Name: preparation and observation of thick biological samples by scanning transmission electron tomography
Uploaded: 2017-03-12T13:00:00
Description: Watch this Scientific Journal Video about Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography at JoVE.com

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.

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 auth...

<ol>
	<li>De Rosier, D. J., Klug, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+three+dimensional+structures+from+electron+micrographs.">Reconstruction of three dimensional structures from electron micrographs.</a> Nature. 217 (5124), 130-134 (1968).</li><li>Gan, L., Jensen, G. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+STEM+for+the+biological+sciences.">Development and application of STEM for the biological sciences.</a> Ultramicroscopy. , 38-49 (2012).</li><li>Ercius, P., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incoherent+bright+field+STEM+for+imaging+and+tomography+of+ultra-thick+TEM+cross-sections.">Incoherent bright field STEM for imaging and tomography of ultra-thick TEM cross-sections.</a> Microsc Microanal. 15, (2009).</li><li>Hyun, J. K., Ercius, P., Muller, D. 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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marker-free+image+registration+of+electron+tomography+tilt-series.">Marker-free image registration of electron tomography tilt-series.</a> BMC Bioinformatics. 10, 124 (2009).</li><li>Kohl, L., Bastin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flagellum+of+trypanosomes.">The flagellum of trypanosomes.</a> Int Rev Cytol. 244, 227-285 (2005).</li><li>Luft, J. 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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 ...

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 &#181;m). Indeed, thick specimens produce too many scattered electrons, generating low signal-to-noise ratio (SNR) images. In addition, tomography involves the collection of images of tilted specimens, with the apparent thickness of the sample increasing with the tilt angle. Even though inelastic scattering can be filtered out using energy filters, the critical amount of electrons needed for high SNR images is barely reached in TEM. Hence, thick biological specimens have only been studied using sectioning4.
Some samples cannot be sliced: some might degrade when they are cut, and others need to be studied in their entirety in order to understand their complexity. An alternative approach is to use TEM in scanning mode5,6,7,8. In scanning transmission electron microscopy (STEM), the optical path of the electrons is different from that in conventional TEM. Electrons passing through the sample without scattering can be collected on the optical axis with a bright-field (BF) detector9, whereas those scattered elastically can be collected at a certain angle from the optical axis with a dark-field (DF) detector. The other advantage of STEM is that the focused electron beam is scanned at the surface of the sample, enabling the pixel-by-pixel collection of the images. Even though the electron beam broadens while passing through the sample10, this particular collection scheme is less sensitive to inelastically scattered electrons than conventional TEM. Furthermore, there is no lens post-specimen in STEM, thereby avoiding the chromatic aberrations that can occur in TEM. The camera length can be adjusted so that the BF detector mainly detects unscattered electrons. Using the DF detector to study thick samples is not recommended because of multiple scattering, which produces inaccurate images. Instead, the BF detector can be used11. While STEM can produce high SNR images, it has a relatively low depth-of-field because of the relatively high beam convergence compared to TEM, decreasing the amount of in-depth information that can be recovered from thick specimens. In the case of aberration-corrected STEM microscopes, where the convergence angle can be as high as 30 mrad, the depth-of-field can be low enough so that the information that is in focus originates from a focal plane of only a few nanometers. Setting up the electron beam in parallel mode enhances the depth-of-field of the electron beam to the detriment of the resolution12. However, this setup is not always possible.
Whenever it is necessary to use a convergent electron beam, one must use techniques that enhance the depth-of-field of the electron beam when studying thick samples. Recent studies have reported the acquisition of multiple images at various focal planes throughout the sample to recover the maximum amount of in-focus information13,14. The two studies describe different ways to handle the information from the different focal planes. Hovden et al. combined in Fourier space the images that were collected at various focal planes, and the final reconstruction was obtained directly from the 3D inverse Fourier transform13. In contrast, Dahmen et al. developed a convergent beam reconstruction engine to reconstruct in real space the 3D volume from the various focal planes14. Our laboratory also developed a method for imaging thick biological specimens. Our strategy was different from the two methods described above in that we merged the information that is in focus in the various focal planes and reconstructed the final 3D volume in real space using a parallel beam projector15. Our aim was to develop a method that can easily be performed in any electron microscopy laboratory. To this end, we aimed to collect the focal images in a limited amount of time, comparable to the time frame of conventional tomography experiments. In addition, our proposed method could be adapted for use with different types of alignment and reconstruction software.
In the context of our publication from 201515, we wanted to visualize and characterize the recovery of the depth-of-field, so we used a large convergence semi-angle of 25 mrad. Here, we present a step-by-step protocol for performing through-focal imaging in STEM according to the method developed in our laboratory in 201515, and we present how the data from 2015 was processed. This method recovers in-focus information from several focal planes throughout a thick (750 nm) biological sample and enables high-quality 3D reconstructions. Where relevant, the differences in this methodology versus the methods used by other groups are also presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>EMS</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Add paraformaldehyde powder to PBS heated at approximately 60 &#176;C. 
			Increase pH by adding 1 N NaOH until no PFA powder is visible.</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxyde</td>
			<td>EMS</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>73943</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Capsule</td>
			<td>EMS</td>
			<td>70110</td>
			<td></td>
		</tr>
		<tr>
			<td>Triming and Histo knives</td>
			<td>LFG Distribution</td>
			<td>Diatome diamond knives</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy copper grid</td>
			<td>LFG Distribution</td>
			<td>G200-Cu</td>
			<td></td>
		</tr>
		<tr>
			<td>Grid Coating Pen</td>
			<td>LFG Distribution</td>
			<td>70624</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Holder</td>
			<td>JEOL</td>
			<td>EM-21311 HTR</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscope</td>
			<td>JEOL</td>
			<td>JEM-2200FS</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and observation of thick biological samples by scanning transmission electron tomography

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.

