Think outside the dot.

1. Feature Description and Application Think outside the dot.

2. IDEAS Image Analysis Software • IDEAS is an image analysis application that performs high content morphometric analysis on tens of thousands of images. • Features are what IDEAS uses to quantify cell morphology. • 162 features in the default template. • 23 features per channel and 6 channels per image or 138 channel features. • 8 additional mask based features. • 16 System features. • Unlimited number of user defined features. • New features are continually being developed. AMNIS CORPORATION – Company Overview

3. Centroid X,Y Intensity Centroid X,Y 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Which of the 162 features do I use? AMNIS CORPORATION – Company Overview

4. Feature Type IDEAS calculates two types of features • Single featuresare automatically calculated by IDEAS and are divided into two categories. • A) Mask basedfeatures, such as Area and Intensity. • B) System features, such as camera timer and flow speed. • Combined Features are created by combining cell based features and are defined by the user. Examples of combined features are radial delta centroid, and nuclear to cytoplasmic ratio. AMNIS CORPORATION – Company Overview

5. Feature Hierarchy 1. Single Features (Require a Mask, 23 per channel, 31 overall) A. Mask Based Features Size: Units in pixels (Area, Perimeter, Major Axis, Minor Axis, Major Axis Intensity, Minor Axis Intensity) Signal Strength: Units are in integrated pixel values (Intensity, Mean Intensity, Minimum Intensity, Peak Intensity) Location: Units in X,Y Coordinates from an origin in the upper left (Centroid X, Centroid Y, Centroid X Intensity, Centroid Y Intensity). Shape: Defines mask shape within the image (Aspect Ratio, Aspect Ratio Intensity, Object Rotation Angle, Object Rotation Angle Intensity, Compactness, Elongatedness, Negative Curvature, Spot Count). Texture: Defines pixel or regional variation (Spot Small Total, Spot Medium Total, Gradient Max, Gradient RMS, Frequency) Correlation: Units in transformed Pearson’s Correlation values (Similarity, Similarity Bright Detail). B. System Features (Do not Require a Mask, 16) Object Rate: (Flow Speed, Camera Timer, Camera Line Number) Channel background: (Background Mean Intensity 1-6, Background Standard Deviation 1-6) Object number 2. Combined Features (User defined): (Radial Delta Centroid, Nuclear to Cytoplasmic ratio, Perimeter ^2/ Area, Peak/Mean Ratio) AMNIS CORPORATION – Company Overview

6. Feature Mask Correlation Single features require a mask • A feature quantifies cell morphology and intensity. • A mask defines a region of interest. • The feature is generated using the pixel values within the mask from either the corrected image file (cif) or from a processed image such as the open residue image. • User defined masks can identify subcellular regions and can enhance the resolution of the feature. • Additional features can be calculated from any user defined mask or by combining features. AMNIS CORPORATION – Company Overview

7. Types of Masks There are three types of masks • The system mask is the default mask and is designed to quantify total fluorescence. M1, M2 and the combined mask are system masks. • A function mask requires user input and there are 5 types of function masks. Dilate, Erode, Fill, Morphology and Threshold. • A combined mask uses Boolean logic to combine and subtract masks. An example is the cytoplasmic mask, created by taking the brightfield mask and not the morphology mask of the nucleus. AMNIS CORPORATION – Company Overview

8. Function Masks There are five types of function masks • Dilate: Adds pixels to the outside of the starting mask. • Erode: Subtracts pixels in from the edge of the starting mask. • Fill: Fills in closed gaps in the starting mask. • Morphology: Uses an algorithm to mask the fine structure within the starting mask. • Threshold: Masks the brightest pixels within the starting mask. AMNIS CORPORATION – Company Overview

9. Pixelated Imagery • 6 Channel Images are collected using a 10 bit CCD camera operated in TDI (time delay integration) mode. • Pixel values from a 10 bit detector range from 0 to 1023. • Each image is a grey scale two dimensional representation of the cell. • The color of the image is determined by the wavelengths of the channel it’s in. • A mask is applied that determines the region of interest. • Features are calculated based on the pixel values underneath the mask. AMNIS CORPORATION – Company Overview

