From pixels to microns

I am a new user to cellprolifer. i am having problems converting pixels to real (e.g micron) values. when i take pics of cell nuclei, i am unable to convert cellprofiler units into microns. i have read the responses on how to do this but still cant figure out the whole procedure using the stage micrometer. how do i get conversion factor using the stage micrometer and then applying to my images?
a way to do it will be greatly appreciated as i am new to the sofware. Thanks

Once you have the stage micrometer, acquire a picture of the micrometer scale markings with the same magnification as for your cell images. You can then measure the pixel distance between marks (which are a set number of microns apart) using the software of choice. For example, you can open the image in CellProfiler by double-clicking on the filename in the lower-left panel, and measure a distance using *Tools > Measure length * in the menu bar; the pixel distance is shown in the lower right of the image. If you are more familiar with using Photoshop or ImageJ for this purpose, feel free to use that. However you chose to do it, the end result is a number of pixels for a given number of microns, so you can determine your conversion factor as (micron distance)/(pixel distance)

This conversion factor can then be used in CalculateMath by selecting the measurement that you want to convert, “None” as the operation and enter the conversion factor in the “Multiply the above operand by” setting. Keep in mind that for area-based measurements, you will need to square the conversion factor before entering it into the setting box.


Thanks Mark. have taken pics of the stage micrometer at same mag. Will try to follow the procedure you’ve outlined. looks clearer to me now.
Thanks and will get back to you.

Thanks alot. Have been able to calculate the surface area (square microns) from pixels as you suggested.