Automatic Cell Counting with ImageJ

Description

This repository contains four ImageJ plugins to count mammalian cells:

• In the pictures of cell suspension in a standard hemocytometer.
  • Bright-field plugins detect ALL cells regardless of their alive/dead status.
  • Phase contrast plugin detects only live cells (obviously in suspension with trypan blue).
• In the pictures of differently stained cells.

The following items are included:

1. Trypan Blue Exclusion [folder]
   • Bright-field [folder]
     • sample images (1x dilution) [folder]
     • CellCount--ProcessingTemplate--BrightField.xltx
     • CellCount--BrightField--Maxima.ijm
     • CellCount--BrightField--Threshold--30.ijm
     • CellCount--BrightField--Threshold--100.ijm
   • Phase contrast [folder]
     • sample images (2x dilution) [folder]
     • CellCount--ProcessingTemplate--PhaseContrast.xltx
     • CellCount--PhaseContrast.ijm
2. Multicolor [folder]
   • sample images [folder]
   • CellCount--Multicolor.ijm
3. README.md (this file)
4. LICENSE (GNU GENERAL PUBLIC LICENSE)

Instructions

0. Here's a VIDEO.
1. DOWNLOAD and install ImageJ.
2. Create a directory (for example, Cell Counting) in the ImageJ plugins directory:
   • Windows
     C:\Program Files\ImageJ\plugins
   • Mac OS X
     /Applications/ImageJ/Plugins
3. DOWNLOAD and unpack the archive with the files:
4. Extract the .ijm files from the archive, and place them into the directory you created.
5. Launch ImageJ.
6. To launch either macro follow:
   Plugins > Cell Counting > [MACRO NAME]
7. The plugin will prompt you for a folder containing the images to be analyzed.
8. Output:
   • Maxima:
     • File “Cell counting results (Maxima).txt” in the folder with the images analyzed.
     • Copy of the results in the system clipboard.
   • Threshold-N:
     • Folder with the processed images in the folder with the analyzed images.
     • File “Cell counting results (Threshold-N).txt” in the folder with the analyzed images.
     • Copy of the results in the system clipboard.
   • PhaseContrast:
     • Folder with the processed images in the folder with the analyzed images.
     • File “Cell counting results (PhaseContrast).txt” in the folder with the analyzed images.
     • Copy of the results in the system clipboard.
   • Multicolor:
     • Folder with the processed images in the folder with the analyzed images.
     • File “Cell counting results (Multicolor).txt” in the folder with the analyzed images.
     • Copy of the results in the system clipboard.
9. For Trypan Blue Exclusion macros, you'll have to normalize the data -- effectively, account for how different the size of your picture is compared to the size of the 1x1 mm square on the hemocytometer panel.
   1. Take a picture of the 1x1 mm square (with or w/o cell suspension).
   2. Locate the picture resolution in picture file properties (X pixels by Y pixels).
   3. Measure the sides of the square in this picture (a by a pixels).
   4. Calculate normalization factor F = (a×a)/(X×Y)
   5. Multiply the cell count reported by macros by F.
10. Excel templates are provided to facilitate the data analysis.