10. Pixelated Imagery • 6 Channel Images are collected using a 10 bit CCD camera operated in TDI (time delay integration) mode. • Pixel values from a 10 bit detector range from 0 to 1023. • Each image is a grey scale two dimensional representation of the cell. • The color of the image is determined by the wavelengths of the channel its in. • A mask is applied that determines the region of interest. • Features are calculated based on the pixel values underneath the mask. AMNIS CORPORATION – Company Overview

11. Pixelated Imagery • These are the pixel values for a single PE image. • Light is quantified for each 0.5 um pixel in the image, and identifies both the intensity and location of the fluorescence. • All 162 features are calculated from the digital image and include everything from total intensity to variation across the image. AMNIS CORPORATION – Company Overview

12. Creating a New Mask and Feature Select New Mask Select Function Mask System Mask Training Cells Membrane Mask Or plot the new feature Morphology Mask Use the new mask to generate a feature Hand tag a training set of cells to test the new feature AMNIS CORPORATION – Company Overview

13. Size Based Features • Size based features are in pixel units. • Area • Major Axis • Minor Axis • Major Axis Intensity • Minor Axis Intensity • Perimeter AMNIS CORPORATION – Company Overview

14. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Area Brightfield Description: Area measures the number of pixels in a mask and is expressed in pixels. • Applications: • Quantify and compares 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 vs. SSC plot for comparing with flow cytometry. Channel 5 PI DNA AMNIS CORPORATION – Company Overview

15. Major Axis and Minor Axis Brightfield Fluo4 Description: Major Axis corresponds to the longest dimension of the ellipse of best fit. Minor Axis is the narrowest dimension of the ellipse of best fit. Major Axis • Applications: • Quantify and compare cell width and height. • Identify small, medium and large cells. • Convert the radius and diameter to um. • Compare particle diameters. Minor Axis Data collected in collaboration with MD. Steven Sollott GRC NIH, Baltimore MD AMNIS CORPORATION – Company Overview

16. Major and Minor Axis Intensity Brightfield Fluo4 Description: Major Axis Intensity is the longest dimension of the ellipse of best fit and is intensity weighted. Minor Axis Intensity is the narrowest dimension of the ellipse of best fit and is intensity weighted. Major Axis Intensity Minor Axis Intensity • Applications: • Quantify and compare fluorescence width and height. • Identify single cells. Data collected in collaboration with MD. Steven Sollott GRC NIH, Baltimore MD AMNIS CORPORATION – Company Overview

17. Perimeter Brightfield Description: The perimeter measures the boundary length of the mask in number of pixels. • Applications: • 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 dendrites vs. those that don’t have them.. Channel 5 PI DNA AMNIS CORPORATION – Company Overview

18. Signal Strength Features • Signal Strength Features are measured in integrated pixel values. • Intensity • Mean Intensity • Minimum Intensity • Peak Intensity AMNIS CORPORATION – Company Overview

19. Intensity Brightfield HLA FITC Description: Intensity is the sum of the pixel values within the mask (total intensity) minus the background intensity and is calculated using the formula; Intensity = Total Intensity – (Background Mean Intensity x Area) • Applications: • Quantify relative levels of fluorescence between cells and within different regions of the same cell. • Immunophenotyping. • Cell cycle analysis. • Protein expression. • Protein activation. AMNIS CORPORATION – Company Overview

20. Peak Intensity RTX FITC CD45 PE Brightfield Description: Peak Intensity is the largest pixel value within the mask. This image is saturated in the FITC channel (peak intensity = 1023) but not in the PE channel. • Applications: • Measure the maximum pixel value within the mask. • Identify cells that saturate the CCD. • Peak to mean ratio identifies bright punctate staining vs. uniform staining. • Plotting peak intensity vs. area of a 30% threshold mask can identify antibody capping. AMNIS CORPORATION – Company Overview

21. Mean Intensity Brightfield CD71 FITC Description: Mean Intensity is the average pixel value within the mask and is calculated using the formula; Mean Intensity = Total Intensity / Area A B • Applications: • Quantifies relative levels of mean fluorescence between cells. • Identify bright punctate spots by calculating the peak to mean ratio. • Track internalization of surface bound antibodies. AMNIS CORPORATION – Company Overview

22. Minimum Intensity Brightfield YoYo1 Brightfield YoYo1 Description: Minimum Intensity is the lowest pixel value within the selected mask. • Applications: • Quantify spectral absorbance using the brightfield image. • Identify over compensated images. • Measure the level of malaria infection in RBCs. AMNIS CORPORATION – Company Overview

