Name: Preparing Retinal Organoid Samples for Transmission Electron Microscopy (Video) | JoVE Journal
Uploaded: 2024-06-07T14:00:00.000+00:00
Duration: 5 min 43 s
Description: Learn an optimized TEM sample preparation protocol for retinal organoids to analyze synaptic ultrastructure at nanoscale resolution. This reproducible method enables detailed subcellular imaging of conventional and ribbon synapses.

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5 minutes

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).

1. Obtaining ROs from iPSCs25
NOTE: ROs were derived from iPSCs by modifying the previously reported procedure.
<ol>
	<li>Dissociate iPSCs at ~90% confluence using a bacterial protease (see Table of Materials). Chop up the colonies into pieces and scrape them with a cell lifter.</li>
	<li>After collection, resuspend the clusters of cells in 0.25 mL of ice-cold Matrigel. Following 20 min of incubation at 37 &#176;C, scatter the solidified gel with the DMEM/F12 medium (0.5% N2 supplement, 1% B27 supplement, 0.1 mM &#946;-mercaptoethanol, 0.2 mM L-glutamine substitute, 100 U/mL penicillin and 100 mg/mL streptomycin). Define this time as day 0 of differentiation.</li>
	<li>After 5 days of floating culture, cells group to form cell cysts. Transfer these cell cysts into a 100 mm Petri dish where they will now adhere and grow.</li>
	<li>On day 15, use bacterial protease to dissociate the adhering cells and culture them in DMEM/F12 medium (2% B27 supplement, 0.1 mM non-essential amino acids).</li>
	<li>On day 21, change the medium with 20 mL of serum-containing medium (DMEM/F12 medium, 8% FBS, 2% B27 supplement, 100 mM Taurine, and 2 mM L-glutamine substitute), and replace the medium every week.</li>
</ol>
2. Anterior fixation of ROs
<ol>
	<li>Gently move one of the cultured ROs (~D180, a size of about 1~2 mm in diameter is optimal) from the Petri dish to 1.5 mL microcentrifuge tubes with a pipette and mark the relevant information about the ROs. 
	NOTE: Cut ROs in half or cut off the poorly structured tissue with a razor under the microscope in the dish containing fixative if the ROs are too large.</li>
	<li>Remove the culture medium from 1.5 mL microcentrifuge tubes with a pipette. Add 1.5 mL of 2% paraformaldehyde-2% glutaraldehyde fixative diluted in 0.1 M phosphate buffer (PB), pH 7.4, and fix at room temperature (RT) for 4 h. Store the microcentrifuge tubes at 4 &#176;C overnight. 
	NOTE: This step can be paused for 1 week but restarting as early as possible is recommended. If ROs are left for longer, it may cause autolysis of cells leading to destruction of the ultrastructure of ROs. Due to the fragility of the ROs, avoid rotating, oscillating, flipping, or harsh operations.</li>
</ol>
3. Post fixation of ROs
<ol>
	<li>Remove the 2% paraformaldehyde-2% glutaraldehyde fixative and wash with 1 mL of 0.01 M phosphate buffered saline (PBS) at pH 7.4 for 3 x 10 min. Gently suspend the ROs in the 1.5 mL microcentrifuge tubes during the washing process for fully rinsing. For every additional day of fixation, increase the number of washes by two to remove&#160;the residual fixative as much as possible.</li>
	<li>Remove any remaining PBS using a pipette and replace it with ~150 &#181;L of 1% osmic acid (OsO4) until the tissue blocks are submerged. Place the microcentrifuge tubes in a dark box to protect them from light and infiltrate at RT for 1 h. 
	NOTE: OsO4 is a strong oxidant and must be handled in a fume hood.</li>
</ol>
4. Staining and dehydration
NOTE: The dehumidifier must be turned on to dry the environment from this step.
<ol>
	<li>Remove OsO4 using a pipette, wash with 0.01 M PBS (pH 7.4) 3 x 10 min, and then wash 3 x 10 min with deionized water. 
	NOTE: To ensure complete rinsing of ROs, we recommend suspending the ROs. Aspirate OsO4 into a dedicated disposal bottle.</li>
	<li>Remove the last deionized water wash and replace with ~150 &#181;L per tube (ensure full infiltration of tissue blocks) of uranium acetate and stain for 1-2 h at RT. Protect the microcentrifuge tubes from light.</li>
