Particle Analysis Question (Screenshot Included)

Hello. The particle analysis tool is not registering cells (on some images but not others), even when I alter the pixel analysis threshold size.

The protocol (right hand side) worked for another image that is very similar to this one.

Thank you very much for your assistance.

I would venture that this image has a calibration that the particle analyzer has issues with.

Notice below the image name it says that the image is calibrated in inches (2.32x1.73 inches). Is this the same for the image that does work?

If not, then you need to either set the real calibration or remove it by going to Image > Properties… and changing Inches to pixels, and the next 3 fields to 1.0

Let us know if this worked for you.

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Thank you sir, that did work.

As a follow-up, how would I go about applying particle analysis to a stack of 288 images (all with different titles)? I attempted to create a batch process macro but the issue seems to be the different names of each image. Surely there is a simple solution but I could not find it after extensive searching. Thank you very much!

Hello,

You could have a look at the proposed solution here

I am sorry but I don’t understand that description or how to apply it to particle analysis (versus fluorescence intensity, which is the other poster’s need).

This is the macro that I created, and I need it to apply to a folder of differently named images (which are paired, DAPI and PI), and I only care about the number of particles in each image to generate a ratio of PI+ to DAPI+ cells:

run("Subtract Background...", "rolling=2000 light sliding disable");
run("Split Channels");
close();
selectWindow("A12 10 A.tif (green)");
close();
selectWindow("A12 10 A.tif (red)");
setOption("BlackBackground", false);
run("Make Binary");
selectWindow("A12 10 A.tif (red)");
run("Analyze Particles...", "  show=Outlines display exclude summarize record");
saveAs("Results", "/Users/Sebastian/Desktop/Results.xls");

Thank you very much!

The idea was to show you how to transform a specific macro (containing verbatim file names) into a generic macro (using variables for the current image titles).

There’s some documentation on the Batch Processing page and a tutorial how to apply a common operation to a complete directory: