<meta charset="utf8"></head><body>Visualizzazione delle strutture sinaptiche retiniche in 3D con la microscopia elettronica volumetrica

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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. 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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. 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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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<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

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.

<meta charset="utf8"></head><body>Autore in primo piano: Studi di neuroscienze retiniche con microscopia elettronica volumetrica

Preparazione di campioni retinici per microscopia elettronica volumetrica

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)

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

Preparation of Retinal Samples for Volume Electron Microscopy

277 Views.  Wenzhou Medical University. Ci concentriamo sull'esame delle caratteristiche strutturali 3D su scala nanometrica dei terminali dei fotorecettori nella retina del topo. Il nostro obiettivo principale è quello di progettare un metodo di preparazione dei campioni EM 3D adatto a piccoli volumi di campioni biologici e abbiamo ricostruito la struttura terminale dei fotorecettori della retina utilizzando FIB-SEM. Utilizzando i campioni EM 3D retinici con il metodo OTO, abbiamo segmentato e ri...