With the later versions of Photoshop CS4 and CS5 extended it is now very easy to add a scale bar to your microscope images. But before we go ahead you will need some information first.
1) The actual Pixel size of the camera attached to your microscope (here is a short list of some common cameras).
2) Did you use any binning when acquiring the image?
3) The lens magnification, C mount, and Objective Magnification (NB: normally lens and C mount are 1x, while objectives are e.g. 63X, 100X)
4) A bit of maths.
Actual Pixel size (APS) = CCD Pixel X Binning / Lens Mag x C mount X Objective Mag
Zeiss AxioimagerZ1 with a Coolsnap HQ2 camera,100X objective, 1×1 binning.
APS = 6450*1 / 1*1*100
Ok so now if you want to add a 5µm (5000nm) scale bar to your image you now do this
5000/64.5= 77.8 pixles So a line that is 77.8 pixels long will be exactly 5µm. Unfortunately the add scale bar feature in Photoshop does not allow for decimal places so its best to round to the closest full integer which in this case is 78 !
So now go to photoshop and from the Analysis menu select “set measurement” then “custom”. Here you can enter 78 for the pixel length and then 5 for the logical length and change the logical units to µm.
Save this as a preset to save time next time you have an image from the same microscope with the same settings.