Comparison of Beam Damage of Hydrated Biological Specimens in High-Pressure Scanning Electron Microscopy and Low-Temperature Scanning Electron Microscopy

Abstract

High Pressure Scanning Electron Microscopy (HPSEM) is a technique in which biological materials are viewed close to their natural state in a saturated water vapour environment and at ambient temperatures. The absence of chemical or cryogenic fixation in this technique can offer considerable advantages in the reduction of preparative artifacts in labile specimens but it is also necessary to consider the effects of the electron beam interaction with the specimen. A comparative study has been undertaken with low temperature scanning electron microscopy (LTSEM) to evaluate the relative damage rates in the labile specimen Narcissus sp. . The results are compared with those expected from theoretical models of the beam-specimen interaction developed by other workers. Full details of the theory and practice of HPSEM have been presented elsewhere.Fresh, untreated perianth segments of Narcissus sp. were excised and examined in HPSEM at a saturated water vapour pressure of 1716 Pa, with a beam voltage of 10 kV and beam current of approximately 100 pA. The beam dose rate was therefore approximately 15 MJ.Kg−1s−1 over the scanned area. Fully frozen hydrated specimens were prepared for LTSEM and examined in an uncoated condition at a beam voltage of 3kV to reduce charging artifacts. No metal films were applied to the LTSEM specimens since this would artificially increase the thermal conductivity of the surface.