23. Location Features • Location Features are in X,Y pixel coordinates from an origin in the upper left corner. • Centroid X • Centroid Y • Centroid X Intensity • Centroid Y Intensity AMNIS CORPORATION – Company Overview

24. (0,0) 32 54 (0,0) 37 35 Centroid X, and Centroid Y Brightfield RTX AF488 Description: Centroid X is the number of pixels from the first column of the image to the center of the mask. Centroid Y is the number of pixels from the first row of the image to the center of the mask. Y X Brightfield RTX AF488 • Applications: • Identify the center of the mask. • Used to calculate the Delta Centroid or the distance between two fluorescent markers. • Used to calculate the Radial Delta Centroid. Y X AMNIS CORPORATION – Company Overview

25. Centroid X,Y Intensity Centroid X,Y Intensity Centroid X,Y Centroid X,Y Centroid X and Y Intensity Description: Centroid X Intensity, is the intensity weighted X centroid and is shifted from the center of the mask toward the center of fluorescence. Centroid Y Intensity, is the intensity weighted Y centroid. • Applications: • Identify the center of peak fluorescence. • Used to calculate the distance between two fluorescent markers. • Used to calculate the intensity weighted Radial Delta Centroid. AMNIS CORPORATION – Company Overview

26. Shape Features • Shape Features define the mask shape and have units that vary with the feature. • Aspect Ratio • Aspect Ratio Intensity • Object Rotation Angle • Object Rotation Angle Intensity • Compactness • Elongatedness • Negative Curvature • Spot Count AMNIS CORPORATION – Company Overview

27. Aspect Ratio Brightfield Composite Description: Aspect Ratio is the minor axis divided by the major axis and describes how round or oblong a mask is. Aspect ratio=0.93 • Applications: • Quantify the roundness of the mask. • Identify single cells vs. doublets. • Cell classification based on shape change. • Identify recently divided cells in mitosis. Brightfield Composite Aspect ratio=0.32 AMNIS CORPORATION – Company Overview

28. Aspect Ratio Intensity Brightfield Draq5 Aspect Ratio Intensity Dq5= 0.31 Description: Aspect Ratio Intensity is the minor axis intensity divided by the major axis intensity. Aspect Ratio Dq5= 0.61 Aspect Ratio Intensity Dq5= 0.31 Aspect Ratio Dq5= 0.61 • Applications: • 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. AMNIS CORPORATION – Company Overview

29. Object Rotation Angle Object Rotation Angle Horizontal Plane Horizontal Plane Major Axis Major Axis Object Rotation Angle and Intensity Brightfield 7AAD Composite Description: Object Rotation Angle is the angle of the major axis from a horizontal plane in radians. Object Rotation Angle Intensity is the angle of the major axis intensity from a horizontal plane in radians. • Applications: • Identify the orientation of an image relative to the image frame. Object Rotation Angle 7AAD = 1.2 Object Rotation Angle 7AAD = 0.83 Object Rotation Angle Intensity 7AAD = 1.2 Object Rotation Angle Intensity 7AAD = 0.81 AMNIS CORPORATION – Company Overview

30. Compactness Nuclear Compactness Brightfield Draq5 Description: Compactness Compactness is computed as the deviation of the object contour from a circle with the same radius and center as the object. A perfect circle has compactness = 0, amoeboid shapes increase in compactness. 0.07 0.11 0.16 0.18 • Applications: • Quantify irregularities in the morphology mask. • Cell classification for identifying cell types based on nuclear morphology. • Discriminates small round shapes from amoeboid shapes. Lymphocytes Neutrophils AMNIS CORPORATION – Company Overview

31. Negative Curvatures Negative Curvatures Brightfield Draq5 Description: Negative CurvatureCorresponds to the number of negative and positive slope changes along the contour of the morphology mask. One positive and one negative change equals a count of 1. 0 1 1 2 2 • Applications: • Enumerate the number of inward folds in the morphology mask. • Identify cell types based on nuclear morphology. • Discriminates small round shapes from amoeboid shapes. 1 3 1 3 2 1 4 4 2 3 AMNIS CORPORATION – Company Overview

