Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Authors

Justin A Courson
Paul T Landry
Thao Do
Eric Spehlmann
Pascal J Lafontant
Nimesh Patel
Rolando E Rumbaut
Alan R Burns

Publication Date

3-26-2021

Journal

Journal of Visualized Experiments

DOI

10.3791/62045

PMID

33843931

PMCID

PMC8225236

PubMedCentral® Posted Date

3-26-2022

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Animals, Face, Microscopy, Electron, Scanning, : Serial Block-Face Scanning Electron Microscopy, SBF-SEM, 3D-EM, 3D-Reconstruction, Tissue Preparation, Specimen Preparation, Serial Sections, Ultrastructure, High-resolution, Volumetric Imaging, Biological, Large Volume

Abstract

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

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