Preparation of Retinal Samples for Volume Electron Microscopy

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 others^3,4,5. 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 signaling^6,7, establish synapses with dendrites of second-order bipolar and horizontal cells in the photoreceptor's terminal to facilitate excitatory signals^8,9. 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 terminals^10,11,12,13. 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 flux^4,14, 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 sample^16,17. 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 contrast^18,19.

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

1. 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) intraperitoneally. Confirm the depth of anesthetization by no retraction of the back paw after pinching the toe or no blinking reflex. Then, decapitate with a guillotine.
2. Remove the eyes with curved scissors and immerse the eyes in a mixed solution containing 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4).
3. Remove the anterior segment and vitreous from the eyes with forceps under the dissecting microscope.
4. Peel the sclera with the two forceps until the retina is completely isolated from the eyecup.
5. Cut the retina rapidly into 100 - 200 µm-thick strips with a razor and handle the retina strips with a Pasteur pipette into a new microcentrifuge (EP) tube containing 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4) for 2 h at room temperature (RT) on the rotator. After completion, place the tubes to fix at 4 °C for 24 h.

2. Sample embedding process

1. After washing in 0.1 M sodium cacodylate buffer (pH 7.4) 5 times for 15 min each, incubate the retina strips with a solution containing 1.5% potassium ferrocyanide and 1% OsO₄(in PB, pH7.4) in 0.1 M sodium cacodylate with 4 mM CaCl₂ for 1.5 h in the dark on ice.
   NOTE: Dark incubation and lack of phosphate can prevent precipitation and artifacts in the sample.
2. Wash the retina strips 3 times in ddH₂O for 10 min each, and then treat with 1% thiocarbohydrazide in ddH₂O for 30 min in the dark at RT.
3. After rinsing in ddH₂O 3 times for 15 min each, incubate the retina strips in 2% OsO4 (in PB, PH 7.4) for 45 min in the dark at RT.
4. After washing in ddH₂O 3 times for 15 min each, immerse the retina strips in 1% aqueous uranyl acetate in the dark at 4 °C overnight.
5. Next day, after washing in ddH₂O 3 times for 15 min each, treat the retina strips with 0.66% lead nitrate in L-aspartic acid (0.03 M, pH 5.5) for 40 min at 65 °C.
6. After washing in ddH₂O 3 times for 15 min each, dehydrate the retina strips with ascending ethanol concentration of 30%, 50%, 70%, 90%, and 100% for 20 min in each solution.
7. Treat retina strips with a mixed solution containing equal ethanol and acetone for 20 min, followed by an acetone wash 2 times for 20 min each.
8. Infiltrate the retina strips in acetone/resin mixed in a ratio of 7:3 in the dark at RT overnight and then acetone/resin at 3:7 in the dark for 24 h at RT.
   NOTE: The precise composition of the resin is as follows: Epon812 10.06 mL, DDSA 4 mL, MNA 5.94 mL, and DMP-30 0.2 mL.
9. Change the tubes the next day and infiltrate the retina strips with pure resin for 24 h at 45 °C. Change the resin again the next day, and embed the retina strips for 48 h at 60 °C.
   NOTE: The purpose of increasing the temperature is to improve penetration.

3. Selection of the area of interest

1. Cut the resin blocks into 1 µm thick sections, stain sections with 1% toluidine-blue, and observe the general structure of the retina under light microscopy to select the rough regions of interest.
2. Coat the resin blocks with platinum using a sputter coater, followed by milling with 70 nm thick section using a focused-ion-beam scanning electron microscopy under a current of 0.23 nA and acceleration voltage of 30 kV.
3. Pre-screen the section using FIB-SEM with 2000x magnification to identify the precise regions of interest.
4. Collect the data using FIB-SEM with a beam current of 0.69 nA and acceleration voltage of 2 kV. The other parameters are as follows: dwell time = 2 µs, voxel size = 1.8 nm x 1.8 nm x 15 nm.

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 by using the OTO method is also clearer.

