One way to characterize the quality of one’s microscope is to measure the point spread function (PSF), that is the image that is created by a point source (which can be a fluorescent bead smaller than the expected size of the PSF embedded in agarose). I recently spent quite some time aligning my multiphoton microscope, and due to some reasons, it took me not only some hours, but several days (or nights). In the end, the PSF again was symmetric, small, sharp and nice, but the way to go there was crowded with all varieties of bad, strange and extremely ugly PSFs, sometimes at points during the alignment when I didn’t expect it.
In scientific publications, one only gets to see the nicest symmetric ‘typical’ PSFs, so I just want to put some really bad PSFs here. Not all the PSFs were as bad as they look like, because I have adjusted the contrast of the gifs in order to better show the shape of the PSF. All gifs are simple z-stacks acquired with variable z-spacing at the same day on the very same microscope, with only minor and rarely predictable changes to the beam path.
Here comes the classical stone in the water-PSF, thrown from the lower right:
This one is thrown from the top right:
This is the bathing in a sea of other beads-PSF. Or, rather than bathing, swimming, because there is a clearly visible upwards direction. This happens when you have to many beads in your agarose:
This astigmatic PSF, on the other hand, is rather indecisive, first going to the right and then upwards.
Here an even more advanced indecisive right-up-goer-PSF:
This one is so undecided that it decides to almost split in two halves. Let’s call it the bifurcating PSF:
This is the banana-PSF, coming from the left and going back again:
And finally, you would not guess this to be a PSF, here comes the flying eagle-PSF. I created it simply by tightening too much one of the screws that held the dichroic beam splitter, the back aperture of the objective still seemed to be properly filled:
Sidenote: In some of these pictures in one or two planes, one can see a diagonal striping pattern. This comes from the pulsing laser, which was unstable during these days, being modulated to the microsecond-timescale. Fortunately, nowadays I have a nice PSF and a stable laser again …