In most microscopy images that are published in research papers, there appears a scale bar. The scale bar is like a ruler that allows you to compare sizes and distances in images from different sources. Although a scale bar is helpful for assessing by eye, many image processing programs allows you to measure distances in the image. The problem is that these measurements are in pixels. That is what I encountered when I wanted to measure certain objects in my images. How to convert from pixels to nanometers (or microns) requires a simple formula and some prior data as follows:
- Objective magnification
- Lens magnification (in some microscopes, it is possible to get extra mag of 1.25x, 1.6x or 2x.
- C mount (is usually 1x)
- Pixel size – is the actual pixel size of the camera that is attached to the microscope.
- Binning – i.e. combining a cluster of pixels to a single pixel. The common options are 1X1, 2X2 and 4X4. Binning is usually used to reduce noise, but at the expense of resolution.
Image pixel size = camera pixel size x binning / (obj. mag x lens mag x C mount)
For Cascade 512 camera (16µm/pixel on CCD), at 60x mag and 1×1 binning:
Pixel size = 16×1/(60x1x1) = 0.2667µm = 266.7nm.
Obviously, the smaller the pixel size of the camera, the better the resolution (i.e. actual pixel size in the image).
Here is a helpful table of pixel sizes (in nanometers) for some common cameras:
Note that all cameras listed here have a square pixel size (e.g. 9300×9300). Some cameras have rectangular pixel sizes (e.g. ExwaveHAD 3CCD with 6350×7400). In such cases, the length and width of the pixel sizes should be calculated separately. However, I am told that microscope-intended cameras today have only square pixels, not rectangular.
For an explanation how to add a scale bar in ImageJ, click here.
For an explanation how to add a scale bar in photoshop, click here.