September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Spectral time sharing to simultaneously measure rhodopsin absorbance and retinol fluorescence while mitigating light scatter in living frog retina suspensions
Author Affiliations & Notes
  • Gabriel Gonzalez-Fernandez
    Ophthalmology, University of Mississippi Medical Center, Jackson, Mississippi, United States
  • Richard J. DeSa
    Research & Development, Olis, Bogart, Georgia, United States
  • Footnotes
    Commercial Relationships   Gabriel Gonzalez-Fernandez, None; Richard DeSa, Olis (P)
  • Footnotes
    Support  Merit Review Award I01BX007080 from the Biomedical Laboratory Research & Development Service of the Veterans Affairs Office of Research and Development, NIH RO1 EY09412; National Center for Research Resources 5G12RR013646-12, National Institutes of Minority Health and Health Disparities (G12MD007591), an Unrestricted Research Grant from Research to Prevent Blindness to the Department of Ophthalmology at SUNY at Buffalo, and NIH grant 5 U42 RR006042 to the NDRI.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4024. doi:
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    • Get Citation

      Gabriel Gonzalez-Fernandez, Richard J. DeSa; Spectral time sharing to simultaneously measure rhodopsin absorbance and retinol fluorescence while mitigating light scatter in living frog retina suspensions. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4024.

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

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Abstract

Purpose : Studies of living photoreceptor cell suspensions and detached retina have been limited by light scatter. Our goal is to monitor retinoid delivery and removal cellular suspensions. We previously described a method to mitigate light scatter allowing spectral measurement of rhodopsin in cellular suspensions and isolated neural retina (F. Gonzalez-Fernandez and R.J. DeSa, 2015, ARVO abstract). This was accomplished by replacing the traditional rectangular cuvette with an integrating sphere. Here we extend the capabilities of the system by adding an emission monochrometer to simultaneously monitor fluorescence

Methods : Light access to an 8.6 ml quartz integrating sphere containing minced or intact Ranna pipiens neural retina suspended in saline was provided via 4 right angle ports allowing for: 1) Light delivery from a selectable (515 or 360 nm) light-emitting diode (LED) cluster firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec (10,000 µsec scan time) via an Olis RSM 1000 UV/Vis rapid-scanning spectrophotometer (RSM), 3) light exit to photomultiplier, and 4) light exit to emission monochrometer. To interleaf the 515 nm pulse between each absorbance spectra, the RSM spectral output slit was narrowed to allow for a dark period of 2,000 µsec when no scanning light was admitted to the cuvette. To monitor all-trans retinol fluorescence the actinic beam was changed to the 360 nm LED allowing fluorescence measurements now time-sharing the 10,000 µsec window.

Results : We began by characterizing the system using purified all-trans retinol in ethanol. Next, rhodopsin bleaching was followed in real time using a bleachin 515 nm LED. Following the bleach, the emergence of retinol fluorescence was monitored by pulsing with the 360 nm LED while monitoring emission at 468 nm. We found that during the 40 min following the bleach, a steady increase in fluorescence could be monitored.

Conclusions : The integrating cavity mitigates light scatter allowing spectra to be obtained from highly light scattering suspensions of minced or intact retina. The system should be valuable to studies of visual cycle biochemical physiology in living tissue where light scatter prevents the use traditional spectroscopic methods.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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