This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.
Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.
The retina, a vital visual sensory organ in humans and mammals, exhibits a distinct laminated structure characterized by three nuclear layers housing neuron somas and two plexiform layers formed by synaptic connections1, including conventional synapses and the specialized ribbon synapse2,3. The ribbon synapse plays a crucial role in transmitting vesicle impulses in a graded manner2,3. The vision process involves electro-optical signal transmission across various levels of neurons and synapses, ultimately reaching the visual cortex4,5.
Retinal organoids (ROs) represent a three-dimensional (3D) culture system derived from induced pluripotent stem cells (iPSCs), mimicking the physiological states of retinal tissue in vitro1,6,7. This approach holds promise for studying retinal diseases8, drug screening9, and serving as a potential therapy for irreversible retinal degenerative conditions such as retinitis pigmentosa10 and glaucoma11. As a powerful in vitro optical conduction system, the synapse within ROs is a crucial structure facilitating effective signal transformation and transfer5.
RO development can be roughly divided into three stages according to their morphological traits and molecular expression profiles6,12. ROs in stage 1 (around D21-D60) comprise neural progenitor cells of the retina, many retinal ganglion cells (RGCs), and a few starburst amacrine cells (SACs), corresponding to the first epoch of human fetal development. In stage 2 (around D50-D150), ROs express some photoreceptor precursors, interneurons, and synaptogenesis-related genes, which represents a phase of transition. Photoreceptors develop maturity in stage 3 ROs (around after D100-D150), corresponding to the third stage of human fetal development6,12,13. Notably, compared with ROs in stage 1 and stage 2, ROs in stage 3 have a distinct lamellar structure whose synapses have matured12, including the presence of ribbon synapses14. Moreover, a recent study has confirmed that the mature synapses exist the transmission of light signals, indicating they are functional13. Thus, ROs in stage 3 are often selected to investigate synaptic structure.
Immunohistochemistry is widely applied to the study of the expression of various molecular proteins. However, the limitation of optical microscopy lies in its ability to observe only a restricted number of specific cells and molecules at a time, resulting in a lack of comprehensive analysis of the relationships between cells and their surrounding environment. Transmission Electron Microscopy (TEM) is characterized by high resolution, with a limited resolution of 0.1-0.2 nm, surpassing the light microscope by ~10-20 times15. It makes up for the defects of optical microscopy and is used to elucidate retinal development and synapse maturation in humans16,17 and various species18,19,20,21. TEM enables the direct distinction of presynaptic and postsynaptic components18,20, and even allows for comprehensive observation of subcellular structures such as ribbons2,3, vesicles22, and mitochondria23. Therefore, TEM is an essential tool for identifying types of synapses and exploring the ultrastructure of synaptic contacts in ROs at the nanoscale.
It is crucial to note that sample preparation is of great importance for acquiring high-quality electron micrographs. Although some studies have performed EM on ROs12,13,24, the detailed procedures are unclear. Since the quality of the electron microscopy image depends on the effect of RO fixation and reagent permeation to a large extent, various important factors need to be considered during preparation. Consequently, to better investigate synaptic contacts in ROs, we present a method with good reproducibility that shows the operation points of RO fixing, embedding, and the identification of observation sites.
1. Obtaining ROs from iPSCs25
NOTE: ROs were derived from iPSCs by modifying the previously reported procedure.
2. Anterior fixation of ROs
3. Post fixation of ROs
4. Staining and dehydration
NOTE: The dehumidifier must be turned on to dry the environment from this step.
