To assess the resolution of your microscope, measuring the point spread function (PSF) is one of the most basic and reliable methods. The PSF essentially shows how large the image of a very small, often sub-diffractive object will be. See this paper for reference on how to measure a PSF, and check out a previous blog post on PSF shapes.

When you align and optimize a microscope, a certain undesired appearance of the PSF (spherical aberration, coma, astigmatism) can rarely be traced back to a specific optical component: does it come from the 10-year-old objective? Or is it the dichroic? Or the misalignment of the Pockels cell? Or is it the laser beam itself? Often, one needs to optimize the optical alignment without understanding where the suboptimal performance comes from.

Recently, I was aligning a system where I observed a clear aberration and – rare enough – could trace it back to a very simple optical component. The main observation was a very strong astigmatism, which occurred when the focus in one dimension (x) did not coincide with the focus of the beam in the other dimension (y):

Technical details: PSFs recorded with a 10x objective at 905 nm using 1 μm diameter fluorescent beads.

This often happens when the dichroic of the microscope is too strongly attached such that it bends, acting as a slightly focusing element in one direction but not the other. In my case, things were even simpler: I had inserted a set of relatively large mirrors (2″ side length) into the beam path. For such large mirrors, even a small strain can bend their surface considerably, leading to the astigmatism observed above. Releasing this strain resulted in a perfectly normal PSF, measured a few minutes after the PSF shown above:

It was great to see how I could reproducibly increase and decrease the strain on the large mirrors back and forth, consistently achieving full-blown vs. negligeable astigmatism. It’s always satisfying to figure out such small technical details.