Counting Cells with ImageJ

Keep in mind that ImageJ processing is not perfect. However, the error of missing cells may be "corrected" by the error of including some noise.

Steps in Video:

Process - Subtract Background
Image - Adjust - Threshold
Process - Binary - Fill Holes
Process - Binary - Convert to Mask
Process - Binary - Watershed
Analyze - Analyze Particles

Basic description of tools:

Subtract Background - cancel out noise of the background
Threshold - change the image to binary image of red, black and white, or blue
Fill Holes - fill empty spaces between rings to make circles
Convert to Mask - allows for subsequent processing
Watershed - automated separation of separate fused cells by a 1 pixel line
Analyze Particles - process the image to acquire a cell count
Size - parameter of what cells to include in data by area (pixels^2)
Circularity - parameter of what cells to include by how close to a circle the shape appears. The maximum bound (1.0) means a perfect circle. The minimum bound deviates from being a circle.
Show - Outlines will trace the outside of the mask images. Masks will show the black and white image similar during thresholding.
Exclude Edges - ImageJ will not include cells that are not fully contained in the boundaries of the image

Macro Code:
run("Subtract Background...", "rolling=12");
setAutoThreshold("Default dark");
setThreshold(35, 255);
run("Convert to Mask");
run("Fill Holes");
run("Convert to Mask");
run("Watershed");
run("Analyze Particles...", "size=120-Infinity circularity=0.00-1.00 show=Outlines display exclude clear summarize");

[vercas.]

Thank you for explaining how to process an image through ImageJ and uploading a video about it!
and I believe you need to revisit your video whenever there are new comments to answer them...again thanks!

[GautamGawande]

Thanks Kevin. It made my job a lot easier.

[majestia]

Thanks! Trying out image J for the first time.

[andrewvirtual]

thanks this makes measurements so much easier

[conglu]

Thanks very much for sharing! this is very helpful!!!!

[HaploMaster]

Thank you very much for that quick tutorial that helped me a lot !

[vidakarimnia]

Thank you very much for the great video

[rizwanurrahmanabeer]

Outstanding Tutorial! It just solved my problem. Thanks a lot, Kevin. :)

[michaelb]

Thanks alot! Fill holes doesn't work well for my particular images - the middle area of cells disappears completely and the outer cells don't change. Eh. It still is working well - I used the 'record' feature to make a macro to automate it and it works like a charm

[mzulfadh]

This is what I looking for.. Thank you

[qianzhu]

Thanks very much! It's really helpful.

[anaghaa]

Thank you so much! ❤️ This really helped me a lot ☺️

[zingo]

Does this work for 3D images as well? As in if i have several slices of pictures? 

Thank you for this!

[alejandroprieto]

Thanks! That's great, I'm trying for geological material!

[zahraahmadi]

Hello I have a question
I want to count hair graft with imageJ
( i have numbers of grafts but when I use this app the results is not correct )
Would you please help me ?

[LikesStrokes]

Thanks for the video! It's excellent!
I have one question: I have to quantify some PI (propidium iodide) images of hippocampal slices... how do I get the percentage of dead cells for the total tissue area? For that, how do I get a sum of the area of ​​all dead cells and divide by the total area of ​​the slice?
Preferably in a quick way without having to mark all dead cells.
Thank you!

[DrsJacksonn]

Hi there. I'm looking for a program to count cells, but this program doesn't seem to do the trick. I have images of a micro-array covered with wells and I only want to count cells _inside_ those wells (which are regularly interspaced as if located on the vertices of a grid). In other words, I need the program to only count cells on particular pre-selected locations of the picture. Any advice there?

[irishguy]

Kevin, How does someone acquire the photograph of a sample that is stand. I mean if I want to count cells per ml, how do I know how much sample is in the photo.
I know people say use a hemocytometer but is there a glass slide with a groove that takes a desired amount of sample or something other than a hemocytometer. I would have thought a hemocytometers lines would mess up the image taken from a microscope.

[irishguy]

Do I buy a glass slide with a well and inject my stained sample into this well prior to taking photo.

[thestella]

Hi what magnification did you get your image on?