I have to prepare the slides of pollen which I have collected from the nests of native solitary pollen bees. The samples are preserved in 70% ethanol as pollen mass itself. I am looking for a standard procedure to prepare pollen slides for taking their pictures in a scanning electron microscope.
DearAnusree Padmanabhan P. S.
The samples must be chemically fixed, dehydrated in a series of solutions of alcohol or acetone with an increasing concentration from 30-50% to 100%, then the alcohol (or acetone) must be removed from the sample in a special apparatus, in which alcohol is replaced by liquid carbon dioxide, which is converted to a gaseous state by passing through the critical triple point.
Some modern microscopes are equipped with a vacuum system capable of maintaining a high (and ultrahigh) 10-3 Pa vacuum in the electron column and a relatively poor vacuum of up to 5-2000 Pa in the sample chamber. As a result, the sample is in an atmosphere (usually consisting of water or nitrogen vapors), although rarefied, but dense enough to neutralize the surface charge. Gas molecules are ionized by the primary electrons emitted from the cathode. The resulting positive ions interact with electrons, which accumulate on the sample and neutralize the surface charge.
As a result, plant pollen samples can be observed without a conductive coating.
Regards, Sergey Viktorovich Pushkin
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