We thank Joanna Hanley, Rebecca Fiadeiro, and Ania Straatman-Iwanowska for expert technical assistance. We also thank Stefan lab members and Ian J. White for helpful discussions. J.J.B. is supported by MRC funding to the MRC Laboratory of Molecular Cell Biology at UCL, award code MC_U12266B. C.J.S. is supported by MRC funding to the MRC Laboratory of Molecular Cell Biology University Unit at UCL, award code MC_UU_00012/6. P.G. is funded by the European Research Council, grant code ERC-2013-StG-337057.

The authors have no conflicts of interest to disclose.

An accessible vEM technique for visualizing organelle structure and interactions in 3D is described in this protocol. The morphology of interorganelle contacts in hepatocytes is presented as a case study here. However, this approach has also been applied to investigate a variety of other samples and research areas, including Schwann cell-endothelial interactions in peripheral nerves45, Weibel Palade Body biogenesis in endothelial cells46, cargo secretion in kidney cells<sup...

Since their invention in the 1930s, electron microscopes have allowed researchers to visualize the structural components of cells and tissues1,2. Most investigations have provided 2D information, as building 3D models requires painstaking serial section collection, manual photography, negative processing, manual tracing, and the creation and assembly of 3D models from sheets of glass, plastic, or Styrofoam3,4. Almost 70 years later, there have been considerable advances in numerous aspects of the process, from microscope performance, serial section collection, automated digital imaging, sophisticated software and hardware for 3D reconstruction, visualization, and analysis to alternative approaches for what is now collectively termed volume EM (vEM). These vEM techniques are generally considered to provide 3D ultrastructural information at nanometer resolutions across micron scales and encompass transmission electron microscopy (TEM) and newer scanning electron microscopy (SEM) techniques; see reviews5,6,7,8.
For example, focused ion beam SEM (FIB-SEM) uses a focused ion beam inside an SEM to mill away the surface of the block between sequential SEM imaging scans of the block&#39;s surface, allowing the repeated automated milling/imaging of a sample and building up a 3D dataset for reconstruction9,10. In contrast, serial block face SEM (SBF-SEM) uses an ultramicrotome inside the SEM to remove material from the block face prior to imaging11,12, while array tomography is a nondestructive process that requires the collection of serial sections, onto coverslips, wafers, or tape, prior to setting up an automated workflow of imaging the region of interest in sequential sections in the SEM to generate the 3D dataset13. Similar to array tomography, serial section TEM (ssTEM) requires physical sections to be collected ahead of imaging; however, these sections are collected on TEM grids and imaged in a TEM14,15,16. ssTEM can be extended by performing tilt tomography17,18,19. Serial tilt tomography provides the best resolution in x, y, and z, and while it has been used to reconstruct whole cells20, it is reasonably challenging. This protocol focuses on the practical aspects of ssTEM as the most accessible vEM technique available to many EM labs who may not currently have access to specialized sectioning or vEM instruments but would benefit from generating 3D vEM data.
Serial ultramicrotomy for 3D reconstruction has previously been considered challenging. It was difficult to cut straight ribbons of even section thickness, be able to arrange and pick up ribbons of the correct size, in the correct order, onto grids with sufficient support, but without grid bars obscuring regions of interest, and most importantly, without losing sections, as an incomplete series may prevent full 3D reconstruction21. However, improvements to commercial ultramicrotomes, diamond cutting and trimming knives22,23, electron lucent support films on grids21,24, and adhesives for aiding section adhesion and ribbon preservation13,21 are just some of the incremental advances over the years that have made the technique more routine in many labs. Once serial sections have been collected, serial imaging in TEM is straightforward and can provide EM images with subnanometer px sizes in x and y, allowing high-resolution interrogation of the subcellular structures-a potential requirement for many research questions. The case study presented here demonstrates the use of ssTEM and 3D reconstruction in the study of endoplasmic reticulum (ER)-organelle contacts in liver hepatocytes, where&#160;ER-organelle contacts were first observed25,26.
While being contiguous with the nuclear envelope, the ER also makes close contacts with numerous other cell organelles, including lysosomes, mitochondria, lipid droplets, and the plasma membrane27. ER-organelle contacts have been implicated in lipid metabolism28, phosphoinositide and calcium signaling29, autophagy regulation, and stress response30,31. The ER-organelle contacts and other interorganelle contacts are highly dynamic structures that respond to cellular metabolic needs and extracellular cues. They have been shown to vary morphologically in their size and shape and the distances between organelle membranes32,33. It is thought that these ultrastructural differences are likely to reflect their different protein/lipid compositions and function34,35. However, it is still a challenging task to define interorganelle contacts and analyze them36. Hence, a reliable yet simple protocol to examine and characterize interorganelle contacts is required for further investigations.
