July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
High resolution two photon scanning fluorescence polarization microscopy for assessing photoreceptor disc membrane structure in living photoreceptors
Author Affiliations & Notes
  • Peter D Calvert
    Center for Vision Research, Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
  • Nycole Maza
    Center for Vision Research, Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
  • Cassandra Barnes
    Center for Vision Research, Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
  • Sungsu Lee
    Center for Vision Research, Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
  • Footnotes
    Commercial Relationships   Peter Calvert, None; Nycole Maza, None; Cassandra Barnes, None; Sungsu Lee, None
  • Footnotes
    Support  NIH Grants R01EY018421 and R01EY028303
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2360. doi:
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    • Get Citation

      Peter D Calvert, Nycole Maza, Cassandra Barnes, Sungsu Lee; High resolution two photon scanning fluorescence polarization microscopy for assessing photoreceptor disc membrane structure in living photoreceptors. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2360.

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

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Abstract

Purpose : Structural stability of photoreceptor discs (rods) and lamellae (cones) is thought to be vital for photoreceptor survival. Currently there is no direct approach for measuring disc membrane stability in living photoreceptors. Here we describe a high spatial resolution, live tissue approach for assessing the highly ordered structure of disc membranes, and how they are locally disrupted in retinal degenerative disease models.

Methods : A highly polarized (10,000:1) Titanium sapphire laser beam (λ=850 nm) was focused to the diffraction limit by a 1.2 NA water immersion objective. Upon x-y scanning of living Xenopus laevis retinal explants, fluorescence emission (λ=590 nm) elicited from rhodopsin was recorded by two hybrid single photon counting detectors placed orthogonally after a polarizing beam splitter in the emission path. Steady-state and time-resolved polarization images were accumulated using a time correlated single photon counting (TCSPC) module. The rhodopsin origin of the polarized fluorescence signal was confirmed by photobleaching and treatment with hydroxylamine.

Results : High resolution fluorescence images of wild type rod photoreceptors showed up to nine-fold polarization of rhodopsin emission. Imaging of rods transgenically expressing opsin possessing the P23H point mutation that is the major cause of autosomal dominant retinitis pigmentosa showed local depolarization of the rhodopsin fluorescence that corresponded with local accumulation of the mutant opsin.

Conclusions : Our results demonstrated that the ordered disc membranes in living rods can be assessed at high spatial resolution and that local disruption of discs by disease-causing mutant rhodopsins are detectable, allowing an assessment of the structural stability of rod outer segments in living photoreceptors.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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