In situ X-ray-assisted electron microscopy staining for large biological samples

Abstract

Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.

Preserving extracellular space for high-quality optical and ultrastructural studies of whole mammalian brains

“…Preserving ECS improves the efficiency of staining by increasing the diffusion efficiency of OsO 4 . Time-lapse X-ray micro-computed tomography (micro-CT) 21 of 3-mm-thick mouse brain slabs during the application of cacodylate-buffered OsO 4 showed that, in ECS-preserved mouse brain slabs, it took 9 h for osmium to penetrate the entire depth ( Figure 2 A). Staining time was extended by 22% to 11 h in a conventionally fixed brain slab ( Figure 2 B).…”

A Scalable Staining Strategy for Whole-Brain Connectomics

“…In particular, the introduction of two rounds of osmication for enhanced membrane contrast ( 29, 40 ), the separation of osmium and ferrocyanide treatments to increase the penetration depth of staining ( 9 ), and the preservation of ECS to improve staining efficiency ( 36 ) have all advanced the field. Moreover, the recent attempts at staining a whole mouse brain have replaced TCH with pyrogallol for tissue conditioning ( 11, 12 ) and employed microCT X-ray monitoring of the staining process ( 42 ), greatly improving large volume staining and its analysis. Nevertheless, multiple challenges persist because, for a sample the size of a mouse brain, improving whole-brain staining quality often has deleterious effects on tissue integrity.…”

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

“…Utilisation of the fs laser for sample processing in life sciences provides a tool for targeted trimming of soft biological tissue that can complement other available mechanical tools in workflows that are highly dependent on precise specimen trimming, such as ultramicrotomy 14 or lathe implementations 2 . To interrogate the ultrastructure at nanometre scale, tissue specimens of dimensions reaching several cubic millimetres need to be contrasted with heavy metals and embedded in resin [15][16][17][18][19][20][21][22] . Studying rare, localised structures with sub-µm detail requires a highly accurate preparation of those volumes of interest to be imaged and analysed 20, [23][24][25] .…”