Fiji Macro for measuring distance of cells to a an helminth parasite

Analysis workflow of neutrophil and monocyte recruitment. Related to Figure 1.

The image analysis was performed with Fiji, using a generic ActionBar to define Region Of Interest (ROI) and a

custom script (ImageJ macro language, available on demand) to automatically threshold and measure the images.

The worm(s) outline(s) were manually drawn by an experimenter for each image, using the generic BIOP

ActionBar “Multi Manual Select” that facilitates ROIs creation.

The two channels of interest (respectively Gr1 or Ly6C and Ly6G markers) were binarized using an automatic

thresholding algorithm (respectively “Intermodes” for 1 hour time point and “Huang” for 6 hours time point,

for both channels).The automatic thresholding algorithm was chosen based on the visual inspection of

output images. The segmented images were denoised with a Median Filter (radius 2 pixels). The resulting masks

of the two channels were used to generate a third mask using the Boolean operator AND (keeping the common

positive pixels).

For each image, the experimenter-defined ROI(s) were used to create a distance map. This distance map was

used to generate measurement bands, with a defined width (defined by experimenter to be 100 microns),

iteratively expanded to a maximum value (defined by experimenter to be 600 microns). Finally, the positive

areas of the masks were measured for each band and output as a table.

As an output image, the three masks (the two channels of interest and the resulting AND operation) were

merged and saved as and RGB image for visualization as thumbnails in different Operating System

independently of any software.

Some data files are attached to allow testing the macro.