June 2023
Volume 64, Issue 9
Open Access
ARVO Imaging in the Eye Conference Abstract  |   June 2023
High resolution imaging of cone pedicles
Author Affiliations & Notes
  • Ellen Townes-Anderson
    Pharmacology, Physiology, and Neuroscience, Rutgers New Jersey Medical School, Newark, New Jersey, United States
  • Ilene Sugino
    Institute of Ophthalmology and Visual Science, Rutgers New Jersey Medical School, Newark, New Jersey, United States
  • Fawad Yousufzai
    Cellular Imaging and Histology Core, Rutgers New Jersey Medical School, Newark, New Jersey, United States
  • Éva Halász
    Pharmacology, Physiology, and Neuroscience, Rutgers New Jersey Medical School, Newark, New Jersey, United States
  • Marco Zarbin
    Institute of Ophthalmology and Visual Science, Rutgers New Jersey Medical School, Newark, New Jersey, United States
  • Luke Fritzky
    Cellular Imaging and Histology Core, Rutgers New Jersey Medical School, Newark, New Jersey, United States
  • Footnotes
    Commercial Relationships   Ellen Townes-Anderson, None; Ilene Sugino, None; Fawad Yousufzai, None; Éva Halász, None; Marco Zarbin, None; Luke Fritzky, None
  • Footnotes
    Support  DoD Research Award W81XWH1910819; NIH High End Instrumentation Grant S10 OD25182
Investigative Ophthalmology & Visual Science June 2023, Vol.64, PB0084. doi:
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    • Get Citation

      Ellen Townes-Anderson, Ilene Sugino, Fawad Yousufzai, Éva Halász, Marco Zarbin, Luke Fritzky; High resolution imaging of cone pedicles. Invest. Ophthalmol. Vis. Sci. 2023;64(9):PB0084.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The first synapse of the visual pathway, between photoreceptors and bipolar cells, is essential for vision. However, these synapses are subject to a variety of diseases and injuries that cause synaptopathy. We have been studying the mammalian retina to understand the structural damage that retinal detachment (RD), which can lead to visual loss, causes to cone pedicles. Compared to conventional confocal microscopy, our morphological assessment has been substantially improved with the use of stimulated emission depletion (STED) microscopy.

Methods : Normal or detached retinas from young adult Yorkshire pigs were fixed in 4% paraformaldehyde and then frozen for sectioning. Sections were either triple-labeled for PSD-95, CtBP2, a ribbon marker, and PNA, a cone specific marker, for conventional confocal (CC) microscopy or double labeled for CtBP2 and PNA for STED microscopy. A Nikon A1R+HD Confocal Microscope or a Leica Stellaris 8 tau-STED Super Resolution Microscope was used respectively. Stacks of 40 images of ~ 0.20 µm optical thickness with CC microscopy and 160 images ~ 0.14 µm thick with STED microscopy, were obtained. 3-D images were surface-rendered with Imaris software.

Results : Conventional confocal images of cone pedicles show multiple ribbons distal to a row of PNA-positive dots surrounded by label for PSD-95 which coats the lateral surfaces of the pedicle. STED microscopy, in contrast, shows PNA as irregular aggregates or islands of label, and the ribbons as a loose network lying just distal to the PNA. Ribbons were single or in branched configurations suggesting an anastomosis of ribbons. Additionally, the arched or horseshoe shape of the ribbon as it borders the synaptic invaginations was clear. After RD, reduction in the length and number of ribbons per pedicles were seen with some pedicles almost completely devoid of ribbons. In addition, the arching of ribbons became shallow, indicating the loss of invaginations. These features, which lead to reduced neurotransmission with RD, are difficult to observe with CC microscopy.

Conclusions : Because of the increased resolution of STED microscopy, features of normal and injured cone pedicles have been observed for the first time. The new microscopic approach will improve our understanding of changes in photoreceptor synapses with injury and disease and help assess proposed treatments to protect or repair the first synapse in the visual pathway.

This abstract was presented at the 2023 ARVO Imaging in the Eye Conference, held in New Orleans, LA, April 21-22, 2023.

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