5. Infiltration
6. Embedding
7. Semi-thin positioning
8. Ultra-thin sectioning
9. Imaging ROs by TEM
The establishment of 3D ROs through iPSC differentiation provides a powerful tool for studying retinal disease mechanisms and stem cell replacement therapy. Although others have demonstrated the synaptic connections in ROs functionally and immunocytochemically, direct evidence of conventional and ribbon synapses is very limited. Here we present a method for investigating the ultrastructure of two types of synapses in ROs by TEM. After 180 days of culture, ROs were fixed, stained, embedded, and ultrathin sliced. TEM observation revealed ribbon synapses at the axonal terminals of photoreceptors (Figure 1 and Figure 2), which are distributed in the OPL. Under TEM, ribbons of rods spherule around the synaptic invagination, forming a horseshoe-shaped profile with horizontal cells (Figure 1). Rods typically have only a single ribbon, but cones have multiple ribbons (Figure 2). Additionally, classical chemical synapses between amacrine cells in the IPL were observed (Figure 3). Under TEM, the chemical synapse consists of three parts: the presynaptic, the synaptic cleft, and the postsynaptic. The cell membranes corresponding to the presynaptic and postsynaptic parts are slightly thicker than the rest of the parts, and the narrow gap between the two membranes is called the synaptic cleft. There are many synaptic vesicles in the cytoplasm of the presynaptic membrane site. This study confirms the presence of complete synaptic connections in 3D ROs derived from iPSC. However, it should be noted that incomplete rinsing between steps would lead to the precipitation of black particles during the process of sample preparation (Figure 4).
Figure 1: TEM images of rod ribbon synapses in ROs. (A,B) Rod and horizontal cells form a horseshoe-shaped profile. A single ribbon (arrowheads) was observed at each rod spherule in the OPL of ROs, surrounded by numerous vesicles. Scale bars = 0.2 µm. Abbreviations: ROs = retinal organoids; HC = horizontal cell; OPL = outer plexiform layer. Please click here to view a larger version of this figure.
Figure 2: TEM images of cone ribbon synapses in ROs. Three ribbon synapses (arrowheads) were obvious at a cone pedicle in the OPL of ROs. Scale bar = 0.2 µm. Abbreviations: TEM = transmission electron microscopy; ROs = retinal organoids; OPL = outer plexiform layer. Please click here to view a larger version of this figure.
Figure 3: TEM image of chemical synapses in ROs. A conventional chemical synapse was observed between amacrine cells in the IPL layer of ROs. The chemical synapse consists of three parts: the presynaptic, the synaptic cleft, and the postsynaptic. There are many synaptic vesicles in the cytoplasm of the presynaptic membrane site. An arrow denotes the direction of synaptic transmission, indicating the transfer of numerous vesicles from one AC1 process to another AC2 profile. Scale bar = 0.2 µm. Abbreviations: TEM = transmission electron microscopy; ROs = retinal organoids; IPL = inner plexiform layer; ACs = amacrine cells. Please click here to view a larger version of this figure.
Figure 4: An example of a problem that can occur during the process of sample preparation. There are a lot of black particles (circles) in the cone pedicle because of incomplete rinsing between steps. Arrows: ribbon synapse. Scale bar = 0.2 µm. Please click here to view a larger version of this figure.
In this article, we presented a detailed protocol for observing conventional and ribbon synaptic ultrastructure in ROs by TEM. This protocol is based on the previously described retinal preparation methods with some modifications20. To improve the success rate of sample treatment and the quality of TEM micrographs, consider the following key points. First, it is important to acknowledge that ROs develop from iPSCs, forming a cell mass lacking vasculature6,26 and glial cells26. This absence may lead to a less compact organization compared to the in vivo retina. Thus, it is crucial to handle Ros gently for maintaining their structure and integrity. Add liquids along the tube wall to minimize disturbance.
Importantly, ensure thorough dehydration of the tissue in protocol step 4 and switch on the dehumidifier to reduce room humidity before starting the dehydration step. When replacing acetone, operate quickly and leave some liquid to submerge the tissue, preventing severe destruction of the RO tissue structure. Undehydrated tissue contracts sharply at high EM altitudes, and water in tissue hampers the penetration of the embedding agent.
Since ROs present as transparent spheres, ensure the correct direction when cutting semi-thin slices to obtain intact lamellar structures. Lastly, ROs in stage 3 (around after D100-D150) are considered mature and possess integral synapses12, as a result of which, the investigation of synaptic contacts, including ribbon synapses and chemical synapses, is recommended to be conducted in those ROs.
