Preparing Retinal Organoid Samples for Transmission Electron Microscopy

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 synapse^2,3. The ribbon synapse plays a crucial role in transmitting vesicle impulses in a graded manner^2,3. The vision process involves electro-optical signal transmission across various levels of neurons and synapses, ultimately reaching the visual cortex^4,5.

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 vitro^1,6,7. 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 profiles^6,12. 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 development^6,12,13. 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 humans^16,17 and various species^18,19,20,21. TEM enables the direct distinction of presynaptic and postsynaptic components^18,20, and even allows for comprehensive observation of subcellular structures such as ribbons^2,3, 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 ROs^12,13,24, 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.