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

Summary

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

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.

Introduction

Serial block face scanning electron microscopy (SBF-SEM) was first described by Leighton in 1981 where he fashioned a scanning electron microscope augmented with an in-built microtome which could cut and image thin sections of tissue embedded in resin. Unfortunately, technical limitations restricted its use to conductive samples, as non-conductive samples such as biological tissue accumulated unacceptable levels of charging (electron buildup within the tissue sample)¹. While coating the block-face between cuts with evaporated carbon reduced tissue charging, this greatly increased imaging acquisition time and image storage remained a problem as ....

Protocol

All animals were handled according to the guidelines described in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Vision and Ophthalmic Research and the University of Houston College of Optometry animal handling guidelines. All animal procedures were approved by the institutions in which they were handled: Mouse, rat, rabbit, guinea pig, and non-human primate procedures were approved by the University of Houston Animal Care and Use Committee, zebrafish procedures were approved.......

Discussion

The purpose of this methods paper is to highlight the tissue preparation and imaging methodology that has allowed our lab to reliably capture high-resolution serial electron microscopy images, and to point out critical steps that lead to this outcome as well as potential pitfalls that can occur when conducting SBF-SEM imaging. Success using this protocol requires proper fixation of tissue, impregnation of heavy metals into the sample, modifications of the embedding resin to reduce charging, as well as an understanding of.......

Materials

Name	Company	Catalog Number	Comments
1/16 x 3/8 Aluminum Rivets	Industrial Rivet & Fastener Co.	6N37RFLAP/1100	Used as specimen pins.
2.5mm Flathead Screwdriver	Wiha Quality Tools	27225
Acetone	Electron Microscopy Sciences	RT 10000	Used to dilute silver paint.
Aspartic Acid	Sigma-Aldrich	A8949
Calcium Chloride	FisherScientific	C79-500
Conductive Silver Paint	Ted Pella	16062
Denton Desk-II Vacuum Sputtering Device equipped with standard gold foil target	Denton Vacuum	N/A	This is the gold-sputtering device used by the authors, alternates are acceptable.
Double-edged Razors	Fisher Scientific	50-949-411
Embed 812	Electron Microscopy Sciences	14120
Gatan 3View2 mounted in a Tescan Mira3 Field emission SEM	Gatan & Tescan	N/A	This is the SBF-SEM device used by the authors, alternates are acceptable.
Glass Shell Vials, 0.5 DRAM (1.8 ml)	Electron Microscopy Sciences	72630-05
Gluteraldehyde	Electron Microscopy Sciences	16320
Gorilla Super Glue - Impact Tough	NA	NA	Refered to as cyanoacrylate glue in text.
Ketjen Black	HM Royal	EC-600JD	Refered to as carbon black in text.
KOH	FisherScientific	18-605-593
Lead Nitrate	Fisher Scientific	L62-100
Microwave	Pelco	BioWave Pro	This is the microwave used by the authors, alternates are acceptable.
Osmium Tetroxide	Sigma-Aldrich	201030
Potassium Ferrocyanide	Sigma-Aldrich	P9387
Silicone Embedding Mold	Ted Pella	10504
Sodium Cacodylate Trihydrate	Electron Microscopy Sciences	12300
Samco Transfer Pipette	ThermoFisher Scientific	202	Used to make specimen pin storage tubes.
Swiss Pattern Needle Files	Electron Microscopy Sciences	62115
Thiocarbohydrazide	Sigma-Aldrich	223220
Uranyl Acetate	Polysciences, Inc.	21447-25
Reconstruction Software
Amira Software	Thermo Scientific	N/A	Used to create the reconstructions found in figures 5-7 and 9.
Fiji (Fiji is Just ImageJ)	ImageJ.net	N/A	TrakEM2 can be added to Fiji to asist in manual segmentation.
Microscopy Image Browser (MIB)	University of Helsinki, Institute of Biotechnology	N/A
Reconstuct Software	Neural Systems Lab	N/A
SuRVoS Workbench	Diamond Light Source & The University of Nottingham	N/A
SyGlass	IstoVisio, Inc.	N/A	Allows for reconstruction in virtual reality and histogram-based reconstruction methods.