Cell counting using ImageJ

Sample preparation

Images should be taken at the right magnification, allowing identification of individual cells in each zone of the growth plate.

Procedure

Watershed algorithm can be used both in ImageJ or Fiji. Make sure java is up to date on the computer as this is a java applet.

To quantify DAPI positive cells open the file and select the zone of interest:

Clear the outline leaving only the zone of interest:

For DAPI and other fluorescence images, invert the colours:

Convert the image into an 8-bit version:

Adjust brightness and contrast (clicking “Auto” and then “Apply” is often enough):

Activate the watershed plugin:

There are certain parameters which need to be set before the analysis:

Radius indicates the predicted radius of the particles to be measured. It works well at 0.5 for the resting and proliferative zones and 1.0 for the hypertrophic zone (larger cells). Min/max level indicates the intensity of greyness which is still recognised as a particle. 0 to 200 is a good setting for the general DAPI staining. If the staining is faint and not enough cells are picked out, in particular for the hypertrophic zone, increase max to 210-220. If too many cells/particles are counted, reduce the max down to 175.

Select “8-connected” particles for the analysis. This accounts for the fact that ellipsoid shapes which will be counted can be touching but will still be included in the analysis (a function which the regular particle counter plugin in ImageJ is lacking):

Select “Overlaid dams” from the bottom menu:

Tick “Show progression messages” and press “Start Watershed”. The output file looks like this:

If too many or not enough particles were counted, adjust the Max value and/or radius.

Note

The analysis will take very long if the Max value is 255 and over. Clicking “Create animaton” slows down the process further.