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 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.