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 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 steps.
1. Preparation of Buffers and Mixtures
<ol>
	<li>Prepare phosphate-buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4).</li>
	<li>Prepare solutions containing 30%, 50%, and 70% ethanol in PBS for sample dehydration. Perform sample dehydration by gradually incubating the sample in increasing ethanol concentrations.</li>
	<li>Prepare epoxy resin by mixing bisphenol-A-(epichlorohydrin) (20 mL), dodecenyl succinic anhydride (9 mL), methyl-5-norbornene-2,3-dicarboxylic (11 mL), and 2,4,6-tris(dimethylaminomethyl)phenol (0.7 mL); other mixing ratios can be used depending on the desired hardness of the resin.</li>
	<li>Prepare solutions containing 30%, 50%, and 70% epoxy resin in ethanol for sample embedding. Perform resin embedding by gradually incubating the sample with increasing resin concentrations. 
	NOTE: The volumes of the prepared solutions depend on the type of sample that is studied. More solutions are needed for tissues than for cell cultures.</li>
</ol>
2. Fixation and Coloration of the Sample
<ol>
	<li>Centrifuge the cell culture for 5 min at 5,000 x g. Perform all of the following centrifugations using the same conditions.</li>
	<li>Discard the supernatant and fix the cell pellet by resuspending it in PBS (1 mL) with 4% paraformaldehyde and 2% glutaraldehyde. Incubate it at room temperature for 30 min (Figure 1A). Alternatively, fix the cells at 4 &#176;C overnight or over the weekend.</li>
	<li>After centrifugation, remove the supernatant and wash the pellet 3 times with PBS (1 mL) to completely remove the fixative agents.</li>
	<li>Post-fix the sample with 1% OsO4 in PBS (1 mL) for 30 min at room temperature.</li>
	<li>After centrifugation, remove the supernatant and wash the pellet 3 times with PBS (1 mL) to completely remove the OsO4. Deactivate the OsO4 in oil and discard.</li>
	<li>Add 2% uranyl acetate in PBS (1 mL) as a contrast agent and incubate the sample for 30 min at room temperature (Figure 1B).</li>
	<li>After centrifugation, remove the supernatant and wash the pellet 3 times with PBS (1 mL) to remove the unbound uranyl acetate; uranyl acetate contains radioactive elements and should be disposed of in a bin dedicated to radioactive waste. 
	NOTE: The incubation times can be modified according to the size of the sample. For example, fixation and staining of thicker samples, such as tissues samples, require longer incubation times. Longer incubation times may also be better for the dehydration and the embedding steps. Uranyl acetate can be replaced by a &#34;uranyl-less&#34; solution that contains Gadolinium salts.</li>
</ol>
3. Sample Dehydration
<ol>
	<li>After centrifugation, discard the supernatant and incubate the sample for 30 min at 4 &#176;C in PBS (1 mL) with 30% ethanol (Figure 1C).</li>
	<li>Repeat step 3.1 using solutions with gradually increasing ethanol concentrations (50%, 70%, and up to 100%). During these dehydration steps, maintain the sample at 4 &#176;C for sample preservation purposes.</li>
	<li>After centrifugation, discard the supernatant and resuspend the pellet in pure ethanol (1 mL); incubate overnight at 4 &#176;C.</li>
</ol>
4. Resin Embedding
<ol>
	<li>After centrifugation, discard the supernatant and resuspend the pellet in ethanol with 30% epoxy resin (1 mL) for 30 min at RT (Figure 1D).</li>
	<li>Repeat step 4.1 using solutions with gradually increasing epoxy resin concentrations (50%, 70%, and up to 100%).</li>
	<li>After centrifugation, discard the supernatant and resuspend the pellet in pure epoxy resin (1 mL); incubate overnight at RT. The presence of hardening DMP-30 might trigger the polymerization of the resin; if this happens, prepare two batches of epoxy resin, one with and one without DMP-30.</li>
	<li>After centrifugation, resuspend the pellet in 200 &#181;L of pure epoxy resin (containing the hardener) in a plastic capsule; the sample should be at the bottom of the plastic capsule.</li>
