Calibration is something people seem to remember after collecting a bunch of data. There is a simple calculation to use if you need to do a quick calibration or want to figure out a resolution/pixel size trade off.
1. Determine the total magnification of your optical system. This includes:
A. The objective magnification
B. Any tube lens magnifiers (Some scopes have 1x and 1.5x switchable tube lenses)
C. The magnification of your camera mount (most often either .63x or 1x – Should be printed on the mount.)
2. The pixel size of your camera’s sensor.
With this info it’s a basic calculation: The calibration for the camera at a specific optical configuration is the size of the pixel at the sample. To determine the pixel size at the sample divide the pixel size by the total magnification. As an example:
– Pixel size 6.45um
– Total Magnification 100x (100x lens, 1x tube lens, 1x adapter)
Pixel Size / Total mag (6.45/100) = 0.0645um.
So when using the 100x lens each pixel is calibrated to 64.5nm in length.
This is a great way to check if a camera binning mode is acceptable, for instance using our 6.45um pixel camera at 100x at 2×2 binning the effective resolution would be (64.5*2) 129nm. This can also come in handy when trying to track objects or make tradeoffs in stage scanning vs. objective magnification.