This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.
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Method Article
Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography
In This Article
Summary
Abstract
This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.
Introduction
Since the early 1970s, tomography approaches in transmission electron microscopy (TEM) have been widely used for the structural characterization of biological specimens1,2,3. The appeal of transmission electron tomography was that it could be used to study a wide range of biological structures at the nanometer scale, from the architecture of cells and the ultrastructure of organelles to the structure of macromolecular complexes and proteins. Nevertheless, transmission electron tomography could not be used to study very thick samples (greater than 0.5 µm). Indeed, thick s....
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Protocol
Caution: Consult the material safety data sheets (MSDSs) of the various reagents before using them. Several of the chemicals used during sample preparation are toxic, carcinogenic, mutagenic, and/or reprotoxic. Use personal protective equipment (gloves, lab coat, full-length pants, and closed-toe shoes) and work under a fume hood while handling the sample. Sectioning the sample with an ultramicrotome involves the use of sharp instruments, and careful use of these tools is mandatory. In the steps described below, the authors assume that the sample is a cell culture sample. The protocol may need to be modified according to the sample type, especially the centrifugation ....
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Results
In our study, electrons were accelerated to 200 kV in the field emission gun transmission electron microscope. Images were collected in STEM BF mode using a 20-µs dwell time. Regarding the design of the through-focal tilt-series, we found that focus intervals of 150 nm gave satisfactory results for the ultrastructural study of a 750 nm-thick biological sample-even though the depth-of-field of the electron beam was only 3 nm-since we used a very large convergence semi-angle of 25 mrad.
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Discussion
In this article, we present a conventional sample preparation protocol along with a step-by-step guide for performing 3D analysis on thick biological samples using STEM through-focal tomography. Resin embedding of biological specimens has been used for decades26, and alternative protocols that are adapted to different kinds of samples can be found throughout the literature27. In contrast, imaging thick specimens in STEM using through-focus methods is a new undertaking, and .......
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Disclosures
We have no conflict of interest to disclose.
Acknowledgements
This work was funded by two ANR grants (ANR-11-BSV8-016 and ANR-10-IDEX-0001-02). We also acknowledge the PICT-IBiSA for providing access to chemical imaging equipment.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Phosphate Buffered Saline | Sigma-Aldrich | P4417 | |
| Ethanol | Sigma-Aldrich | 2860 | |
| Epoxy resin | EMS | 14120 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Add paraformaldehyde powder to PBS heated at approximately 60 °C. Increase pH by adding 1 N NaOH until no PFA powder is visible. |
| Glutaraldehyde | Sigma-Aldrich | G5882 | |
| Osmium Tetroxyde | EMS | 19150 | |
| Uranyl Acetate | Sigma-Aldrich | 73943 | |
| Gelatin Capsule | EMS | 70110 | |
| Triming and Histo knives | LFG Distribution | Diatome diamond knives | |
| Electron Microscopy copper grid | LFG Distribution | G200-Cu | |
| Grid Coating Pen | LFG Distribution | 70624 | |
| Specimen Holder | JEOL | EM-21311 HTR | |
| Electron Microscope | JEOL | JEM-2200FS |
References
- De Rosier, D. J., Klug, A. Reconstruction of three dimensional structures from electron micrographs. Nature. 217 (5124), 130-134 (1968).
- Gan, L., Jensen, G. J. Electron tomography of cells. Q Rev Biophys. 45 (1), 27-56 (2012).
- Lucic, V., Rigort, A., Baumeister, W.
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