</ol>
5. Resin Polymerization
<ol>
	<li>Incubate the plastic capsule containing the sample in an incubator set at 60 &#176;C for 48 h; other kinds of resin might polymerize with different settings.</li>
	<li>Remove the capsule from the incubator and verify that the resin has polymerized; the resin should be hard if pressed against a hard surface.</li>
	<li>Add pure epoxy resin (containing the hardener) on top of the polymerized resin containing the sample; this extra layer of resin will make it easier to cut the sample. Incubate the capsule at 60 &#176;C for 48 h.</li>
</ol>
6. Sample Sectioning
<ol>
	<li>Mount the sample upside-down in a specimen holder so that the sample is towards the top and fix it on the trimming block of the ultramicrotome. Tightly lock the lever to secure the trimming block to the ultramicrotome.</li>
	<li>Using a razor blade or a similar sharp cutting instrument, cut out the plastic capsule to separate it from the polymerized resin. Cut the resin so that the sample is contained in resin in roughly the shape of a pyramid. There is no need to remove the entire plastic capsule; only remove the part covering the polymerized sample.</li>
	<li>Take the capsule and the specimen holder off of the trimming block and install them on the moving arm of the ultramicrotome.</li>
	<li>Install a trimming knife on the knife block and accurately trim the faces of the pyramid. The use of binoculars is advised to give a perfect shape to the pyramid; the better the pyramid, the better the sections will be.</li>
	<li>Take off the trimming knife and replace it with a histology knife that can be used to cut sections that are thicker than 0.5 &#181;m.</li>
	<li>After filling the cuvette of the histology knife with the correct amount of water, bring the cutting edge of the knife to the surface of the sample and use the binoculars to adjust the capsule pitch angle so that the knife will slice perpendicularly to the pyramid.</li>
	<li>Adjust the cutting speed according to the desired section thickness to recover homogeneous sections.</li>
	<li>Let the section float over the water and fish it with a loop.</li>
	<li>Move the loop towards an electron microscopy copper grid that has been placed on top of filter paper. 
	NOTE: The grid should have been treated previously to increase its hydrophilicity. This can be performed with a grid-coating pen.</li>
	<li>Due to water absorption by the filter paper, let the section stick to the grid. Let it dry for few minutes before inserting it into the electron microscope.</li>
</ol>
7. Design of the Through-focal Tilt-series
<ol>
	<li>Collect through-focal images at constant focus intervals. 
	NOTE: The through-focal tilt-series consist of a set of through-focal images that are collected at each tilt angle of the tilt-series. The whole focus amplitude should be sufficient to cover the whole sample thickness.</li>
	<li>Determine the value of the focus interval. If the interval is equal to the depth-of-field, collect the entire sample in focus; this setting represents the highest sampling possible. If the interval is greater than the depth-of-field, some parts of the sample will not be acquired in focus, meaning that some parts of the sample will not be collected in focus.</li>
	<li>Calculate the depth-of-field of an electron beam from the electron&#39;s wavelength and the convergence semi-angle of the electron beam. Whereas the wavelength of electrons is directly linked to the acceleration voltage, use the convergence semi-angle provided by the electron microscope manufacturer or determine it experimentally from the diffraction pattern of a known specimen16. Because electrons accelerated at a wavelength &#955; hit the sample with a convergence semi-angle of &#945; in an electron microscope, use the following equation to determine the depth-of-field (DOF): 
	 
