Germinal center (GC) Analysis

We use Fiji to identify distinct regions within the Germinal center (GC) using follicular dendritic cell (FDC) markers: CD23 and CD35. We use Stardist to identify T cells there.

Overview

Given an image with four channels, CD23, CD35, Dapi and T cells staining, and an ROI file marking the borders of the entire GC within the image, the macro does the following:

1. Generates an ROI for CD35 and CD23 staining within the fitting ellipse of the GC's ROI
2. Detect T-CELLS using Stardist
3. Generate a histogram along the main diagonal of the GC on top of CD35 and CD23 channels

Generate an ROI for CD35/CD23

We first blur the CD35/CD23 channel using Fiji's "Gaussian Blur..." operation with sigma = 15. We then identify CD35/CD23 regions withing the GC to be the region with intensity threshold above 5000 for CD35 and above 4000 for CD23. Left: CD35 channel. Right: The identified CD35 region within the GC that is contained within the fitting ellipse of the user provided GC’s ROI

Detect T Cells using Stardist

To detect T cells we first run out-of-the-box Stardist on the T-cells Dapi's channel with the following parameters: args=['input':'Dapi', 'modelChoice':'Versatile (fluorescent nuclei)', 'normalizeInput':'true', 'percentileBottom':'1.0', 'percentileTop':'99.8', 'probThresh':'0.5', 'nmsThresh':'0.4', 'outputType':'Both', 'nTiles':'1', 'excludeBoundary':'2', 'roiPosition':'Automatic', 'verbose':'false', 'showCsbdeepProgress':'false', 'showProbAndDist':'false'], process=[false]"). We then filter out identified nuclei with low mean intensity (lower than 3000), or non-regular size (smaller than 10 m^2 or bigger than 100 m^2)

Generate histograms

To generate the histograms, we define a rectangle along the main diagonal of the GC's Fitting ellipse. The rectangular captures also regions before and after the GC as to capture the CD35/CD23 intensity there too.

The width of the rectangle is set to 20% of the GC fitting ellipse’s minor diagonal. The Length of the rectangular is set to include extra 20% of the GC fitting ellipse’s major diagonal residing before and after the GC region (around 0-100 m and 600-700 m in the image above).