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.

All animals were housed in accordance with the UK Home Office guidelines, and the tissue harvesting was carried out in accordance with the UK Animal (Scientific Procedures) Act 1986.
1. Specimen fixation and preparation
<ol>
	<li>Dissect the liver tissue into appropriate size pieces, approximately 8 mm x 8 mm x 3 mm, and place the pieces in warm phosphate-buffered saline (PBS, 37 &#176;C).</li>
	<li>Inject room temperature (20-25 &#176;C) fixative (1.5% glutaraldehyde in 1% ...

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 ...

This protocol allows 3D reconstruction of organelles in a simple and robust manner, whilst being accessible to laboratories that do not currently have specialist volume electron microscopes. This technique is extremely flexible, providing excellent X/Y resolution, whilst being able to re-image samples if necessary. It's also compatible with tilt tomography when very high Z resolution is required.This technique allows interrogations of organelle morphology in inter-organelle context. Therefore, it can be extended to examine any pathological conditions with organelle defects. This approach can provide insight into organelle interactions and cellular trafficking events.This method can also be applied to other tissues, organoid models, and cell culture systems, in addition to hepatocytes. First-time users may find serial sectioning and pick up challenging without loss. It's recommended to be proficient at regular ultra-thin sectioning before applying this protocol to a precious sample.Begin by carefully trimming the resin-embedded tissue using a razor blade with the sample locked in the trimming adapter. Transfer the specimen ark together with the chuck and the sample to the specimen arm of the microtome and position the specimen ark so that the ark's travel range runs from top to bottom. Place and lock the diamond knife in the knife holder, ensuring that the knife's cutting angle is set appropriately.Lock the knife holder into the stage securely. Turn off the stage downlighting, turn on the stage uplighting, cautiously move the knife towards the specimen, continually adjusting the knife's lateral angle, specimen tilt, and specimen rotation by adjusting the relevant knobs until the block face is aligned to the knife's edge. Set the top and bottom of the cutting window of the specimen arm and leave the specimen just below the knife edge.Fill the knife boat with clean, double-distilled water and ensure the water surface is level with the knife's edge and slightly concave. Then, start cutting sections. Stop the cutting just after the sample has passed the knife's edge.Use an empty slot grid to estimate the number of sections to collect on each grid. Using an eyelash in each hand, gently break the ribbon into smaller ribbons that can fit in the length of the slot grid. Make a mental note of their relative positions within the sample.Using a glass applicator rod, hover a drop of chloroform over the sections to flatten them out. Pick up the first empty slot grid with the first numbered forceps and gently dip it in the Triton X-100 and then two times in the distilled water. Use a piece of filter paper to remove the excess water from the forceps'edge.Using forceps, gently lower approximately 2/3 of the formvar-coated slot grid into the water of the knife boat. Ensure that the formvar side is facing down and the long right hand edge of the slot is at the surface of the water and parallel to the water's edge. Gently waft the grid in the water towards the ribbons so that upon the return stroke, the sections drift towards the grid.Continue to do this in smaller and smaller wafts, until the right hand edge of the ribbon lines up with the right hand edge of the slot. Then, with the last waft, gently bring the grid up to pick the sections up into the slot grid. Place several pellets of sodium hydroxide under a Petri dish lid to provide a carbon dioxide-free environment.Then, carefully pipette 40-microliter drops of Reynold's lead citrate on the parafilm, one drop for each grid. Invert each grid onto the lead citrate drop and leave protected by the Petri dish lid for 7 to 10 minutes. While the grids are staining, place five approximately-300-microliter drops of distilled water for each grid on the parafilm.At the end of the lead citrate incubation, transfer each grid to a droplet of distilled water to wash for one minute without breathing directly on the grids. Repeat this four more times. Use numbered crossover forceps to pick up the first grid.Touch the edge of the grid to filter paper to wick away most of the water and allow to dry in the forceps for at least 20 minutes. Repeat this for each grid. At low magnification, observe the order, location, and position of the serial sections.Navigate to the middle section of the series on the grid. Browse the sample and identify a region-of-interest. Then, observe the sample at the desired magnification.Consider collecting the series at a slightly lower magnification, as sections are often not perfectly aligned, and images may need to be cropped later. Next, take reference images at lower magnifications. This will help to appreciate the context of the region-of-interest, it's rough location at different magnifications in relation to section boundaries, and landmark features within the sample.Use these to signpost the region-of-interest in other sections. For screen referencing, use reusable adhesive putty, stickers, or a piece of overhead projector paper taped to the screen to place temporary markers on the screen. This will allow routine re-imagining of the same features of the region-of-interest in the center of the image throughout the dataset.Open the image stack, specify the voxel measurements, and select the Segmentation tab to start segmentation. Click New in the segmentation editor panel to define new objects in the material list. Right click to change the color of the object and double click to rename the object.For manual segmentation, choose the segmentation tool below the material list and select the default brush tool to highlight the pixels. The electron microscopy image of two opposed hepatocytes is shown here. Higher magnification imaging allows the observation of the morphological details of the different organelles with nanometer resolution.The representative image shows the segmented version of the same tomogram, trace of the endoplasmic reticulum, mitochondria, and the intermembrane contacts between the ER and the mitochondria of different spaces are shown here. The representative image shows a tilted ortho-slice overlaid with segmentation traces and its relative position within the whole mitochondrion. 3D reconstruction of the segmented organelles and the endoplasmic reticulum-mitochondria contacts at different angles are shown here.As this protocol is compatible with tilt tomography when very high Z resolution is needed, it helps to resolve small or convoluted structures. This technique is perfect for the initial assessment of the spatial relationship between different organelles, and, therefore, particularly useful in the progressing field of membrane contacts at membrane homeostasis.

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Research

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Biology

5.3K vistas.  University College London. Este protocolo permite la reconstrucción 3D de orgánulos de forma sencilla y robusta, a la vez que es accesible a laboratorios que actualmente no cuentan con microscopios electrónicos de volumen especializados. Esta técnica es extremadamente flexible, proporcionando una excelente resolución X / Y, al tiempo que puede volver a obtener muestras si es necesario. También es compatible con la tomografía basculante cuando se requiere una resolución Z muy alta.Esta técnica permite in...