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 cells47, and synapse morphology in hippocampal neurons48. To better understand liver biology, this approach can be used to investigate hepatocyte morphology in 2D cell culture, 3D hepatic organoid model systems49, and liver tissue. This robust and flexible approach can be used by many laboratories with access to a conventional 120 kV transmission electron microscope and ultramicrotome and does not require expensive sample preparation techniques.
While this is an accessible protocol, it is important to note that there are alternative vEM techniques that can provide certain advantages to the researcher, depending on the research question; see Table 1 for an overview6,50,51. Often the resolution and volume of the data needed directly influence the researchers&#39; choice of the vEM technique and the ease and speed at which the project can progress. FIB-SEM, for example, can remove very thin layers from the sample prior to imaging, resulting in excellent z-resolution (z thickness as low as 4 nm) and potentially providing volumetric 3D data with isotropic voxels. This allows visualization of the data with equal resolution from every angle, which can sometimes provide distinct advantages when contact points are difficult to discern52,53. FIB-SEM can generate large volumes but is often costly and time-consuming, while SBF-SEM is an excellent technique to use when larger volumes are required, e.g., 100s to 1000s of sections54. SBF-SEM and array tomography provide similar xy resolution as FIB-SEM but with reduced z-resolution. As array tomography involves collecting physical sections, the z-resolution is perhaps the poorest of its competitors (z thickness 50+ nm) but has several distinct advantages. Foremost, because it is a nondestructive technique, the sample can be revisited at different xy resolutions, with easy montaging, at various times, and across numerous regions, allowing full context and detail to be appreciated in a single sample. Serial section TEM also shares this benefit; however, as sections need to be collected onto TEM grids, it is better suited to smaller volumes that require fewer sections to be collected prior to imaging. However, it is compatible with TEM tilt tomography, a technique that provides very high z-resolution (z thickness as low as 2 nm) of very small volumes, enabling further interrogation of specific contact sites of interest if required.
Regardless of the vEM approach taken, sample preservation is critical to the success of the project. The fixation medium should be an isotonic match to the tissues, and the transfer to the fixation medium should be done rapidly to avoid drying of the tissues55. Liver tissue can be challenging to preserve, and while it offers excellent preservation, allowing retention of liver tissue for additional experiments, whole liver perfusion for electron microscopy is often not possible. Liver-wedge needle perfusion is an excellent alternative56. Cryoimmobilization is also an option but requires immediate high-pressure freezing to achieve homogeneous vitrification of fresh vibratome sections; thus, this approach is quite challenging. After initial fixation, there are a variety of EM specimen preparation protocols that yield satisfactory results. A routine TEM protocol is presented here57, but &#34;enbloc megametal&#34; protocols have also been successfully employed with other biological samples45,58. While these &#34;enbloc megametal&#34; protocols provide more contrast in the sample, giving some structures a more heavily stained appearance, they also have the advantage of removing the requirement to stain sections, thus reducing the risk of adding unwanted stain precipitate on samples and damaging grids in the process.
Another critical step in this protocol is ultrathin sectioning and ribbon collection. Collecting serial sections from the water surface of the cutting boat is a manual process that could lead to damaged and missing sections. For example, should sections fail to adhere to each other and form stable ribbons, contact cement can be applied to the leading edge of the blockface to stabilize the ribbons13. The duration and routine success of this step are skill-dependent59. Although a plethora of model-specific section-picking methods60,61 may confuse beginners, some simple, practical guides55,62 for routine sectioning, together with practice, can significantly improve sectioning skills. This protocol requires minimal specialist equipment, and with the aid of video footage showing the practical aspects of manual manipulation techniques during sectioning, ribbon detachment, and ribbon handling and pickup, provides an excellent beginner&#39;s guide to serial sectioning.
Volume EM image analysis continues to be an area of development with a variety of open-source and commercial software options currently available, and the list is ever-growing. A selection of software for alignment (TrakEM241), 3D segmentation, and reconstruction (Amira42) are presented here; however, many alternatives are available for alignment (e.g., Fiji, Register Virtual Slices63, Atlas5, and 3D reconstruction (e.g., MIB64; Reconstruct65; IMOD66). For relatively small analysis projects, regardless of whether the data are generated locally or sourced from openly shared datastores (EMPIAR67, Open Organelle68), manual segmentation described here is an accessible and feasible option. However, for larger datasets, many users are turning to machine learning and artificial intelligence for automation of these tasks69,70, as well as crowd-sourcing volunteers71 or combining both72.
Ultimately, after alignment and 3D segmentation, 3D reconstruction reveals not only the morphology of the structures in question but, importantly, also their relationship with other organelles, including the unique characteristics of any contact sites present. These organelle contacts can be analyzed further, and features such as the number of contacts, contact area, volumes of organelles, and morphometric parameters of the organelles can be extracted and compared between experimental models70. Quantitation options are numerous, and their relevance varies hugely depending upon the exact research question. Hence, they have not been covered in depth as part of this protocol.
Pathological processes affecting organelle contacts have been implicated in both the rare (e.g., Wilson&#39;s disease) and common (viral hepatitis and non alcoholic steatohepatitis) disorders73,74,75. For example, the ER-mitochondria and mitochondria-lipid droplet contact sites appear to regulate lipid metabolism in hepatocytes; therefore, the study of these contacts is of great interest. In conclusion, serial section TEM and subsequent 3D reconstruction and analysis is a powerful technique to interrogate interorganelle contacts and probe their relative importance in liver functions and diseases.

