What question did this study set out to answer?

The aim is to evaluate ultrastructure expansion microscopy (U-ExM) for better structural and molecular analysis of photoreceptor ribbon synapses in retinal tissues.

June 3, 2026Open Access

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

Key Points

The aim is to evaluate ultrastructure expansion microscopy (U-ExM) for better structural and molecular analysis of photoreceptor ribbon synapses in retinal tissues.
Cryosections from canine retinas were immunolabeled with 44 antibodies targeting 29 proteins.
U-ExM processed formaldehyde-fixed sections were analyzed for spatial resolution and compartment morphometry.
Multi-orientation visualization and line-profile measurements were used to interpret synaptic architecture.
U-ExM improved antibody compatibility in fixed tissues, enhancing detectability across targets.
Refined analysis revealed multiple expression sites of PSD-95 in synaptic compartments.
Enabled detailed mapping and quantitative measurements of photoreceptor ribbon structures across developmental stages.

Abstract

Purpose: Immunofluorescence-based mapping of photoreceptor ribbon synapse nanostructure in extended formaldehyde-fixed specimens is challenging due to diffraction-limited resolution and fixation-associated loss of antigen accessibility that restrict reliable molecular localization and structural interpretation. To circumvent these physicochemical limitations, we tested whether ultrastructure expansion microscopy (U-ExM) could overcome these barriers and enable compartment-resolved structural and molecular analysis of photoreceptor ribbon synapses in archival canine retinal tissues. Methods: Cryosections from non-fixed and formaldehyde-fixed canine retinas were immunolabeled by conventional immunohistochemistry to screen 44 antibodies targeting 29 ribbon synapse-associated proteins. The same antibody set was then evaluated in U-ExM-processed formaldehyde-fixed cryosections. U-ExM datasets were analyzed using multi-orientation visualization, line-profile measurements, and three-dimensional segmentation for ribbon morphometry in mature and developing retinas. Results: Antibody performance differed markedly between non-fixed and formaldehyde-fixed sections, indicating fixation-dependent loss or gain of detectability across targets. U-ExM increased the fraction of compatible antibodies in formaldehyde-fixed tissue and improved effective spatial resolution within synaptic compartments. Multi-orientation visualization refined interpretation of rod spherule architecture and revealed multiple postsynaptic density 95 (PSD-95) subcellular sites of expression, including a postsynaptic pool in horizontal cell axon terminals within the invaginated compartment. U-ExM further enabled subcompartment-level visualization of active zone proteins, extended compartment-resolved mapping to the more complex cone pedicle, and supported quantitative ribbon measurements in mature retina and across postnatal development. Conclusions: U-ExM provides a practical framework for compartment-resolved molecular mapping and quantitative phenotyping of photoreceptor ribbon synapses in formaldehyde-fixed/frozen archival retinal tissue, enabling systematic comparisons across conditions and developmental stages in translational and comparative vision research.

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