This work was supported in part by Grants from National Key Research and Development Program of China (2022YFA1105503), State Key Laboratory of Neuroscience (SKLN-202103), Zhejiang Natural Science Foundation of China (Y21H120019).

Animal care and use protocols were approved by the Ethics Committee of Wenzhou Medical University and followed the guidelines established by the Association for Research in Vision and Ophthalmology (ARVO). All mice were maintained in a 12-h light and 12-h dark cycle and supplied with a standard chow diet.
1. Retina dissection and fixation
<ol>
	<li>Anesthetize the mice (C57BL/6J, Male, 8-week-old, 20-25 g) by injecting 2,2,2-tribromoethanol (0.25 mg/g of body weight) intrape...

Visualizing Retinal Synaptic Structures in 3D with Volume Electron Microscopy

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B., Witkovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+at+retinal+ribbon+synapses.">Synaptic transmission at retinal ribbon synapses.</a> Prog Retin Eye Res. 24 (6), 682-720 (2005).</li><li>Thoreson, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+at+rod+and+cone+ribbon+synapses+in+the+retina.">Transmission at rod and cone ribbon synapses in the retina.</a> Pflugers Arch. 473 (9), 1469-1491 (2021).</li><li>Moser, T., Grabner, C. P., Schmitz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+processing+at+ribbon+synapses+in+the+retina+and+the+cochlea.">Sensory processing at ribbon synapses in the retina and the cochlea.</a> Physiol Rev. 100 (1), 103-144 (2020).</li><li>Schmitz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+synaptic+ribbons:+how+they+are+built+and+what+they+do.">The making of synaptic ribbons: how they are built and what they do.</a> Neuroscientist. 15 (6), 611-624 (2009).</li><li>Kantardzhieva, A., Peppi, M., Lane, W. S., Sewell, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+composition+of+immunoprecipitated+synaptic+ribbons.">Protein composition of immunoprecipitated synaptic ribbons.</a> J Prot Res. 11 (2), 1163-1174 (2011).</li><li>Zampighi, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conical+tomography+of+a+ribbon+synapse:+structural+evidence+for+vesicle+fusion.">Conical tomography of a ribbon synapse: structural evidence for vesicle fusion.</a> PLoS One. 6 (3), e16944 (2011).</li><li>Barnes, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+diversity+of+the+rod+spherule:+A+study+of+serially+reconstructed+electron+micrographs.">Morphological diversity of the rod spherule: A study of serially reconstructed electron micrographs.</a> Plos One. 11 (3), e0150024 (2016).</li><li>Xu, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+FIB-SEM+systems+for+large-volume+3D+imaging.">Enhanced FIB-SEM systems for large-volume 3D imaging.</a> Elife. 6, e25916 (2017).</li><li>Crewe, A. V., Lin, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+backscattered+electrons+for+imaging+purposes+in+a+scanning+electron+microscope.">The use of backscattered electrons for imaging purposes in a scanning electron microscope.</a> Ultramicroscopy. 1 (3), 231-238 (1976).</li><li>Richards, R. G., Gwynn, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Backscattered+electron+imaging+of+the+undersurface+of+resin-embedded+cells+by+field-emission+scanning+electron+microscopy.">Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy.</a> J Microsc. 177, 43-52 (1995).</li><li>Wergin, W. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+thin+and+thick+sections+of+biological+tissue+with+the+secondary+electron+detector+in+a+field-emission+scanning+electron+microscope.">Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope.</a> Scanning. 19 (6), 386-395 (1997).</li><li>Willingham, M. C., Rutherford, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+osmium-thiocarbohydrazide-osmium+(OTO)+and+ferrocyanide-reduced+osmium+methods+to+enhance+membrane+contrast+and+preservation+in+cultured+cells.">The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.</a> J Histochem Cytochem. 32 (4), 455-460 (1984).</li><li>Buravkov, S. V., Chernikov, V. P., Buravkova, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+method+of+specimen+preparation+for+scanning+electron+microscopy.">Simple method of specimen preparation for scanning electron microscopy.</a> Bull Exp Biol Med. 151 (3), 378-382 (2011).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+diversity+in+the+retina.">Neuronal diversity in the retina.</a> Curr Opin Neurobiol. 11 (4), 431-436 (2001).</li><li>Svara, F. N., Kornfeld, J., Denk, W., Bollmann, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+EM+reconstruction+of+spinal+cord+reveals+wiring+specificity+in+speed-related+motor+circuits.">Volume EM reconstruction of spinal cord reveals wiring specificity in speed-related motor circuits.</a> Cell Rep. 23 (10), 2942-2954 (2018).</li><li>Kubota, Y., Sohn, J., Kawaguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+volume+electron+microscopy+and+neural+microcircuit+analysis.">Large volume electron microscopy and neural microcircuit analysis.</a> Front Neural Circuits. 12, 98 (2018).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+approach+using+cryofixation+for+high-resolution+3D+analysis+by+FIB-SEM.">An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM.</a> J Struct Biol. 212 (1), 107600 (2020).</li><li>Seligman, A. 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The authors have no disclosures.

