Springtime for two-photon microscopy

Today, the fields and forests around Basel are full of flowers that try to disseminate their pollen. Fixed pollen are, apart from sub-diffraction beads and the convallaria rhizome, one of the most commonly used test/reference samples for fluorescence microscopy. This is both due to their fine, spiky structures and their strong autofluorescence. The scientific study of pollen (and other small things), palynology, provides us with elaborate protocols on how to collect, clean, stain and fix pollen (example 1example 2) with glycerol jelly between two glass slides.

For two-photon microscopy, these protocols are not ideal since the objectives typically have no correction for glass cover slips between the sample and the objective. Therefore I tested whether it would be possible to look at pollen with a much simpler protocol, using a two-photon microscope.

Here comes the sample, a dandelion flower (one of the most common flowers right now around Basel), with its anthers carrying the precious pollen (photographed through the eyepiece of a dissection microscope):


To make the pollen ready for imaging, I warmed up some low-melting agarose, created an agarose drop on a petri dish and briefly dipped the flower into the agarose drop. A lot of pollen detached from the anthers and started to float on the agarose surface. This process can be clearly seen with the eyes under a dissection microscope. Next, I waited until the agarose became solid.

Under the two-photon microscope (20x objective, resolution around 3 micron FWHM in z) two dandelion pollen look like this in a z-stack (the diameter of one pollen grain is around 30 micron):


That’s a quite complex structure. The roundish bright blobs are probably nothing but dust. The smaller bright spots are spikes; the spikes on the side and on the bottom side cannot be recognized well due to degraded resolution. A 3D projection makes the structure a bit more evident:


I leave it up to any interested reader to google for “dandelion pollen”, which will give you a much nicer picture of the pollen grain based on electron microscopy.

Here’s also a picture of a pollen from a flower that I picked together with the dandelion, a daisy. The size of the pollen is a bit smaller (20 micron diameter) and exhibits a less complex structure: it is basically a sphere starred with a lot of spikes. It is very similar to the pollen reference slides that I used to have in previous labs:


This “agarose-fixed” sample will of course dry out after some time and can not be used as a reference sample. But, for example, if you have a friend (who is not so much into science) visiting your lab and you want to show him your two-photon microscope, it might be inappropriate to sacrifice a transgenic animal for that purpose – but it does not require much effort to pick up some flowers in spring and dip them in molten agarose.

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