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

Abstract

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.

Can MRI measure myelin? Systematic review, qualitative assessment, and meta-analysis of studies validating microstructural imaging with myelin histology

“…In studies with human tissue, a further complication is that post-mortem time also influences histological staining. The length of the post-mortem time indicates the advance of the tissue autolysis and therefore the microstructural intactness of the tissue ( Sele et al., 2019 ), but it can vary between a few hours and a few days between samples. While a considerable number of assessed papers did not report post-mortem times, in those who did, it varied between 4 hours and 3 days.…”

Perisomatic innervation and neurochemical features of giant pyramidal neurons in both hemispheres of the human primary motor cortex

“…However, the subjects they investigated were considerably older (between 74 and 102 years). Furthermore, they used materials with 12 h PMI, and post-mortem degradation could cause similar changes in brain tissue (Sele et al, 2019 ). Therefore, we can assume that the changes they described occur later in life or are the results of poorer preservation.…”

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

“…Frozen samples were pre-trimmed similar to the specimen preparation on a dry-ice-cooled metal plate and the obtained frozen tissue pieces prepared as previously described (Sele et al 2019 ). In brief, samples were incubated for 8 h at − 90 °C in 2% osmium tetroxide/0.2% uranyl acetate (diluted in water free acetone) within an AFS2 (Leica Microsystems, Vienna, Austria) (Fig.…”

“…Semi-thin and ultrathin sectioning was conducted as previously described (Sele et al 2019 ). In brief, semi-thin sections were acquired and stained with a 1% toluidine blue solution (Sigma-Aldrich, USA) to confirm the area of interest under the light microscope (Olympus BX63) for trimming.…”