Summary

Preparation of Retinal Samples for Volume Electron Microscopy

Published: May 24, 2024
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Summary

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

Abstract

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.

Introduction

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'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 < 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'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.

Protocol

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 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…

Representative Results

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…

Discussion

We implemented the OTO's Volume EM sample preparation protocol to analyze the photoreceptors' 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…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

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).

Materials

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

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Zhao, W., Yang, Q., Lai, X., Zhang, J. Preparation of Retinal Samples for Volume Electron Microscopy. J. Vis. Exp. (207), e66589, doi:10.3791/66589 (2024).

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