	<li>Remove the uranium acetate and replace it with 1 mL of 50% acetone in the fume hood, dehydrating for 10 min. Then, use 70%, 80%, 90%, 100%, and 100% acetone for gradient dehydration of the ROs successively. Each dehydration lasts 10 min. 
	NOTE: Leave some liquid to soak the ROs for each exchange to prevent exposure to air and moisture. Additionally, operate quickly as soon as possible because acetone is volatile. Aspirate uranium acetate into a dedicated disposal bottle.</li>
</ol>
5. Infiltration
<ol>
	<li>Discard the acetone and add ~150 &#181;L of the mixture consisting of acetone and Epon-812 resin at a ratio of 1:1. Place it in a 37 &#176;C oven for 1 h.</li>
	<li>Remove the above mixture and replace it with a mixture consisting of acetone and Epon-812 resin at a ratio of 1:4. Place it in a 37 &#176;C oven overnight.</li>
	<li>Transfer the ROs into the new tubes containing ~500 &#181;L of pure epoxy resin carefully and place them in a 45 &#176;C oven for 1 h.</li>
</ol>
6. Embedding
<ol>
	<li>Prepare the 30-50 mL of pure epoxy resin in a 50 mL centrifuge tube by mixing the reagent kit according to the manufacturer&#39;s instructions in advance. Rotate the pure epoxy resin upside down slowly for 30 min to reduce air bubbles introduced by the mixer. Then, leave it in a 37 &#176;C oven for 20 min. Add 2/3 volume of pure epoxy resin to the groove of the embedding mold.</li>
	<li>Use a toothpick to pick ROs into the embedding mold equipped with pure epoxy resin.</li>
	<li>Fill the groove with pure epoxy resin until it slightly protrudes or bulges.</li>
	<li>Adjust the position of ROs at both ends of the embedding mold for directional embedding. Place the embedding mold in a 45 &#176;C oven for 1-2 h and then put it in a 65 &#176;C oven for 48-72 h.</li>
</ol>
7. Semi-thin positioning
<ol>
	<li>Sand the excess epoxy resin around the tissue from the ends of the longer four sides of the embedding block to form a trapezoidal face. Install the embedding block into the sample mounting stand and screw down the bolt with an L screwdriver. Then, install glass knives on the knife stand and tighten the nut manually.</li>
	<li>Trim away the excess resin until the desired area is exposed.</li>
	<li>Fill the trough of the glass knife with water. Cut semi-thin slices to a thickness of 1 &#181;m using a semi-thin microtome and lay them on a microscope slide with a needle.</li>
	<li>Add 50 &#181;L of 1% toluidine blue to dye semi-thin slices and incubate for 1 min at 95 &#176;C on the heating plate. Rinse with distilled water for 2 min. Use a 20x ordinary optical microscope to observe the structure of the sections.</li>
</ol>
8.&#160;Ultra-thin sectioning
<ol>
	<li>Install the embedding blocks and glass knives as described in step 7.1. Adjust the distance between the knife and the tissue block, the liquid level of the sink, and the slicing speed. Cut the ultrathin sections with a thickness of ~70-100 nm with an ultrathin microtome and scoop the slices onto the copper mesh (200 mesh) with the supporting film.</li>
	<li>Stain ultrathin sections with 3% lead citrate for 15 min to enhance contrast in a dual staining procedure.</li>
</ol>
9. Imaging ROs by TEM
<ol>
	<li>Launch the software (see the Table of Materials).</li>
	<li>Click on the Memory mode button and mark each ultrathin section roughly.</li>
	<li>Search for synaptic structures.
	<ol>
		<li>Distinguish the laminated structure of ROs at low magnification.</li>
		<li>Locate the outer nuclear layer (ONL), which shows a nuclear structure with a deeper electron density. Subsequently, navigate to the terminal of the photoreceptor where numerous vesicles accumulate. Search for the rod terminals with a single synaptic ribbon and the cone terminals with multiple synaptic ribbons.</li>
		<li>Move to the inner plexiform layer (IPL) and seek out a similar structure with a small ribbon.</li>
	</ol>
	</li>
	<li>Capture the microscopic image.
	<ol>
		<li>Click Start and input the corresponding magnifying power.</li>
		<li>Save the images.</li>
	</ol>
	</li>
</ol>

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

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</ol>

The authors have no conflicts of interest to disclose.