As ER-organelle contacts can range from 10 to 30 nm in membrane-to-membrane separation, the gold standard for identification has historically been TEM. Thin-section TEM has revealed specific subdomain localization for resident ER proteins at distinct membrane contacts37. Traditionally, this has revealed ER-organelle contacts with nm resolution but often only presented a 2D view of these interactions. However, vEM approaches reveal the ultrastructural presentation and context of these contact sites in 3D, enabling full reconstruction of contacts and more accurate classification of contacts (point vs. tubular vs. cisternal-like) and quantification38,39. In addition to being the first cell type where ER-organelle contacts were observed25,26, hepatocytes have an extensive system of other interorganelle contacts that serve vital roles in their architecture and physiology28,40. However, thorough morphological characterization of ER-organelle and other interorganelle contacts in hepatocytes is still lacking. Accordingly, how interorganelle contacts form and remodel during regeneration and repair is of particular relevance to hepatocyte biology and liver function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m syringe filter</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum trays</td>
			<td>Agar Scientific</td>
			<td>AGG3912</td>
			<td></td>
		</tr>
		<tr>
			<td>Amira v6</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>https://www.thermofisher.com</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>C/4960/PB08</td>
			<td></td>
		</tr>
		<tr>
			<td>DDSA/Dodecenyl Succinic Anhydride</td>
			<td>TAAB</td>
			<td>T027</td>
			<td>Epon ingredient</td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>DiaTOME</td>
			<td>ultra 45&#176;</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30/2,4,6-tri (Dimethylaminomethyl) phenol</td>
			<td>TAAB</td>
			<td>D032</td>
			<td>Epon ingredient</td>
		</tr>
		<tr>
			<td>Dumont Tweezers N5</td>
			<td>Agar Scientific</td>
			<td>AGT5293</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>https://imagej.net/</td>
		</tr>
		<tr>
			<td>Fiji TrakEM2 plugin</td>
			<td></td>
			<td></td>
			<td>https://imagej.net/</td>
		</tr>
		<tr>
			<td>Formaldehyde 36% solution</td>
			<td>TAAB</td>
			<td>F003</td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar coated slot grid</td>
			<td>Homemade</td>
			<td></td>
			<td>Alternative: EMS diasum (FF2010-Cu)</td>
		</tr>
		<tr>
			<td>Glass bottle with applicator rod</td>
			<td>Medisca</td>
			<td>6258</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Fisher Scientific</td>
			<td>15364769</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde 25% solution</td>
			<td>TAAB</td>
			<td>G011</td>
			<td></td>
		</tr>
		<tr>
			<td>MNA/Methyl Nadic Anhydride</td>
			<td>TAAB</td>
			<td>M011</td>
			<td>Epon ingredient</td>
		</tr>
		<tr>
			<td>Osmium Tetroxide 2% solution</td>
			<td>TAAB</td>
			<td>O005</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P-8131</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene oxide</td>
			<td>Fisher Scientific</td>
			<td>E/0050/PB08</td>
			<td></td>
		</tr>
		<tr>
			<td>Reuseable adhesive</td>
			<td>Blue Tack</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reynolds Lead Citrate</td>
			<td>TAAB</td>
			<td>L037</td>
			<td>Section stain</td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Sigma-Aldrich</td>
			<td>C-0250</td>
			<td>to make 0.1 M Caco buffer</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>RS Components</td>
			<td>918-6872</td>
			<td>Cyanoacrylate glue, Step 1.3</td>
		</tr>
		<tr>
			<td>TAAB 812 Resin</td>
			<td>TAAB</td>
			<td>T023</td>
			<td>Epon ingredient</td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>TAAB</td>
			<td>T046</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Two part Epoxy Resin</td>
			<td>RS Components</td>
			<td>132-605</td>
			<td>Alternative: Step 2.13</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>UC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica</td>
			<td></td>
			<td>100 &#181;m thick slices, 0.16 mm/s cutting at 1 mm amplitude .</td>
		</tr>
		<tr>
			<td>Weldwood Original Contact cement</td>
			<td>DAP</td>
			<td>107</td>
			<td>Contact adhesive: Step 3.1.4</td>
		</tr>
	</tbody>
</table>

three-dimensional characterization of interorganelle contact sites in hepatocytes using serial section electron microscopy

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. 
This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. 
Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

For this technique, regions of interest are selected based on the biological research aim and identified prior to the trimming and sectioning of embedded tissue. Similarly, the size of the block face may be dictated by the research question; in this case, the sample was trimmed to leave a block face of approximately 0.3 mm x 0.15 mm (Figure 4A). This allowed for two grids of 9 serial sections per grid, providing 18 serial sections and incorporating a volume of liver tissue of a volume of app...

Watch this Scientific Journal Video about Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy at JoVE.com

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is ...

three-dimensional-characterization-interorganelle-contact-sites

Research

JoVE Journal

Biology

5.3K Views.  University College London. A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

Video: Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

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 imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

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

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

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, 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.

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

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.

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

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

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.