Posted by
Alex Brown on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Measuring-PSFs-using-water-dipping-objective-tp7578494p7578505.html
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Stephan,
I tried this precise thing recently - measuring the PSF of a water dipping
objective (in a two-photon microscope). I used tetraspeck beads, as they are
bright and perfect for my application.
I found that the beads move a lot. To prevent this:
Embed the beads in a thin layer of agar in a small petri dish. Leave the dish
overnight (in the dark). The beads will tend to move to the bottom of the layer,
so make sure it is thin - though I presume you're using a fairly long WD
objective?
Use agar instead of agarose, and use a high (5%) concentration. Plain agar
contains agarose as well as agaropectins, which are a collection of smaller
molecules. This seems to really cut down the movement. I haven't tried gelatin,
but that may well be a good alternative.
You could also image the (settled) beads at the bottom of the dish by flipping it
over. Obviously this may introduce some aberration, as you are now imaging
through a layer of plastic, but it seems to work pretty well.