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Preparing Retinal Organoid Samples for Transmission Electron Microscopy

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Revisiting the Molecular Architecture of Photoreceptor Ribbon Synapses Using Ultrastructure Expansion Microscopy.

Kei Takahashi¹, Natalia Dolgova¹, Raghavi Sudharsan¹

¹Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States.

Investigative Ophthalmology & Visual Science

|June 1, 2026

Summary

Ultrastructure expansion microscopy (U-ExM) enhances molecular mapping of photoreceptor ribbon synapses in fixed retinal tissues. This technique improves structural and molecular analysis for vision research.

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Area of Science:

Neuroscience
Cell Biology
Ophthalmology

Background:

Photoreceptor ribbon synapse nanostructure analysis is hindered by limitations in standard immunofluorescence.
Formaldehyde fixation reduces antigen accessibility and diffraction limits resolution, complicating molecular localization and structural interpretation.

Purpose of the Study:

To evaluate ultrastructure expansion microscopy (U-ExM) for overcoming physicochemical limitations in analyzing photoreceptor ribbon synapses.
To enable compartment-resolved structural and molecular analysis in archival formaldehyde-fixed canine retinal tissues.

Main Methods:

Screened 44 antibodies against 29 ribbon synapse proteins in conventional immunohistochemistry and U-ExM on fixed and non-fixed canine retinal cryosections.
Analyzed U-ExM datasets using multi-orientation visualization, line-profile measurements, and 3D segmentation for ribbon morphometry.
Assessed antibody performance differences between fixation methods and U-ExM processing.

Main Results:

U-ExM increased compatible antibodies in formaldehyde-fixed tissue and improved effective spatial resolution within synaptic compartments.
Refined interpretation of rod spherule architecture and identified multiple postsynaptic density 95 (PSD-95) expression sites.
Enabled subcompartment visualization of active zone proteins and mapping in cone pedicles, supporting quantitative ribbon measurements.

Conclusions:

U-ExM offers a practical framework for compartment-resolved molecular mapping and quantitative phenotyping of photoreceptor ribbon synapses.
Facilitates systematic comparisons across conditions and developmental stages in archival fixed retinal tissues.
Supports translational and comparative vision research by enhancing analysis of synaptic nanostructure.