Observation of Biological Specimens Using Dark Field STEM

Abstract

Unstained biological specimens were observed in the dark field STEM mode using a JEM-100CX TEMSCAN equipped with an annular type dark field detector. It was confirmed, by comparing the micrographs obtained in the above mode with micrographs obtained in the dark field CTEM mode, that the former were superior in regard to observable detail. In electron microscopy, it is a well known fact that the observation of unstained biological specimens consisting of light elements such as carbon, nitrogen, oxygen, etc., is extremely difficult due to the small scattering factor involved. In general, although negative or positive staining by heavy metallic elements is feasible for such specimens, the possibility of structural, morphological and chemistry related (e.g., bonding) degradation, as a result of such staining, is always present. Accordingly, freeze etching and freeze sectioning, in spite of the complicated preparation procedure involved, are becoming quite popular in that a specimen so treated, becomes highly resistant to degradation resultant upon staining. A further popular means for surmounting the above problems relating to specimen degradation, poor contrast, etc. is the so-called “dark field method”, the salient feature of which is that it is simple to utilize and dispenses with the problems involved in the freeze etching and freeze sectioning techniques. The main reason why dark field STEM is more effective than dark field CTEM is that, in the case of former, there are no lenses below the specimen, thus permitting thicker specimens to be observed without having to take into consideration lens chromatic aberration caused by inelastic scattering. Another reason Is that increased space availability allows a large angle acceptance detector to be used, thereby enhancing the collection efficiency1) of the elastically scattered electrons which in turn ensures a high contrast image and low-dose specimen observation.