Fast Homogeneous En Bloc Staining of Large Tissue Samples for Volume Electron Microscopy

Abstract

Fixation and staining of large tissue samples are critical for the acquisition of volumetric electron microscopic image datasets and the subsequent reconstruction of neuronal circuits. Efficient protocols exist for the staining of small samples, but uniform contrast is often difficult to achieve when the sample diameter exceeds a few hundred micrometers. Recently, a protocol (BROPA, brain-wide reduced-osmium staining with pyrogallol-mediated amplification) was developed that achieves homogeneous staining of the entire mouse brain but requires very long sample preparation times. By exploring modifications of this protocol we developed a substantially faster procedure, fBROPA, that allows for reliable high-quality staining of tissue blocks on the millimeter scale. Modifications of the original BROPA protocol include drastically reduced incubation times and a lead aspartate incubation to increase sample conductivity. Using this procedure, whole brains from adult zebrafish were stained within 4 days. Homogenous high-contrast staining was achieved throughout the brain. High-quality image stacks with voxel sizes of 10 × 10 × 25 nm³ were obtained by serial block-face imaging using an electron dose of ~15 e^-/nm². No obvious reduction in staining quality was observed in comparison to smaller samples stained by other state-of-the-art procedures. Furthermore, high-quality images with minimal charging artifacts were obtained from non-neural tissues with low membrane density. fBROPA is therefore likely to be a versatile and efficient sample preparation protocol for a wide range of applications in volume electron microscopy.

Keywords: BROPA; EM; SBEM; block-face; connectomics; protocol; sample preparation; zebrafish.

Figure 1

Unsuccessful staining attempts. (A) Telencephalon of adult zebrafish stained with a protocol that reduced incubation times of the original BROPA protocol to a total duration of 2 weeks. Note severe tissue damage. (B) Telencephalon of adult zebrafish stained with a protocol that reduced incubation times of the original BROPA protocol to a total duration of 5 days without lead aspartate. Note charging artifacts in nuclei and neuropil.

Figure 2

Application of fBROPA to the adult zebrafish brain. (A) Coronal section through the telencephalon of adult zebrafish at the level of Dp (posterior zone of the dorsal telencephalon). Note homogeneous staining. Black particles outside the tissue are silver particles in the surrounding resin to optimize conductivity. (B) Neuropil close to the surface. (C) Neuropil 300 μm below the surface.

Figure 3

Application of fBROPA to the adult zebrafish brain. (A) Section through the tectum near the location where the diameter is maximal (1.1 mm). Image is a mosaic of 6 × 6 tiles. (B–D) Three images acquired at different depths. Approximate locations of images are indicated by outlines in (A). (E) Examples of images showing synapses (5 nm pixel size). Vesicle pools close to the presynaptic membrane and a thickening of both membranes are visible. Synapse detection can be performed in 3D as shown in Supplementary Data S1 (movie). Note uniformly high contrast. The partial damage on the right side of the tectum occurred during dissection and is unrelated to fixation or staining.

Figure 4

Application of fBROPA to early differentiated organoids (two cells). Insert shows details of the membrane ultrastructure between the two cells.