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 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 computational 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.

Sample preparation and warping accuracy for correlative multimodal imaging in the mouse olfactory bulb using 2-photon, synchrotron X-ray and volume electron microscopy

“…2e). It is similar to previously reported artefacts associated with insufficient staining times (Hua et al, 2015;Mikula and Denk, 2015;Ströh et al, 2021), and it was completely absent in slices with thickness smaller or equal than 0.6 mm (Fig. 2f).…”

“…A number of variations were implemented to this protocol backbone with the aim to optimise the yield of samples holding optimal staining quality. Staining quality was routinely assessed with X-ray tomography using a laboratory-based micro-CT (LXRT), a technique well-suited to resolve the different histological layers (Bosch et al, 2021) as well as the dynamics of heavy metal diffusion when staining brain tissue (Ströh et al, 2021).…”

“…Heavy metals passively diffuse through soft tissue following a quadratic kinetic (Mikula and Denk, 2015; Ströh et al, 2021), i.e. staining three times the depth takes nine times longer.…”

“…Further, albeit more difficult to implement improvements are: (1) finding optimal formulations for fixative solutions for a specific sample, (2) replacing TCH for a reagent that does not create bubbles - such as pyrogallol (Mikula and Denk, 2015), and (3) minimising the sample expansion processes by e.g. counteracting osmium-derived effects with suitable additives (Hua et al, 2015; Ströh et al, 2021).…”

“…In order to prevent such charging artefacts, SBEM-specific SPP optimisations have incorporated elements that increase the conductivity of the sample -such as by increasing the total metal load employed in staining, in terms of both the number of metallic compounds and their concentrations (Tapia et al, 2012). The diffusion mechanism used to deliver those metals through the tissue remains passive, and therefore incubation time is subject to a quadratic relationship with the distance each compound should travel (Ströh et al, 2021), imposing severe temporal burdens for staining specimens of dimensions beyond 1 mm (Mikula and Denk, 2015). Moreover, abnormalities arising from individual staining steps can interact with each other (Hua et al, 2015; Mikula and Denk, 2015), creating an additional challenge to ensure all compounds optimally stain the tissue across all dimensions.…”