Low temperature backscattered electron imaging in the study of human tissues

A scanning electron microscopic technique for the examination of bulk, fresh, hydrated human tissue is described. Samples of fresh tissue are frozen in liquid nitrogen against a mirror-finished copper block and planed in a cryoultramicrotome before transfer to a low temperature scanning electron microscope. After sublimation of water from the specimen surface, the tissue is examined in secondary electron and backscattered electron modes. Adjacent pieces of tissue, and those retrieved after backscattered electron observation, can be readily prepared for and examined by light and by conventional transmission electron microscopy. The method has been tested with multiple blocks taken from 6 cases of human breast carcinoma. In the backscattered electron mode, the infiltrating columns of neoplastic cells can be distinguished from mammary adipose and fibrous tissue. Within a carcinoma, the collagenous stroma, carcinoma cells, and perivascular and perineural infiltrates can be identified. These features have been contrasted with those obtained by light microscopy, by low temperature scanning, and by transmission electron microscopy. This use of backscattered electron imaging for the investigation of unfixed hydrated tissue offers the possibility that the technique could be of considerable value in the microscopy of very small samples in which, because of a need for subsequent biochemical, histochemical, and immunologic investigation, fixation and dehydration are to be avoided.