1. Cell-counting with ImageJ
By Nate Honold, David Li, Andrew Marx, Joseph I. Wang, and Jimmy Zhang

2. Opening a Picture
File >> Open
Select Image

3. Removing Background and Noise
Process>>Subtract Background
Image>>Type>>8-bit
The ball size depends on the relative size of the cells in the image. For smaller cells, choose a higher size (i.e- 15~20)

4. Altering Color-scheme
Image>>Adjust>>Threshold
Choose Default and B&W
Adjust the top slider until only the desired cells are left (Left – More cells, Right – Less cells)
Check Light Background if the picture has a black background.
Do NOT move the bottom slider and click Apply.

5. Refining the Cells Process>>Binary>>Fill Holes
This will color the cytoplasm black
Process>>Binary>>Convert to Mask
Process>>Binary>>Watershed
This adds lines to clusters of cells to distinguish between each one.

6. Analysis Parameters Analyze>>Analyze Particles
Approximate size of cells can be determined by using the wand tool and clicking on cells to find their individual area with Analyze>>Measure.
Circularity determines what shapes ImageJ will search for (Low – not circular, High – Circular)

7. Result
All objects that match the size and shape specified will be highlighted.
The Summary will show the total number and area of the cells.
Ex) 709 cells were detected
One can add a scale to convert the pixel area to actual area if desired