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ImageJ Mitochondria Analysis Guide

To analyze stained cells in an image using ImageJ: 1. Download and install the Color Deconvolution plug-in to split the image into colors representing all cells, stained cells, and a control. 2. Apply thresholding to the stained cell image to highlight cells and exclude background, and use the Watershed feature to separate touching cells. 3. Analyze the stained cell image by counting particles between 6-60 pixels to quantify the number and percentage of stained cells.

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Peter Hong
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47 views4 pages

ImageJ Mitochondria Analysis Guide

To analyze stained cells in an image using ImageJ: 1. Download and install the Color Deconvolution plug-in to split the image into colors representing all cells, stained cells, and a control. 2. Apply thresholding to the stained cell image to highlight cells and exclude background, and use the Watershed feature to separate touching cells. 3. Analyze the stained cell image by counting particles between 6-60 pixels to quantify the number and percentage of stained cells.

Uploaded by

Peter Hong
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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ImageJ Instructions

First you have to download ImageJ


Then you have to add the Color Deconvolution feature. To do so, you have to
download the plug-in from a website. Save the plug-in to the plug-in folder of the
ImageJ filed (saved wherever you decided).
To load an image, simply open the file from ImageJ.
REPETITION STARTS NOW
From there, you must click on the Color Deconvolution option.
Plug-in Color Deconvolution
Select the H DAB vector and press OK. This will split the Image into
three colors:
Purple- All cells, Colour 1
Brown- Stained Cells, Colour 2
Green- control (dont need to analyze this image), Colour 3

We have to analyze the purple and brown images


First thing we have to do is to convert the image into 8-bit (so the image can
be counted)
Image Adjust Threshold
This will open up a pop-up menu. Here we can select which cells to
highlight and which to exclude. Compare to the original image for optimal
calculation.
The brown filter usually requires parameters of 0 as the
minimum and 60 as maximum.

Once you are satisfied with the amount of highlighted cells click Apply
and Set
Next we have to differentiate between clumps of cells. To do so, we
have to use the Watershed feature.
Process Binary Watershed

Finally we are ready to analyze, or count the number of particles in the


Image.
Analyze Analyze Particles
This will lead you to this pop-up menu

The optimum sizes for analyzing cells would be 6-60 pixels (this
lets ImageJ avoid the small meaningless debris, while disregarding the
giant chunks of stains not bound to cells). Dont need to change
anything else. Just click Ok, and ImageJ should have counted the
number of particles that match the parameters you set.

You can get the total percentage stained by simply dividing


number of particles in Brown/Purple

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