Figure 2 illustrates the experimental workflow employed for reconstructing the ultrastructural characteristics of photoreceptor terminals. The reconstruction focused on four types of photoreceptor terminals, including M-cone pedicles, S-cone pedicles, rod1 spherules, and rod2 spherules. The main emphasis was on essential components, such as mitochondria, synaptic ribbons, and synaptic vesicle pools within the photoreceptor terminals.

Figure 3 displays the reconstructed mitochondria and synaptic ribbons in the rod photoreceptor terminals. Notably, the synaptic ribbons of the rod2 spherules exhibit a distinctive horseshoe shape, typically featuring a single ribbon per spherule. Moreover, Figure 2 highlights the presence of two mitochondria in rod spherules, with a consistent position within the central space of the terminals, located above the synaptic ribbons. All the structures were traced manually, page by page.

Figure 1: Examples of images sampled using FIB-SEM for traditional chemical double fixation method and OTO method. (A) Traditional chemical double fixation method. (B) OTO method. Scale bar: 1000 nm. Please click here to view a larger version of this figure.

Figure 2: 3D reconstruction workflow for photoreceptor terminals using FIB-SEM. Following alignment using 3D data visualization software, the serial images were manually segmented and rendered for further analysis. (A) Original serial images. (B) Aligned images. (C) Segmented images. (D) Reconstructed images. Please click here to view a larger version of this figure.

Figure 3: 3D reconstruction. The representative 3D reconstruction images depict photoreceptor terminals in white, mitochondria in orange, and synaptic ribbons in blue. Scale bar: 500 nm. Please click here to view a larger version of this figure.

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 synaptic connections, the retina exhibits a regular and hierarchical structure despite the complexity of its neuronal and synaptic components²⁰. In dissecting the retina, it becomes imperative to ensure that the thin strips are minimized to guarantee uniform contrast in subsequent sample preparation stages. The photoreceptor axon terminals, critical structures of interest, are situated in the outer plexiform layer (OPL) of the retina. The hierarchical and organized nature of the retina allows for efficient localization of the target structures, facilitating precise sampling during EM and ensuring the accuracy of subsequent 3D reconstruction results.

Volume EM facilitates three-dimensional visualization of local neural circuits and synaptic connections within the retina at the nanoscale, utilizing serial sections^21,22. This technique enables the automatic generation of high-resolution 3D images, and FIB-SEM holds the potential to unveil intricate details of both physiological and pathological activities. Therefore, maintaining stable image contrast throughout the entire block is crucial during the sample preparation process. To achieve uniform conductivity, we employ the pre-embedding OTO method, a classical approach widely acknowledged for its effectiveness in enhancing contrast in scanning electron microscopy (SEM)^18,23,24. This method involves a double osmium coating combined with thiocarbohydrazide, ensuring optimal bulk conductivity for large samples. Additionally, the double osmium treatment contributes to the superior preservation of lipid membrane structures, adding a layer of precision to our investigations. The retinal samples are hierarchical, the tissue blocks are small and easy to penetrate, and the OTO method can maintain better cell membrane contour and uniform image contrast.

Several critical factors contribute to the success of this protocol. Firstly, swift retinal harvesting and precise fixation times are paramount for maintaining optimal subcellular structure. Secondly, ensuring appropriate concentrations of osmium and thiocarbohydrazide is essential to preserve contrast in the membrane structure of the series images. Osmium, in particular, plays a crucial role by binding with lipid molecules, enhancing the BSE signal.

Our retinal sample preparation protocol is user-friendly, requires no additional equipment, and minimizes processing time. This characteristic highlights its potential applicability to other Volume EM techniques, suggesting versatility and ease of adoption in diverse experimental setups. The OTO's method notably enhances the contrast of FIB-SEM images. However, the details about the cell membrane, especially the bilayer structure, may still appear somewhat blurred. Our current sample preparation scheme involves some sacrifice of small details in cell structure to a certain extent. We aim to develop optimized methods in the future to address and improve this limitation.

Materials

Name	Company	Catalog Number	Comments
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