Scanning Microscopy


Double Layer Coating for backscattered electron imaging is a coating and imaging method especially suitable for high resolution scanning electron microscopy (SEM) of large biological samples. Since the backscattered electron (BSE) signal from thin metal coating layers is quite low, field emission SEM's and very sensitive BSE-detectors are required for this method. In this study an improved BSE-detector of the YAG type was used with an in-lens type field emission SEM. Two samples were investigated in order to demonstrate and to improve the potential of this new approach: (1) cryo-prepared cultured kidney cells were shadowed by electron beam evaporation with platinum-carbon (unidirectionally at a fixed angle of 45°) and then coated with an additional 10 nm carbon layer; and (2) cryo-prepared trichocyst matrixes (paracrystalline structures contained in secretory granules, the trichocysts, found in Paramecium) were coated by ion beam sputtering with about 1 nm of platinum. This sample was rotated and tumbled during coating in order to obtain as uniform a metal layer as possible and then an additional 10 nm carbon layer was evaporated over the metal. When these samples were viewed at a primary beam accelerating voltage (Vo) of 10 kV or higher, contrast was good on the unidirectionally coated cell culture samples. However, trichocyst matrixes with the thinner and more uniform coating showed very poor contrast because most of the BSE detected represented beam-specimen interactions from the bulk of the sample and not in the thin platinum layer. The situation was improved by using low Vo (4 kV). Under these conditions the penetration depth of the electrons is reduced and a greater proportion of the BSE electrons are scattered by the platinum layer. The results were compared with freeze-fracture and deep-etch transmission electron microscope studies of the trichocyst matrixes from the literature: Almost similar resolution is achieved on the biological structures but a better impression of the three dimensional arrangement of the whole trichocyst matrix is obtained with the SEM. The globular particles form disc-like structures that are connected with each other by thin fibers.

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