32. Elongatedness Brightfield Composite AF488 Description: Elongatedness is the ratio of the maximum width to the minimum width of the bounding rectangle of the object. 488 Elongatedness = 6.4 • Applications: • Quantify the roundness of the morphology mask. • Identify single cells vs. doublets. • Cell classification based on shape change. • Identifies recently divided cells in mitosis. 488 Elongatedness = 2.0 488 Elongatedness = 1.2 AMNIS CORPORATION – Company Overview

33. Spot Count Brightfield Babesia YOYO1 Description: Spot Count is an integer corresponding to the number of connected components within a mask. Single Parasite • Applications: • Enumerate the number of fluorescent particle inside a cell. • FISHIS chromosomal polysomy. • Parasitic protozoan enumeration. • Counting phagocytosed particles. Two Parasites Three Parasites Data collected in collaboration with PhD Henry Wortis Tufts Dept. of Pathology, Boston MA AMNIS CORPORATION – Company Overview

34. Texture Features • Texture Features measure pixel or regional variation and indicate the granularity or complexity of the image. • Spot Small Total • Spot Medium Total • Gradient Max • Gradient RMS • Frequency AMNIS CORPORATION – Company Overview

35. Intensity Spot Small and Medium Total Transferrin PE Description: Spot Small Total is the local background subtracted intensity of spots smaller then 7 pixels in diameter. Spot Medium Total is the local background subtracted pixel intensity of spots smaller then 14 pixels in diameter Pixel values in blue are integrated into the Spot Small Total value. • Applications: • Quantify the amount of light in small spots. • Identify cells with bright punctate staining. • Used to distinguish apoptotic cells from live cells. • Quantifies the total intensity of small FISH spots. Transferrin Topo Map Line Profile AMNIS CORPORATION – Company Overview

36. Gradient Max and RMS Brightfield Draq 5 Description: Gradient Max is the largest slope between any three by three adjacent pixels in the image. Gradient RMS is the over all magnitude of all the gradient values in the image and reflects gradient quadratic mean. • Applications: • Quantify image crispness. • Identify cells with high contrast • Used to eliminate out of focus events. • Identify apoptotic events, or cells with crisp bright staining. A histogram of a line that transects the image shows high Gradients for in focus cells, and low gradients for out of focus Cells. AMNIS CORPORATION – Company Overview

37. Frequency Brightfield Scatter AnxnV_7AAD Description: Frequency is the standard deviation of the pixel intensities under the mask, and is an indicator of texture. • Applications: • Quantify light variation within a mask. • Identify images with a high degree of variation. • Apoptotic cells may have very different scatter frequencies then live cells. • Granular cells may have higher scatter frequency. Apoptotic Cells with high scatter frequency Live Cells with low scatter frequency AMNIS CORPORATION – Company Overview

38. Correlation Features • Correlation Features compare two channel images using a log transformed Pearson’s correlation coefficient. • Similarity • Similarity Bright detail AMNIS CORPORATION – Company Overview

39. NF-kB Pixel Intensity NF-kB Pixel Intensity 7-AAD Pixel Intensity 7-AAD Pixel Intensity Similarity • Applications: • Quantify translocation. • Identify copolarization of two probes. Description: Similarity is the log transformed Pearson’s Correlation Coefficient. Untranslocated Translocated 7-AAD image NF-kB image 7-AAD image 7-AAD image AMNIS CORPORATION – Company Overview

40. Similarity Bright Detail Description: Similarity Bright Detail is the log transformed Pearson’s correlation coefficient that is non mean normalized, and is applied to the open residue image. ADC Image Endosomes image • Applications: • Quantify the degree of colocalization between two probes. • Used to track internalization and intracellular trafficking of antibody drug conjugates to either the endosomes or the lysosomes. • Colocalization of Rituxan and compliment C3b. ADC Image Endosomes image AMNIS CORPORATION – Company Overview

41. SBD Open Residue Image • To remove the contribution of background, an image processing step called the “opening residue” is performed on each image of the image pair prior to calculation of SBD. • First, bright details are eroded with a 7 pixel-wide structuring element followed by a dilation to create the ‘Detail Eroded’ images. • Next, the detail eroded images are subtracted from the originals to produce the ‘Bright Detail’ images. • SBD measures the correlation of this final image pair. Original Image Detail Eroded Image Bright Detail Image AMNIS CORPORATION – Company Overview