Although limited ultrastructural proof of RO synapses has been provided in previous studies, there is no detail about the complete procedure of RO sample preparation as well as the identification of various types of synaptic structures12,13,24. Capowski et al. used different techniques to investigate the characteristics of ROs induced by different hPSC lines at different stages, including electron micrographs showing the existence of vesicle-laden ribbon synapses in the cone pedicle, which revealed that ROs in stage 3 induced by various hPSC lines are mature and functional12. Using TEM and two-photon imaging, Cowan et al. confirmed the formation of functional ribbon synaptic structures in mature ROs13, but the type of synapse was not indicated (cone or rod synapses). However, our several batches of micrograph results showed the membrane structure of the ribbon synapses with an ideal clarity and contrast, which illustrates that our method is feasible and reproducible. In addition, we identified different types of synapses according to their different morphological characteristics: cone synapses are usually larger and have several ribbons while rod synapses are usually smaller, showing only one ribbon; In chemical synapses, vesicles accumulate in the presynaptic structures, and the electron density of the synaptic cleft is enhanced.
The ultrastructural integrity of the synaptic contacts in ROs is vital for their function. Employing TEM to investigate these contacts offers broad and significant advantages in various fields, such as studying the pathogenesis of the retina in vitro, conducting drug screening in ROs, and evaluating differentiation techniques of ROs, among other applications. Limited by 2D TEM imaging, only a specific aspect of the ROs ultrastructure is observable, making it challenging to analyze the synapse's entire structure stereoscopically. In addition, the boundedness of differentiation contributes to misplaced cells13,26, further complicating the observation process. Thus, combining volume EM with 3D reconstruction may overcome these shortcomings in the future.
The authors have nothing to disclose.
This work was supported in part by grants from the National Key Research and Development Program of China (2022YFA1105503), the State Key Laboratory of Neuroscience (SKLN-202103), and the Zhejiang Natural Science Foundation of China (Y21H120019), the Natural Science Foundation of China (82070981).
100 mm Petri dish | Corning | 430167 | |
Acetone | Electron Microscopy Science | 10000 | |
B27 supplement | Gibco | A3582801 | |
Cell lifter | Santa Cruz | sc-395251 | |
Copper grids | Beijing Zhongjingkeyi Technology Co., Ltd. | AZH400HH | |
DigitalMicrograph Software | Gatan, Inc. | Software | |
Dispase | StemCell Technologies | #07913 | Bacterial protease |
DMEM/F12 medium | Gibco | #11320033 | |
Embedding mold | Beijing Zhongjingkeyi Technology Co., Ltd. | GZ10592 | |
Epon-812 resin | Electron Microscopy Science | #14900 | |
Fetal Bovine Serum (FBS) | Biological Industries | #04-0021A | |
Glutaraldehyde | Electron Microscopy Science | 16020 | |
hiPSC | Shownin Biotechnology Co. Ltd. | RC01001-A | |
Lead citrate | Beijing Zhongjingkeyi Technology Co., Ltd. | GZ02618 | |
L-GlutaMax | Life Technologies | #35050061 | L-glutamine substitute |
Matrigel | Corning | 356234 | |
Microscope slide | CITOTEST | 80312-3161 | |
N2 supplement | Gibco | 17502048 | |
Na2HPO4· 12H2O | Sigma | 71650 | A component of PB/PBS |
NaH2PO4· H2O | Sigma | 71507 | A component of PB/PBS |
Non-essential amino acids | Sigma | #M7145 | |
Optical microscope | Lab Binocular Biological Microscope | Xsz-107bnii | |
OsO4 | TED PELLA | 4008-160501 | |
Oven | Bluepard | BPG9040A | |
Paraformaldehyde | Electron Microscopy Science | 157-8 | |
Penicillin-Streptomycin | Gibco | #15140-122 | |
Semi/ultrathin microtome | Reichert-Jung | 396649 | |
Taurine | Sigma | #T0625 | |
Toluidine blue | Sangon Biotech | E670105-0100 | |
Transmission Electron Microscopes | HITACHI | H-7500 | |
Uranyl acetate | TED PELLA | CA96049 | |
β-mercaptoethanol | Sigma | 444203 |