We implemented the OTO&#39;s Volume EM sample preparation protocol to analyze the photoreceptors&#39; terminal structure in retinal tissue. The focus was on detailing the entire procedure, starting from the detachment and fixation of the retina to showcasing the results of 3D reconstruction of photoreceptor axon terminals.
The distinctive feature of retinal tissue, unlike brain tissue, lies in its lack of regional differences. Comprising three layers of neuronal cell bodies and two layers of s...

The retina is densely packed with intertwining neuronal axons and dendrites that form synapses between them1. Microscopy is an indispensable tool for studying retinal anatomy as it has fine, intricate, and small structures. Although electron microscopy (EM) provides unparalleled power to investigate the ultrastructure of subcellular organelles and the accurate localization of specific proteins at the nanometer level2, it produces images limited to the two-dimensional (2D) plane, leading to potential loss of key information.
The development of emerging high-resolution volume electron microscopy (Volume EM) techniques supports the provision of more comprehensive and larger-scale three-dimensional (3D) structural information. Some 3D EM methods have been recently reviewed by others3,4,5. 3D EM allows for the reconstruction of neuronal shape and connectivity details, enabling precise quantitative analysis of structures of interest. This demonstrates that the data obtained by volume EM are more systematic, complete, and accurate.
Retinal photoreceptors, constituting the initial neurons in visual signaling6,7, establish synapses with dendrites of second-order bipolar and horizontal cells in the photoreceptor&#39;s terminal to facilitate excitatory signals8,9. These terminals, referred to as cone pedicles and rod spherules, encompass three crucial components: mitochondria, synaptic ribbons, and synaptic vesicles. While previous studies have predominantly concentrated on the general structure of ribbon synapses, there has been a notable absence of investigation into the fine structure of major components, including mitochondria, ribbon, vesicle pools, and their spatial organization in terminals10,11,12,13. A precise and systematic analysis of each component, along with an understanding of their inter-association within photoreceptor terminals, is vital for unraveling spatial organization and comprehensively grasping visual processing functions. In photoreceptors, mitochondria are mainly present in the inner segment, cell body, and terminal. We focused here on the mitochondria in the terminal photoreceptors. Focused ion beam scanning electron microscopy (FIB-SEM), a type of volume EM boasting high resolution (x, y, and z resolution &#60; 5 nm) and a relatively large volume flux4,14, stands as a potent tool for accurately visualizing the 3D structure of photoreceptor terminals.
Both FIB-SEM and Serial Block Face Scanning Electron Microscope (SBF-SEM) are Volume EM based on SEM for obtaining the image of tissues by scanning the surface of the sample. The ultrastructural features of a specimen&#39;s surface are revealed through the contrast created by the intensity of secondary or backscattered electrons (BSE) when the electron beam scans the sample15. Essentially, detecting BSE or secondary electrons from the cross-section surface of a resin-embedded tissue sample in SEM allows for obtaining images of the embedded sample16,17. When BSE or secondary electrons are less generated, information on the sample surface can only be obtained. Achieving consistent contrast and high-quality serial images necessitates sufficient deposition of heavy metals in the sample. Therefore, specific sample preparation protocols are crucial for subsequent segmentation, 3D reconstruction, and analysis when utilizing SEM for serial imaging. The osmium-thiocarbohydrazide-osmium (OTO) method is a typical sample preparation scheme for 3D electron microscopy of biological samples, preserving the structure of lipid-containing membranes and maintaining good contrast18,19.
Here, we developed the OTO method for the preparation of retinal samples for the use of Volume EM. This process particularly focuses on dissecting the retina, determining the optimal fixation time for retinal tissue, and detailing the specific procedures and precautions in 3D sample preparation. Additionally, segmentation and 3D reconstruction of the target structure are integral steps in this extended application. The retina, being a small and challenging structure to obtain materials from, requires swift and precise operations for EM, with fixed times and fresh reagents prepared for immediate use.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,2,2-Tribromoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Electron Microscopy Science</td>
			<td>10000</td>
			<td></td>
		</tr>
		<tr>
			<td>Amira 6.8</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C-2661</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding mold</td>
			<td>Beijing Zhongjingkeyi Technology</td>
			<td>GP10590</td>
			<td></td>
		</tr>
		<tr>
			<td>Epon resin</td>
			<td>Electron Microscopy Science</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>16020</td>
			<td></td>
		</tr>
		<tr>
			<td>Helios NanoLab 600i dual-beam SEM</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>Sigma</td>
			<td>56-84-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>Sigma</td>
			<td>10099-74-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4.12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>A component of phosphate buffer</td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>71507</td>
			<td>A component of phosphate buffer</td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>TED PELLA</td>
			<td>4008-160501</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide</td>
			<td>Sigma</td>
			<td>14459-95-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Sigma</td>
			<td>6131-99-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Leica</td>
			<td>ACE200</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Sigma</td>
			<td>2231-57-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>TED PELLA</td>
			<td>CA96049</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of retinal samples for volume electron microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

