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

Opening a Picture File >> Open Select Image

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)

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.

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.

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)

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