Fiji to Icy conversion?

S8R2 ABA (20 uM 45 min) Treated Col-0 DCF (4.3 uM 15 min)_Linear unmixing_Maximum intensity projection.tif (6.1 MB)

Background

I wrote a macro in Fiji, and I want to covert it to a “protocol” in Icy. It’s not beautiful, but it does the job

//open, split, name channels
orgName = getTitle();
run ("Split Channels");
close("C3" +orgName)
selectWindow ("C1-"+ orgName);

//find somata
run("Duplicate...", "title=BW");
run("Gaussian Blur...", "sigma=5");
setOption("ScaleConversions", true);
run("8-bit");
run("Brightness/Contrast...");
run("Enhance Contrast", "saturated=0.35");
run("Auto Threshold", "method=Triangle white");
run("Duplicate...", "title=stomata");
run("Analyze Particles...", "size=100-500 circularity=0.50-1.00 add");

//find chloroplasts
selectWindow ("C2-" +orgName)
run("Gaussian Blur...", "sigma=5")
setOption("ScaleConversions", true);
run("8-bit");
run("Auto Threshold", "method=Otsu white");

//chloroplasts within stomata
run("Duplicate...", "title=chloroplasts");
imageCalculator("AND create", "stomata","chloroplasts");
selectWindow("Result of stomata");
run("Analyze Particles...", "size=10-100 circularityy=0-1.00 add");

//measure DCF
selectWindow ("C1-" +orgName)
roiManager("Measure");
saveAs("Results", "C:/Users/Anthony/Desktop/results.csv")

//close down windows
close("C2" +orgName)
close("chloroplasts")
close("BW")

Challenges

The goal is to compare the signal in the whole stomata (ROI’s in C1) to the chloroplasts (C2) that are only in the stomata. In Fiji I used the image calculator and AND function. Is there anything similar in Icy, or do I need to figure out a different way to do this?

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Hey @Heather_BrownHarding,

interesting question! Let’s try together. This is how far I come: Channel splitting, Gaussian blur, automatic thresholding. Do you by chance know how one can make a Particle Analyser as Icy protocol?

dual_channel_analysis.zip (1.4 KB)

Furthermore, what does this code snippet in your macro do?

setOption("ScaleConversions", true);
run("8-bit");
run("Brightness/Contrast...");
run("Enhance Contrast", "saturated=0.35");

Cheers,
Robert

Hi @haesleinhuepf,

Thanks for your help. I guess I should have also posted how far I had made it in the protocol. It is working ok so far and it’s less individual steps, but I don’t know how robust it is yet. I still can’t quite figure out how to filter only the chloroplast ROIs (step 7) that follow within the ROIs of step 2.

leaf take 2.protocol (10.6 KB)

I’m only on day 3 of using the program so I’m pretty useless right now. Give me another week to work it out :wink:

The lab likes to look at the images coming up so they can make sure it’s doing what I say it will, so that is for visualization only. I should have pulled it out since it wasn’t relevant at all but, meh :woman_shrugging:

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I was being a bit dense. The answer is simple-- Boolean Operation ROI (using AND) will pull just the chloroplasts in the stomata. Time to refine protocol and send back to users. How this helps someone.

leaf take 3.protocol (8.4 KB)

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