Application of backscattered electron imaging for observation of biological specimens using resin-embedded and de-embedded samples.

To obtain stereographic images of tissues containing both free and sectioned surfaces, backscattered electron (BS) imaging was tested with both resin and de-embedded specimens. Although the ultrastructure of several organelles in cells could be identified on a sectioned plane of the resin-embedded tissues, three-dimensional images of tissues were difficult to obtain. On the other hand, three-dimensional images showing both the free and sectioned surfaces of tissues could be obtained by scanning electron microscopic observation of the de-embedded specimens stained with heavy metals in BS mode. Organelles in the sectioned plane could be recognized with enough contrast to discriminate the different cellular components even after the de-embedding process. Our present method would make it easier to obtain images of rare structures barely detectable by the conventional cracking methods and will contribute to a three-dimensional analysis of biological specimens.