I am doubting if the scaling that I use for measuring areas is indeed correct and if it is the best way to do.
I first drew a line from the left to the right corner in my .czi file and measured the distance in µm with the zen blue software.
To write the macro, I opened one of the tiff files (czi converted to tiff) drew the same line and set the µm distance that I got from the czi image.
Each image consists of several tiles (4x4) stitched together taken with a 5x objective.
Is this a good way to do the scaling? If so, can I check somewhere in the summary table if indeed areas are expressed in µm2 rather than pixels or inches?
This is my macro:
run("Set Scale...", "distance=5000.4052 known=6455.941 unit=inch"); run("8-bit"); setAutoThreshold("Default dark no-reset"); //run("Threshold..."); setThreshold(11, 255); //setThreshold(11, 255); setOption("BlackBackground", true); run("Convert to Mask"); run("Set Measurements...", "area mean standard min median limit display redirect=None decimal=2"); run("Analyze Particles...", "size=100-Infinity circularity=0.01-1.00 display exclude clear include summarize add");
The summary table gives me these numbers for the image attached
ARPE19IFNy-4dpi-Scene-01-B1-B01-Stitching-01-Image Export-01_c1.tif 265 1877700.887 7085.664 9.311 250.426 255
which would mean the average size of the plaques in this image is 7085 µm2?
Thank you for your help!