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><tbody><tr><td>0.22 &amp;micro;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&amp;deg;</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 &amp;micro;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 approximately 62 &#181;m3 (316 &#181;m x 150 &#181;m x 1.3 &#181;m). This volume was sufficient to allow the complete 3D reconstruction of individual mitochondria in liver tissue.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63496/63496fig04.jpg" /> 
Figure 4: Segmentation and 3D reconstruction reviewing the morphology of ER and associated ER-mitochondria contacts. (A) Overview of ribbon of serial sections in the TEM. (B) Low-magnification view of region of interest and contextual landmarks for relocation. Scale bars = 40 &#181;m (i), 20 &#181;m (ii), 5 &#181;m (iii), 1 &#181;m (iv). (C) Left: EM tomogram of two apposed hepatocytes. Right: segmented version of the same tomogram. Trace of the ER (yellow), mitochondria (cyan), and intermembrane contacts between the ER and mitochondria of different space: 0-20 nm (magenta); 21-100 nm (blue). (D) An orthoslice isolated from the reconstructed model showing only the ER and the different membrane contacts, corresponding to the arrow annotated region in the inset. (E) 3D reconstruction of the segmented organelles and ER-mitochondria contacts at different angles. Abbreviations: ER = endoplasmic reticulum; TEM = transmission electron microscopy; mt = mitochondria. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63496/63496fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Visualization of the serial sections by TEM at low magnification helps to identify the designated area of interest and its context within the rest of the tissue (Figure 4B). These images can be used to find the same region of interest in subsequent sections. A region of interest showing good preservation at a boundary between two apposed hepatocytes was selected and presented here. Higher magnification imaging allows the observation of the morphological details of the different organelles (Figure 4C) with nm resolution. To illustrate two types of ER-organelle associations, selected mitochondria and the ER were segmented, manually tracing the edge of the membranes presenting with enhanced electron density. Afterward, the brush tool was set to a fixed nm/px thickness (Figure 3B) and used to follow the boundary of the organelle of interest to highlight the regions between the two targeted organelles that were within a specified contact distance to one another and could be designated as a particular class of organelle contacts. Figure 4D shows a tilted orthoslice overlaid with segmentation traces and its relative position (arrowhead in the inlet 3D model) within the whole mitochondrion.
The traces were assigned different colors, reconstructed into a 3D model after segmentation, and displayed at different orientations (Figure 4E). The ER (yellow) structure was shown to be partially transparent in the middle panels to visualize the ER-mitochondria contacts and mitochondria (cyan) underneath. The front and rear views of the model reveal an asymmetrical distribution of interorganelle contacts of different intermembrane spacings. Intermembrane space smaller than 21 nm was assigned as ER-mitochondria contact (pink) because functions such as lipid transfer have been reported to be feasible at this distance43. The intermembrane space (blue) between 21 and 100 nm was also annotated because this region may represent the recently reported mitochondria-rough-ER associations44. Wrapping ER structures of similar curvature were observed in the 3D model (Figure 4E). Figure 4D shows an example of changing intermembrane distance (~40 nm &#8594; ~20 nm &#8594; ~40 nm) between the wrapping ER cisternae and the mitochondria. The method allows follow-up patch analysis of these two distinct intermembrane spaces, such that quantitative analysis of their abundance, distribution, and topology is possible.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Concerns</td>
