Basic study on high-resolution SEM images using replica films.

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Ultrastructures of the replica films of biological specimens were observed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and scanning tunnel microscopy (STM). They were not observed by a routine SEM method because of severe contaminations, but could be revealed at ultrahigh-magnifications without contamination using a heating stage. Intramembranous particles were demonstrated as regular round protrusions by TEM,but were observed as irregular depressions by SEM.Complementary comparison of the ultrastructures were performed by TEM and SEM using the newly developed double replication method with Cr and Pt-C.Minute depressions which were not discerned by TEM were clearly noted by ultrahigh-resolution SEM.The present ultrahigh-resolution SEM method using replica films was proved to be useful for studying ultrastructures of intramembranous particles, gap junctions, tight junctions. Furthermore, the present method is applicable for the specimens of AFM and STM,although problems to be overcome were remained.