Optimization of ultrastructural preservation of human brain for transmission electron microscopy after long post-mortem intervals

Electron microscopy (EM) provides the necessary resolution to visualize the finer structures of nervous tissue morphology, which is important to understand healthy and pathological conditions in the brain. However, for the interpretation of the micrographs the tissue preservation is crucial. The quality of the tissue structure is mostly influenced by the post mortem interval (PMI), the time of death until the preservation of the tissue. Therefore, the aim of this study was to optimize the preparation-procedure for the human frontal lobe to preserve the ultrastructure as well as possible despite the long PMIs. Combining chemical pre- and post-fixation with cryo-fixation and cryo-substitution (“hybrid freezing”), it was possible to improve the preservation of the neuronal profiles of human brain samples compared to the “standard” epoxy resin embedding method. In conclusion short PMIs are generally desirable but up to a PMI of 16 h the ultrastructure can be preserved on an acceptable level with a high contrast using the “hybrid freezing” protocol described here.