			<td>Serial Tomography</td>
			<td>Serial Section TEM</td>
			<td>FIB-SEM</td>
			<td>SBF-SEM</td>
			<td>Array Tomography</td>
		</tr>
		<tr>
			<td colspan="3">Lateral (x,y) resolution</td>
			<td rowspan="2">0.5 nm</td>
			<td rowspan="2">0.5 nm</td>
			<td rowspan="2">2 nm</td>
			<td rowspan="2">2 nm</td>
			<td rowspan="2">2 nm</td>
		</tr>
		<tr>
			<td colspan="3">(Minimum px size)</td>
		</tr>
		<tr>
			<td colspan="3">Axial (z) resolution</td>
			<td rowspan="2">1 nm</td>
			<td>50 nm</td>
			<td rowspan="2">4 nm</td>
			<td rowspan="2">20 nm</td>
			<td>50 nm</td>
		</tr>
		<tr>
			<td colspan="3">(Minimum px depth)</td>
			<td>Limited to section thickness</td>
			<td>Limited to section thickness</td>
		</tr>
		<tr>
			<td colspan="3">Volume ranges of data in typical applications</td>
			<td>0.1 - 50 &#181;m3</td>
			<td>10 - 250 &#181;m3</td>
			<td>10 - 1 x 104 &#181;m3</td>
			<td>10 &#8211; 1 x 1012 &#181;m3</td>
			<td>10 - &#8734;&#160; &#181;m3</td>
		</tr>
		<tr>
			<td colspan="3">Revisit or reimage of the samples</td>
			<td>Possible</td>
			<td>Possible</td>
			<td>Not possible</td>
			<td>Not possible</td>
			<td>Possible</td>
		</tr>
		<tr>
			<td colspan="3">Cost of the equipment</td>
			<td>&#163;&#163;</td>
			<td>&#163;</td>
			<td>&#163;&#163;</td>
			<td>&#163;&#163;</td>
			<td>&#163;&#163;</td>
		</tr>
		<tr>
			<td colspan="3">(instrument examples)</td>
			<td>(200 kV TEM)</td>
			<td>(120 kV TEM)</td>
			<td>(FIB-SEM)</td>
			<td>(SBF-SEM)</td>
			<td>(SEM+AT)</td>
		</tr>
		<tr>
			<td colspan="3">Cost of equipment maintenance</td>
			<td>&#163;&#163;</td>
			<td>&#163;</td>
			<td>&#163;&#163;</td>
			<td>&#163;&#163;</td>
			<td>&#163;</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of volume electron microscopy techniques. Abbreviations: EM = electron microscopy; TEM = transmission EM; FIB-SEM = focused ion beam SEM; SBF-SEM = serial block face SEM; SEM + AT = SEM with Array Tomography.

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

Nanyang Technological University

Research

JoVE Journal

Biology

5.4K 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

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Biochemistry

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Developmental Biology

3D Technique to Visualize Mitochondrial Cristae in Pancreatic Cancer Cells

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Author Spotlight: A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells  

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

Cancer Research

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Medicine

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 ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

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

Summary

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Chapters in this video

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

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

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics