Microscope Calibrations

This post is to document how and why we calibrate the microscopes. To begin with a microscope's most basic function is to change the size of something (i.e. make it bigger) for us to view. This does not mean the object changes size, just what we see does (the image is bigger). But we may want to use the image to measure a real size or distance. For example, in the image shown below, we might want to know far it is from the bug's belly to the ruler (or the bug's leg length or width or something).


Currently we usually store an image as a digital photograph on a computer, which means the image is stored as a collection of pixels (the far right part of the image above). If I were to measure a distance I would count how many pixels from place to place. Using the example above I might say that the bug's belly is one pixel away from the ruler, at its closest point. The question is what this distance is in the 'real world', i.e. the real distance from the bug to the ruler. To say, I need to know the 'calibration factor' for my microscope.

The calibration factor is a unit conversion, usually from pixels to microns for an ordinary light microscope. To calculate the conversion factor, all we need is a known distance. In the example above, the ruler will have real distances marked on it, so we have a 'known' distance in our image. All we need to do is measure how many pixels are between two markings on the ruler, and note the dimensions of the markings. For example, say I measure 5 pixels between two millimeter marks on the ruler. My calibration factor is then given by: 1000 um / 5 pixels = 200 um / pixel. I can then take this calibration factor and convert the bug-ruler distance: 1 pixel x 200 um/pixel = 200 um. So my bug is 200 um from the ruler.