Author Spotlight: Retinal Neuroscience Studies with Volume Electron Microscopy

Preparation of Retinal Samples for Volume Electron Microscopy

We focus on examining the nanoscale 3D structural features of photoreceptor terminals in the mouse retina. Our primary goal is to design 3D EM sample preparation method suitable for small volume of biological samples, and we have reconstructed the terminal structure of retina photoreceptors using FIB-SEM. Using the retinal 3D EM samples with the OTO method, we segmented and reconstructed the 3D structure of four types of photoreceptor terminals and subsequently analyze the relationship between their components.Our retinal sample variation protocol is user friendly, requires no auditory equipment and reduce sensor processing time. However, there may be some in the details of the cell membrane, which we will In the future, we hope to further explore the function of each component within the retinal terminal and its role(indistinct)

Figure 1A shows the image of retinal photoreceptor terminals prepared using the traditional chemical double fixation method, and Figure 1B shows the image of retinal photoreceptor terminals prepared using the OTO method. Both were sampled by FIB-SEM. It can be clearly seen that the cell membrane structure can be retained as much as possible by using the OTO method, and even the outline of vesicles can be clearly seen. In addition, the contrast of images obtained...

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level ...

preparation-of-retinal-samples-for-volume-electron-microscopy

Research

JoVE Journal

Neuroscience

Retina Dissection and Fixation for High-Resolution 3D Volume Electron Microscopy

To begin, place the euthanized mouse on the operating table. Using curved scissors, excise the eyes under dim red light and immediately place them in the fixation solution. After removing the anterior segment and vitreous from the eye, isolate the retina completely.Under a dissection microscope, quickly slice the retina into 100 to 200 micrometer thick strips and transfer them into a new micro centrifuge tube containing fixation solution. Incubate the tube in a rotator for two hours at room temperature and then place it at four degrees Celsius for 24 hours.

Embedding Retina Samples to Study Photoreceptor-Synapses Using Volume Electron Microscopy

To begin, obtain the retinal strips from mice and wash them five times in 0.1 molar sodium cacodylate buffer for 15 minutes. Then incubate the strips with potassium ferrocyanide and osmium tetroxide solution in the dark on ice for 1.5 hours. Wash the strips three times in double distilled water and incubate them with 1%thio-carbo-hydrazide.After rinsing with double distilled water, sequentially treat the retina strips with osmium tetroxide, uranyl acetate, and lead nitrate. Dehydrate the retina strips with increasing concentrations of ethanol. Then, treat retina strips with a solution containing equal ethanol and acetone for 20 minutes, followed by two acetone washes for 20 minutes each.Sequentially incubate the retina strips in acetone resin mixtures in the dark at room temperature. Infiltrate the retina strips with pure resin for 24 hours at 45 degrees Celsius, followed by fresh resin for 48 hours at 60 degrees Celsius. The focused ion beam scanning electron microscopy of retinal photoreceptor terminals using this method preserves cell membrane structure and vesicle outlines more clearly than the traditional double fixation method.

247 Views.  Wenzhou Medical University.  This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

Visualizing Retinal Synaptic Structures in 3D with Volume Electron Microscopy

Özet