42. System Features • System Features do not require a mask, and tend to deal with system wide metrics. • Object Number • Flow Speed • Camera Line number • Camera Timer • Background Mean Intensity 1-6 • Background Standard Deviation 1-6 AMNIS CORPORATION – Company Overview

43. Camera Timer and Flow Speed Description: Camera Timer is the camera clock reading that starts with data acquisition and ends when the file is written. Multiplying by 0.00182 converts the units to seconds. Flow Speed is the velocity of the core stream in mm/sec. • Applications: • Track kinetic changes in the sample during data acquisition. • Track changes in fluorescent intensities over time. • Flow speed vs. Camera Timer can track velocity changes over time. 23 Flow Speed vs. Camera Timer shows the periodic variation in the flow speed that gets corrected out when data is analyzed. Cells with large areas tapered off over the run While small cells remained at a constant concentration. AMNIS CORPORATION – Company Overview

44. Camera Line Number and Object Number Description: Camera Line Number counts the number of pixel rows that is written off the camera during acquisition. Object number is the order in which the images are collected • Applications: • Track kinetic changes in the sample during data acquisition. • Track changes in sample concentration over time. Drop in cell concentration Subtle changes in the sample concentration can be observed by plotting the Object number vs. the Camera Line Number and is indicated by the slight variation in this line.. Surge in cell concentration AMNIS CORPORATION – Company Overview

45. Background Mean Intensity and Standard Deviation Description: Background Mean Intensity is calculated by adding the intensities in the top 4 and bottom 4 rows of the image and dividing by the number of pixels. Background Standard Deviation is the square root of the variance in the top and bottom 4 rows. • Applications: • Measure detector variation in the background of the image. • Used to subtract background from features that calculate intensity. BG Mean = 30.2 30.1 30.0 30.1 191.7 30.3 BG Stnd Dv = 1.0 0.96 1.1 1.3 1.7 1.5 AMNIS CORPORATION – Company Overview

46. Combined Features • Combined features are user defined and are generated by combining base features and mathematical functions. • Radial Delta Centroid • Nuclear to Cytoplasmic Ratio • Peak Intensity / Mean Intensity • Perimeter ^2/ Area AMNIS CORPORATION – Company Overview

47. 44.3 44 0.3 46 46 0.0 C=√(Delta Centroid X)2+(Delta Centroid Y)2 34.5 56.3 56.5 23.3 8.3 48.2 Centroid X Pixels Centroid X Pixels Delta X and Y Centroid in Pixels Radial Delta Centroid Description: Radial Delta Centroid is the radial distance from the center of one mask to another measured in pixels. • Applications: • Quantifies the spatial relationship between two fluorescent probes. • Identify false apoptotic positive cells with the TUNEL and Annexin V assay. • Quantify shape change AMNIS CORPORATION – Company Overview

48. Nuclear To Cytoplasmic Ratio Brightfield Composite Description: Nuclear to Cytoplasmic Ratio is the area of the nuclear morphology mask divided by the area of the brightfield mask eroded 3 pixels and not the nuclear mask (cytoplasmic mask). • Applications: • Compare the nuclear area to the cytoplasmic area. • Identify cells in metaphase. • Quantify changes in cell volume relative to nuclear volume over time. Brightfield Composite Brightfield Composite Metaphase cells have lower nuclear to cytoplasmic ratios with small nuclear area and large cytoplasmic area. AMNIS CORPORATION – Company Overview

49. Peak to Mean Ratio Description: Peak to Mean Ratio is the background subtracted Peak intensity divided by the background subtracted mean intensity Brightfield GFP Composite • Applications: • Identify bright punctate staining from diffuse staining. • Quantify cells with incomplete capping. • Identify endosomal internalization. • Identify LC3 clustering at the autophagolysosome. Peak to Mean = (Peak Intensity – Background Mean) / (Mean Intensity – Background Mean) AMNIS CORPORATION – Company Overview

50. Internalization Description: Internalization uses a complex mask to calculate the ratio of the brightest 50% of the pixels in the cytoplasm, divided by the brightest 50% of the fluorescence everywhere in the cell. When the brightest fluorescence is inside the cell the ratio is 1. Brightfield EGF Brightfield EGF Brightfield EGF • Applications: • Quantify the fluorescence internalization. • Internalization of EGF at 37 over time. Intensity (Th50% and Center Mask) / Intensity Th50% AMNIS CORPORATION – Company Overview