This works for a multichannel z-stack containing fluorescently stained microglia cells. Only one channel is processed and can be chosen via a pop up dialog. The processing steps are as follows:

1. Open full dataset, raw input file (use virtual stack if too big for RAM)

2. Pre-processing

   • Maximum intensity projection
   • duplicate
   • apply Grays LUT
   • enhance contrast: CLAHE (https://imagej.net/plugins/clahe)
   • Threshold using Li
   • save mask in input file directory {originalFileName-mask.tif}

3. Area Fraction measurement

   • duplicate mask
   • Analyze particles: size 2-Inf µm^2; summarize, add to manager [the size filter should exclude objcts caused by background/noise but still include cell segments; adjustable via initial user dialog]
   • save summary table (containing %Area = area fraction based on whole FOV) and ROI set in input file directory: {originalFileName-Summary_results.csv} & {originalFileName-area-fraction-intensity-ROISet.zip}

4. Intensity measurements

   • for original image, substract background: rolling ball = 5, process whole stack
   • Sum Slices projection
   • activate all intensity-based parameters: area mean standard modal min integrated median display
   • Measure all ROIs
   • save results in input file directory {originalFileName-Measurement_results.csv}
   • clear ROI manager

5. Ramification index measurements

   • select original mask
   • Analyze particles: size 35-Inf µm^2; summarize, exclude from edges, add to manager [the size filter should include only objects that are large enough to be whole cells; adjustable via initial user dialog]
   • save ROI set in input file directory: {originalFileName-cell-ROIs.zip}
   • set measurement parameters to area only
   • loop through all ROIs:
     • measure area
     • create convex hull
     • measure area of convex hull
     • calculare ramification index: area / convex hull area
   • collate all results in a results table and save table in input file directory {originalFileName-Ramification_results.csv}

6. Clean up

   • close results file
   • reset ROI manager
   • close all image windows

The starting point is a generated segmentation mask. Once the binary mask has been generated/opened, select it and hit [Run] at the bottom of the script editor.