staining, dewatering, and infiltration of fixed retinal organoids for tem sample preparation

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

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.

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Our research primarily focuses on analyzing the synaptic ultrastructure in mature retinal organoids. We aim to develop a reproducible and straightforward transmission electron microscope, or TEM, sample preparation method for retinal organoids, which could greatly benefit the field of retinal organoid research. Compared to other techniques such as optical microscope, transmission electron microscope offers the ability to characterize the ultrastructure of integral synapse and the subcellular organelles in retinal organoids at a nanoscale level.Our protocol offers optimized conditions for transmission electron microscope sample preparation of retinal organoids, returning simple and user-friendly stats. Our findings clearly show the ultrastructure morphology of synapse within retinal organoids, including both conventional synapse and ribbon synapse. This establishes a solid foundations and presents a reproducible methods for subsequent functional investigations of retinal organoids, along with the evaluation of their clinical applications such as drug screening.

Staining, Dewatering, and Infiltration of Fixed Retinal Organoids for TEM Sample Preparation

To begin staining the fixed retinal organoids, or ROs, for transmission electron microscopy, use a pipette to remove the osmium acid previously added to the organoids in the microcentrifuge tube during post-fixation. Then, wash the ROs with 0.01 molar PBS at pH 7.4 three times for 10 minutes each, followed by washing with deionized water three times for 10 minutes each. Remove the last deionized water wash, replace it with about 150 microliters of uranium acetate and stain for one to two hours at room temperature.Next, in a fume hood, remove the uranium acetate and replace it with one milliliter of 50%acetone to dehydrate the samples for 10 minutes. Then, perform gradient dehydration using 70%80%90%and two times 100%acetone successively, each for 10 minutes. After the gradient dehydration, discard the residual acetone and add approximately 150 microliters of a mixture of acetone and Epon-812 resin.Place the tube in a 37-degree Celsius oven for one hour. After removing the previous acetone-resin mixture, replace it with a different composition of acetone and Epon-812 resin mixture. This time, incubate the tube at 37 degrees Celsius overnight.Finally, transfer the retinal organoids into a new tube containing approximately 500 microliters of pure epoxy resin, carefully. And incubate at 45 degrees Celsius for one hour before proceeding with organoid embedding.

staining-dewatering-infiltration-fixed-retinal-organoids-for-tem

Research

JoVE Journal

Neuroscience

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.

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 ...

Retinal Organoid Fixation and Post Fixation Procedure for TEM Sample Preparation

To begin the anterior fixation of the induced pluripotent stem cell-derived retinal organoids or ROs. Using a pipette, gently move one of the cultured ROs from the Petri dish to a 1.5-milliliter microcentrifuge tube. With the help of a pipette, remove the culture medium from the microcentrifuge tube and add 1.5 milliliters of 2%paraformaldehyde, 2%glutaraldehyde fixative.After the ROs are fixed, remove the paraformaldehyde-glutaraldehyde fixative before proceeding to wash the organoids. Gently suspend the organoids in the microcentrifuge tube by adding 1 milliliter of 0.01 molar PBS to ensure complete washing. Then pipette out any remaining PBS and replace it with approximately 150 microliters of 1%osmic acid until the tissue blocks are submerged.Place the microcentrifuge tube in a dark box.

Retinal Organoid Embedding and Semi-Thin Slice Positioning for Transmission Electron Microscopy

To begin, add pure epoxy resin to the groove of the embedding mold up to two thirds of its volume. Use a toothpick to pick retinal organoids and transfer them into the embedding mold, equipped with pure epoxy resin. Then fill the groove with pure epoxy resin until it slightly protrudes or bulges.Adjust the position of the organoids at both ends of the embedding mold for directional embedding. 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.Trim away the excess resin until the desired area is exposed. Fill the trough of the glass knife with water. Using a semi-thin microtome, cut semi-thin slices to a thickness of one micron, and lay them on a microscope slide with a needle.Add 50 microliters of 1%toluidine blue to dye the semi-thin slices, and incubate for one minute at 95 degrees celsius on the heating plate. Rinse with distill water for two minutes. Use a 20x ordinary optical microscope to observe the structure of the sections.