Using the Analyze Particles function in ImageJ to count fluorescent particles
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- Опубликовано: 12 сен 2024
- Quick tutorial for how to threshold your image to make everything binary and then how to use the Analyze Particles function for automatic counting of all circular particles. This can be used for counting fluorescent particles, cells, etc.!
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Here is a link to the documentation on this function (just ctl+f to find the analyze particles section): imagej.nih.gov/ij/docs/menus/analyze.html
And here is that link to download imageJ if you needed it! imagej.nih.gov/ij/download.html
Hi, I have a question, if the size of the particles you're interested in is in the 10-20 pixel range, do you input that range as is in the size (pixel^2) window or do you square that first and input 100-200?
Hi! Awesome question -- if you are looking for particles that have an area of 10-20 pixels total then you would input 10-20 in the pixel size.
Thank you for the video! How difficult is it to measure mean intensity rather than size? I have a three channel fluorescence image, I'd like to use the DAPI channel to isolate individual nuclei, then measure the mean intensity nucleus by nucleus in the other two channels. Is this possible?
One thing this analyze particles function doesn't do is provide the xy coordinates of each of the isolated particles, so you won't have an easy way of assigning a mean intensity to each actual nucleus in the image (unless the sizes are obviously different and then you can just manually assign).
However if you wanted to use the DAPI channel to create a mask (black and white image of the nuclei) you could use that to subtract the non-nuclei background of the other images, and then just use the basic measure tool to get a mean intensity measurement of each of the nuclei - unless you split the color channels you'll get three mean intensities by doing this, one for each Red Blue and Green channel. Less automatic, but sounds like it may suit your purposes.
@@diybiosensors Thanks for your response! I've managed to work things out, here is my method for anyone else interested:
1. Open image
2. Image -> Duplicate (Ctrl+Shift+D)
3. Image -> Color -> Split Channels
4. Select DAPI channel
5. Image -> Adjust -> Threshold (Ctrl+Shift+T)
6. Set bottom slider to maximum, and top slider to a point where all nuclei are red
7. Click apply
8. Analyze -> Analyze Particles
9. Set minimum size to desired size, make sure "Add to manager" is selected
10. Click Ok
11. The ROI Manager will automatically open
12. Analyze -> Set Measurements...
13. Make sure the desired measurements are selected (such as Mean gray value)
14. Select the window of the channel you wish to measure
15. Click "Measure" in the ROI Manager
16. Save the results from the Results window
17. Repeat as needed for the other channels
Could we differentiate the particle like metal, nonmetal, fiber etc..
Depends on if they have a visible property to differentiate them with - if they have a different shape, different color, or different general reflection of light then definitely!
how beautiful she is
The fluorescent particles are the real beauties here.
@@diybiosensors Agreed! There's nothing like a good fluorescence microscope image! Especially if it's part of an experiment you've been trying to get working for some time!