PT - JOURNAL ARTICLE AU - Sebastian Ströh AU - Eric W. Hammerschmith AU - David W. Tank AU - H. Sebastian Seung AU - Adrian A. Wanner TI - <em>In situ</em> X-ray assisted electron microscopy staining for large biological samples AID - 10.1101/2021.06.19.448808 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.06.19.448808 4099 - http://biorxiv.org/content/early/2021/06/20/2021.06.19.448808.short 4100 - http://biorxiv.org/content/early/2021/06/20/2021.06.19.448808.full AB - 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 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.Competing Interest StatementA.A.W., D.W.T. and H.S.S. are the inventors of US Patent Application 16/681,028.