SEM techniques cannot substitute LM but they can provide a great deal more information, especially about ornamentation. Methods of sample preparation for SEM should preserve the living condition of a pollen grain as far as possible. In addition, pollen coatings should be removed from the pollen surface in such a way that no details of the pollen grain are lost. For the SEM, ace-tolysis is not an optimal method for cleaning the pollen surface, as it will often destroy apertural details. Pollen with fragile exines may also be destroyed.
As a routine, all pollen grains should be observed in an air-dried condition, which gives the best information about the pollen grains at anthesis and their harmomegathic situation.
The best results have been obtained using 2,2-dimethoxypropane (DMP) (HALBRITTER 1998). This method can be used for fresh material (pollen grains should be collected when anthesis starts) and for herbarium samples after short rehydration in water. Unless stated otherwise, the pollen grains shown in "Pollen Terminology. An illustrated Handbook" represent the turgescent state.
Fresh anthers are put into a pouch made of filter paper and immediately transferred into acidified 2,2-dimethoxypropane (a drop of 0.2 M HCl added to 30 ml DMP). After 20-30 min in DMP (or up to 24 h) samples are transferred to pure acetone for a few minutes and critical-point dried in CO2 using acetone as intermediate fluid. The dried pollen samples are then mounted on stubs using double-sided adhesive tape, sputter-coated with gold and observed with the SEM.
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