application has previously been installed the previous version will be removed during installation TO INSTALL IDEAS SOFTWARE 1 Insert the CD or DVD that is labeled IDEAS application Or download the appli cation Setup file from your account at www amnis com View the contents in My Computer or Windows Explorer Double click Setup exe Follow the instructions until the installation process has completed MadCap help viewer is installed and opened during installation or upgrade nN UO A WY N An ic
193. 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 feature under Texture For more information see Spot Area Min Feature on page 140 Spot Count Feature on page 170 Spot Intensity Min and Spot Intensity Max Features on page 183 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 UNDERSTANDING THE IDEAS FEATURES AND MASKS 151 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 on page 205 and Skeleton Mask on page 200 In the figure below 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
194. ut mask shape Selecting an input mask that can accurately capture the object shape 1s important See the Shape Ratio Feature on page 161 Thickness Min Feature on page 141 and Thickness Max Feature on page 141 for more information Thickness max shape ratio Thickness min Length Thickness min 136 UNDERSTANDING THE IDEAS FEATURES AND MASKS 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 on page 154 for more information inor Axis ajor Axis APPLICATION EXAMPLES Quantify and compare cell shape Identify small medium and large cells UNDERSTANDING THE IDEAS FEATURES AND MASKS 137 MAJOR AXIS INTENSITY AND MINOR AXIS INTENSITY FEATURES The Major Axis Intensity is the longest dimension of an ellipse of best fit and is intensity weighted The Minor Axis Intensity is the narrowest dimension of the ellipse of best fit and is intensity weighted The figure below illustrates the difference between intensity weighted and non intensity weighted Major or Minor Axis and Aspect Ratio See the Aspect Ratio Intensity Feature on page 156 for more information tr fm 1 4 Ci q m p i po ajo iF hsp ner ir tn i mm D Fi to 4 bo xis Intensity Aspect Ratio M4 4 Aopen Ratio 0 76 Major A Minar
195. which allows you to use the proce dure for analyzing other files Refer to Using the Data Analysis Tools on page 59 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 51 GETTING STARTED WITH THE IDEAS APPLICATION On the File menu click Save as Data Analysis File Used Features Only and follow the instructions 2 3 above SAVING A COMPENSATED IMAGE FILE CIF The IDEAS application creates and saves a cif file when a rif file is opened By default the application names the cif file with the same name that the rif file has replacing the rif extension with cif The application also places the 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
196. wo Parasites Three Parasites APPLICATION EXAMPLES Counting parasites Counting phagocytosed particles FISH spot counting Counting punctate spots in images 170 UNDERSTANDING THE IDEAS FEATURES AND MASKS STD DEV FEATURE The Std Dev feature describes the overall distribution of pixel intensities The Std Dev is the standard deviation of the pixel intensity values in the mask The Std Dev value provides an indication of the texture or complexity of an object The following illustrates that apoptotic cells AnxnV positive exhibit higher Std Dev values in the darkfield channel scatter and higher brightfield Modulation values than non apoptotic cells AnxnV negative Brightfield Scatter AnxnV_7AAD a 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 IDEAS FEATURES AND MASKS 171 UNDERSTANDING THE SIGNAL STRENGTH FEATURES Signal Strength features include the following e Bkgd Mean and Bked StdDev features describe the background of the image e Intensity and Raw Intensity features quantify the intensities in the region of interest 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 subtracte
197. y clicking Add Files and browsing for the no brightfield control files for the experiment Hold down the control key to select multiple files at once 40 GETTING STARTED WITH THE IDEAS APPLICATION 3 When all of the control files for the experiment have been added to the list click Next The control file s are merged and loaded 4 Step 2 Verify the channels for each control in the experiment by checking the channel boxes gt Create Compensation Matri Step 2 Select remove channels for compensation Venfy that the selected channels are appropriate for your selected controls Make any appropriate changes and click Next Chaz Cho3 ChO5 F chos chos E chos Ch11 Ch12 The following tables are provided as a guide for each instrument configuration TABLE 2 FIRST GENERATION IMAGESTREAM CH 1 CH 2 CH3 CHG CH5 CH 6 470 500nm 400 470nm 500 560nm 560 595nm 595 660nm 660 735nm TABLE 3 IMAGESTREAM 1 CAMERA CH1 CH 2 CH3 CH5 CH 6 430 505nm 505 560nm 560 595nm 595 660nm 660 745nm 745 800nm DAPI PE Texas AF647 APC Cy7 Red GETTING STARTED WITH THE IDEAS APPLICATION 41 TABLE 4 IMAGESTREAM 2 CAMERA CH 1 CH 2 CH 3 CH5 CH 6 430 480nm 480 560nm 560 595nm 595 660nm 660 745nm 745 800nm Red 430 505nm 505 570nm 570 595nm 595 660nm 660 745nm 745 800nm DAPI Pacific BF Texas Red AF647 APC Cy7 Orange 5 Background and spatial offset corrections are performed the
198. y direct product thereof are exported or re exported directly or indirectly in violation of or used for any purposes prohibited by such laws and regulations 10 General This Agreement will be governed by and construed in accordance with the laws of the State of Washington without regard to or application of conflict of laws rules or principles The United Nations Convention on Contracts for the International Sale of Goods will not apply You may not assign or transfer this Agreement or any rights granted hereunder by operation of law or otherwise without Amnis prior written consent and any attempt by you to do so without such consent will be void Except as expressly set forth in this Agreement the exercise by either party of any of its remedies under this Agreement will be without prejudice to its other remedies under this Agreement or otherwise All notices or approvals required or permitted under this Agreement will be in writing and delivered by confirmed facsimile transmission by overnight delivery service or by certified mail and in each instance will be deemed given upon receipt All notices or approvals will be sent to the addresses set forth in the applicable ordering document or invoice or to such other address as may be specified by either party to the other in accordance with this section The failure by either party to enforce any provision of this Agreement will not constitute a waiver of future enforcement of that or any ot

IDEAS User Manual 4.0 - Department of Flow and Image Cytometry

Contents

Download Pdf Manuals

Related Search

Related Contents