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3.22. Can I calculate the calibration properties of an image post Acquisition?

If you are going to be performing any form of Quantitative analysis on your image data then it would be more accurate to retrieve the calibration properties from the Acquisition system or re-acquire the data having first calibrated using the specific objective lens.

That said, the basic equation to derive the calibration properties (um/pixel) for your image is as follows:

image properties um/pixel = camera cell size/magnification*

e.g. for an image acquired using a Hamamatsu C9100-13 camera and a 40X objective lens (no other intermediate magnification) the calculation would be as follows:

cell size of the camera (taken from the Hamamatsu C9100-13 spec sheet) = 16um x 16um

magnification = 40

16/40 = 0.4 um/pixel

*magnification includes the Objective magnification used to acquire the data but may also include your C-mount magnification along with the magnification introduced by any other intermediate lens e.g. an Optovar.