The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts – and knowing how to avoid them – takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a ‘before-and-after’ experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animalsin vivowhere the ECS typically becomes nearly depleted.Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.Significance StatementChemical fixation of biological samples using PFA is a standard step routinely performed in neuroscience labs. However, it is known to alter various anatomical parameters ranging from protein distribution to cell morphology, potentially affecting our interpretation of anatomical data. With the increasing use of super-resolution microscopy, understanding the extent and nature of fixation artifacts is an urgent concern.Here, we use live STED microscopy to monitor in real time the impact of PFA on the microanatomy of organotypic hippocampal brain slices. Our results demonstrate that while PFA has little impact on the extracellular space and astrocytes, it compromises cell membranes and dendritic structures. Our study provides a strategy for a direct characterization of fixation artifacts at the nanoscale, facilitating the optimization of fixation protocols.

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