	<img alt="Equation" src="/files/ftp_upload/55215/55215eq1.jpg" /></li>
	<li>Design the focal series so that the whole focus amplitude will cover the sample at its maximal apparent thickness (i.e., at its highest tilt). 
	NOTE: A 0.75 &#181;m-thick sample will have an apparent thickness of 2.19 &#181;m at a 70&#176; tilt, so the focus amplitude should be greater than 2.19 &#181;m. For this sample, if the focus interval determined above is equal to 50 nm, it will require 44 images (2.19 &#181;m/50 nm) to cover the sample at its maximum apparent thickness. The whole focus amplitude is equal to the value of the focus interval multiplied by the number of focal steps. At the highest tilt (&#952;), the sample apparent thickness is defined as the following: 
	 
	<img alt="Equation" src="/files/ftp_upload/55215/55215eq2.jpg" /></li>
</ol>
8. Sample Imaging
NOTE: It is beyond the scope of this protocol to describe how to set up an electron microscope in STEM mode; most of this information can be found in the reference manual provided by the electron microscope manufacturer. However, note the following: i) the spot size should be chosen according to the desired magnification; ii) the Ronchigram must be well-aligned; and iii) in BF mode, the camera length must be adjusted to select unscattered electrons while maintaining good illumination of the detector. Resin sections deposited on electron microscopy copper grids should be illuminated before image acquisition to prevent shrinkage. Resin shrinkage and charging effects might be confounded during image acquisition. The use of an objective lens aperture might improve sample stability when charging effects occur. However, in the specific case of STEM setups, the objective aperture is not compatible with HAADF imaging mode, and very small objective apertures are not compatible with BF imaging mode.
<ol>
	<li>Using low magnification (around 20,000X in STEM), browse through the sample and locate the region of interest.</li>
	<li>Adjust the eucentric height (Z-axis) so that the region of interest does not drift away while rotating the sample.</li>
	<li>Verify that the object of interest will remain visible throughout the entire tilt range.</li>
	<li>Because of the substantial number of images acquired during a through-focal tilt-series, use an automatic collection software. Either use a commercial solution or design a custom data collection software if desired. Ensure that the software program is able to perform the three following steps:
	<ol>
		<li>Track and focus as in a standard tilt-series collection scheme.</li>
		<li>Ensure that the through-focal images overlap with the Z-position of the sample.</li>
		<li>Automatically acquire a series of through-focal images at each tilt angle.</li>
	</ol>
	</li>
	<li>Give the software program the main parameters of the through-focal tilt-series (i.e., focal intervals, focal step, minimum tilt angle, maximum tilt angle, tilt step, and scanning speed).</li>
	<li>Start the image collection process and wait for the software program to finish.</li>
</ol>
9. Image Processing
NOTE: In this protocol, the through-focal tilt-series processing is performed using ImageJ plugins17. Supplementary Data 1 shows an ImageJ macro for the alignment step (Turboreg) and for merging in-focus information (Extended Depth of Field) for a through-focal tilt-series designed with three focus values.
<ol>
	<li>Align the images collected at the same tilt angle (i.e., the through-focal images). Since images do not have the same in-focus information, they do not harbor the same features for alignment; perform alignment by aligning neighboring images two by two (i.e., following a pyramidal hierarchy, with the closest focus values first) using Turboreg18, an ImageJ plugin.</li>
	<li>Consistently verify the alignment of the through-focal images at each tilt angle. Stack the aligned through-focal images to improve visual inspection. 
	NOTE: Only the zone that is in focus should move while browsing through the stack. Accurate alignment is the most important thing, since the following step consists of the recovery of the information that is in focus from different images.</li>
	<li>Merge the information that is in focus using Extended Depth of Field19, an ImageJ plugin; this will eventually generate a single image from the stack of images. Several settings can be used during the depth-of-field recovery, but use the highest settings to preserve the image information. 
	NOTE: The tilt-series is now composed of a single image per tilt angle, each image containing the in-focus information recovered from the through-focal images.</li>
	<li>Process the data. 
	NOTE: Standard tools for alignment and reconstruction can be used since the data has now been reduced to a standard tilt-series. In this protocol, TomoJ is used for data alignment and data reconstruction20,21. More details about how to use TomoJ can be found in the original publication22.</li>
	<li>Perform fine alignment of the tilt-series. 
	NOTE: A good strategy is to use gold beads as fiducial markers to accurately track shifts between images. Local minima can also be used if gold beads cannot be added to the sample.</li>
	<li>Perform a 3D reconstruction of the aligned data; several algorithms are available for performing the reconstructions in real space or Fourier space, each with advantages and limitations.</li>
	<li>If the final alignment of the tilt-series is poor, make some improvements by optimizing the initial steps. Reiterate the alignment steps if the final 3D reconstruction appears blurred or deformed; in practice, the greater the number of collected focal planes, the better the contrast and details of the 3D reconstruction. 
	NOTE: Several software programs can be used to process the through-focal tilt-series. However, the tools used in this protocol were chosen because, in our experience, they performed the best. Turboreg18 performed better in terms of aligning the images when a gradient of focus was present. More information can be found on the dedicated website23. The Extended Depth of Field plugin19 performed very well in terms of recovering the in-focus information from different images, whereas other tools resulted in visible pixels modifications. More information, especially regarding script writing, can be found on this dedicated website24.</li>
</ol>

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We have no conflict of interest to disclose.

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><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 &amp;deg;C.&lt;br/&gt; 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&amp;nbsp;</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...

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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 ...

preparation-observation-thick-biological-samples-scanning

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.2K Views.  Campus Universitaire d'Orsay. This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

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

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie &#38; Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study&#39;s transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

Environment

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Engineering

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Bioengineering

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 complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

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

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 Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

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

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.

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

Summary

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Chapters in this video

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Miniaturized Sample Preparation for Transmission Electron Microscopy

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

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Visualization of Organelles In Situ by Cryo-STEM Tomography