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&#160;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&#160;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&#39;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 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&#160;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&#160;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.

<table><tbody><tr><td>100 mm Petri dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Acetone</td><td>Electron Microscopy Science</td><td>10000</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>A3582801</td><td></td></tr><tr><td>Cell lifter</td><td>Santa Cruz</td><td>sc-395251</td><td></td></tr><tr><td>Copper grids</td><td>Beijing Zhongjingkeyi Technology Co., Ltd.</td><td>AZH400HH</td><td></td></tr><tr><td>DigitalMicrograph Software</td><td>Gatan, Inc.</td><td></td><td>Software</td></tr><tr><td>Dispase</td><td>StemCell Technologies</td><td>#07913</td><td>Bacterial protease</td></tr><tr><td>DMEM/F12 medium</td><td>Gibco</td><td>#11320033</td><td></td></tr><tr><td>Embedding mold</td><td>Beijing Zhongjingkeyi Technology Co., Ltd.</td><td>GZ10592</td><td></td></tr><tr><td>Epon-812 resin</td><td>Electron Microscopy Science</td><td>#14900</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biological Industries</td><td>#04-0021A</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Electron Microscopy Science</td><td>16020</td><td></td></tr><tr><td>hiPSC</td><td>Shownin Biotechnology Co. Ltd.</td><td>RC01001-A</td><td></td></tr><tr><td>Lead citrate</td><td>Beijing Zhongjingkeyi Technology Co., Ltd.</td><td>GZ02618</td><td></td></tr><tr><td>L-GlutaMax</td><td>Life Technologies</td><td>#35050061</td><td>L-glutamine substitute</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356234</td><td></td></tr><tr><td>Microscope slide</td><td>CITOTEST</td><td>80312-3161</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot; 12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71650</td><td>A component of PB/PBS</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot; H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71507</td><td>A component of PB/PBS</td></tr><tr><td>Non-essential amino acids</td><td>Sigma</td><td>#M7145</td><td></td></tr><tr><td>Optical microscope</td><td>Lab Binocular Biological Microscope</td><td>Xsz-107bnii</td><td></td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt;</td><td>TED PELLA</td><td>4008-160501</td><td></td></tr><tr><td>Oven</td><td>Bluepard</td><td>BPG9040A</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Science</td><td>157-8</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>#15140-122</td><td></td></tr><tr><td>Semi/ultrathin microtome</td><td>Reichert-Jung</td><td>396649</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>#T0625</td><td></td></tr><tr><td>Toluidine blue</td><td>Sangon Biotech</td><td>E670105-0100</td><td></td></tr><tr><td>Transmission Electron Microscopes</td><td>HITACHI</td><td>H-7500</td><td></td></tr><tr><td>Uranyl acetate</td><td>TED PELLA</td><td>CA96049</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>444203</td><td></td></tr></tbody></table>

preparing retinal organoid samples for transmission electron microscopy

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

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 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).
<img alt="Electron microscopy image of retinal cells showing rod and HC structures, cellular anatomy study." class="xfigimg" src="/files/ftp_upload/66590/66590fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/66590/66590fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electron microscopy; cellular structure; detailed cone cell diagram; research; scale 0.2 μm." class="xfigimg" src="/files/ftp_upload/66590/66590fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/66590/66590fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electron microscope image of cellular structure." class="xfigimg" src="/files/ftp_upload/66590/66590fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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. <a href="https://www.jove.com/files/ftp_upload/66590/66590fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electron microscope image, retinal cell structure; labeled cone; cellular ultrastructure analysis." class="xfigimg" src="/files/ftp_upload/66590/66590fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
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. <a href="https://www.jove.com/files/ftp_upload/66590/66590fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

preparing-retinal-organoid-samples-for-transmission-electron

Research

JoVE Journal

Neuroscience

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