A.

Region Selection using PhenochartTM

1. Select and designate ROIs 2. Define ROI coordinates 3. Scan ROIs at higher
magnification, export
Select ROIs utilizing: image as .im3 file
#1 1) computer assisted or
#2 2) user defined methods
X: 50.92 mm, Y: 6.00 mm

#3 Hovering the cursor in

#4 ...[50961, 6064].im3
the middle of the ROI will
provide approximate coordinates
File name contains exact
Number ROIs according to position PhenochartTM coordinates

B. Spectral Unmixing & Visualization using inForm®

1. Build spectral library 2. Spectrally unmix .im3 files 3. Examine signal fluorescence
across all channels
Opal 520 Opals 540-690

DAPI

X
CD3 CD4 B220
FoxP3 CD8 F4/80
Autofluorescence Signal No signal
ex: 520 single stain
CD3 Examine signal across
FoxP3
CD4
CD8
Assign IDs and 4. Confirm signals make biological sense all channels to identify
B220 pseudocolors spectral bleedover
F4/80
DAPI for each stain
Autofluorescence Unexpected colocalization
of markers suggests
Utilize appropriate single
staining artifacts and/or
stain, unstained, and DAPI
issues with panel
controls for spectral unmixing F4/80 CD3
design/staining protocol

C. Cell Segmentation using inForm®

1. Empirically define settings to
Default Settings
identify cellular compartments
(i.e. nucleus, membrane)

Change settings while Unsegmented Image Fill holes Fill holes Fill holes Fill holes Fill holes
visualizing the results Roundness Roundness
Min-Max size
Roundness Roundness
Min-Max size
Roundness
Min-Max size Min-Max size Min-Max size
of segmentation Refine splitting Refine splitting Refine splitting Refine splitting Refine splitting

2. Apply segmentation settings to all images

Nucleus
Membrane

Green = predicted nucleus

Red = predicted membrane
3. Examine segmentation results on all ROIs
Retrain iteratively as needed