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-&#181;s 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...

Watch this Scientific Journal Video about Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography at JoVE.com

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

The overall goal of this procedure is to obtain a three-dimensional reconstruction with depth of field recovery of a thick biological specimen using convergent beam scanning transmission electron tomography. This method can answer key questions in the field of structural biology about electron tomography of thick biological specimens. The main advantage of this technique is that images can be acquired in convergent bit mode and the data processing can be performed on most software dedicated to tomography.To begin preparing cell culture sample, centrifuge the sample at 5000 x g for five minutes. Use these centrifugation parameters throughout sample preparation. Discard the supernatant and resuspend the cells in one milliliter of PBS enriched with 4%paraformaldehyde and 2%gluteraldehyde.Incubate the suspension for 30 minutes at room temperature. Centrifuge the mixture, discard the supernatant, and wash the pellet three times with one milliliter portions of PBS. Using the same incubation conditions and washing procedure, incubate the pellet in one milliliter of PBS, enriched with 1%osmium tetroxide, and in one milliliter of PBS enriched with 2%uranyl acetate.Next, cool the sample to four degrees Celsius. Dehydrate the pellet by successive incubations in increasingly concentrated ethanol solutions. Maintaining the sample temperature of four degrees Celsius throughout incubation, centrifugation, and washing.Resuspend the dehydrated pellet in one milliliter of pure ethanol, and incubate overnight at 4 degrees Celsius. Then, allow the sample to warm to room temperature, and centrifuge the sample. Incubate the pellet in increasingly concentrated epoxy resin solutions at room temperature.After the final wash, resuspend the pellet in one milliliter of pure epoxy resin, and incubate overnight at room temperature. Centrifuge the sample, and remove the supernatant, and then resuspend the pellet in 200 microliters of epoxy resin and hardener. Transfer the suspension into the capsule.Incubate the plastic capsule at 60 degrees Celsius for 48 hours to polymerize the resin. Remove the capsule from the incubator and verify resin polymerization. Add a layer of epoxy resin and hardener over the embedded sample and incubate the capsule again under the same conditions.Mount the sample embedded in resin upside down in a specimen holder. Securely fix the sample on the trimming block of an ultramicrotome. Cut the plastic capsule away from the sample in the resin.Shape the exposed resin into a pyramid. Then transfer the capsule and specimen holder to the moving arm of the ultramicrotome. Install a trimming knife and guided by a microscope, refine the pyramid shape.Replace the trimming knife with a histology knife. Under the microscope, adjust the capsule pitch angle so the pyramid is perpendicular to the knife edge. Set the cutting speed and section the sample.After sectioning the sample, transfer the chosen section to a treated copper grid over filter paper. Allow the sample to dry, and then load the sample into the electron microscope. To design the through focal tilt series, first calculate the electron beam depth of field and the maximal apparent thickness of the sample.Based on these values, select a focus interval and determine the number of focal steps needed to image the entire sample at the maximal apparent thickness. Then, at low magnification, locate the region of interest within the sample. Adjust the eucentric height so that the ROI does not appear to move when the sample is rotated.Verify that the ROI remains visible throughout the tilt range. Fill in the through focal tilt series parameters for automated image collection and acquire the images. Import the image series into an image processing program, and align the images collected at the same tilt angle in a pyramidal hierarchy.Find the alignment at each tilt angle by stacking the aligned images. Browse through the stack to ensure that only the focused region moves from image to image and realign the images as needed. Once alignment is verified throughout the sample, merge the in-focus information at the high settings to yield the series with a single image per tilt angle.Perform find alignment of the tilt series based on local minima or fiducial markers. Perform a 3-D reconstruction and inspect the resulting image. If the reconstruction appears blurred or deformed, realign the original image series.A sample of t. brucei was imaged and analyzed using this method. At a zero degree tilt angle, several details of the flagellar pocket are visible.At the higher tilt angle of 70 degrees, only a portion of each image collected in the through focal series is in focus. This position shifts throughout the series. By merging the in-focus sections, a single image with a larger in-focus zone is produced.This effect is even more pronounced when the high frequency areas of the through focal tilt series are highlighted. The high frequency information spans most of the combined image, indicating that the through focal tilt series has been processed well. Once mastered, the image processing part can be done in around three to four hours, depending on the chosen alignment and reconstruction parameters.While attempting this procedure, it's important to remember that the quality of the final reconstruction greatly depends on the quality of the previous alignments tests. After watching this video, you should have a good understanding of how to perform the collection and the image processing of a through focal tilt series.

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Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

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

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

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...