June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Two-photon fluorescence lifetime ophthalmoscopy of intrinsic fluorophores on a cellular scale in the living macaque
Author Affiliations & Notes
  • James Feeks
    The Institute of Optics, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Sarah Walters
    The Institute of Optics, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Christina Schwarz
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Jennifer J Hunter
    Flaum Eye Institute, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Footnotes
    Commercial Relationships   James Feeks, University of Rochester (P); Sarah Walters, None; Christina Schwarz, None; Jennifer Hunter, University of Rochester (P)
  • Footnotes
    Support  This research was supported by the National Eye Institute of the National Institutes of Health under Awards P30 EY001319 and EY022371. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1419118. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. This work was also supported by an unrestricted grant to the University of Rochester Department of Ophthalmology from Research to Prevent Blindness, New York, New York.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3431. doi:
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    • Get Citation

      James Feeks, Sarah Walters, Christina Schwarz, Jennifer J Hunter; Two-photon fluorescence lifetime ophthalmoscopy of intrinsic fluorophores on a cellular scale in the living macaque. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3431.

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

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Abstract

Purpose : Fluorescence lifetime imaging quantifies the decay times of fluorophores and is sensitive to the environment of the fluorophore, which may be useful for early detection of changes in cell health. By combining fluorescence lifetime imaging with adaptive optics two-photon excited fluorescence ophthalmoscopy, we provide a novel technique to interrogate at a cellular level the properties of intrinsic fluorophores involved in cellular metabolism and the visual cycle.

Methods : For adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO), a single photon counting detector and time-correlated single photon counting module were added to the fluorescence detection path of a two-photon adaptive optics scanning light ophthalmoscope. AOFLIO was performed in 2 macaques. Fluorescence was excited using 7 mW of 730 nm light and emission <550 nm collected for 150 s. Fluorescence lifetimes were determined for each pixel containing >500 photons by fitting exponential decay curves to photon arrival time histograms. Cones and rod regions were identified using the simultaneously collected fluorescence intensity image. For comparison, fluorescence lifetime images of fixed macaque retina were captured using the microscope arm of the same instrument. Wilcoxon rank-sum test was used to test for significance.

Results : AOFLIO of the photoreceptor mosaic revealed significantly different (p = 0.001) mean lifetimes for cones (260 ± 60 ps; n = 731 cones) and rod regions (200 ± 18 ps; 8 locations in 2 macaques) (fig. 1). Some cones were difficult to distinguish in the intensity image but were identifiable in the lifetime image. In contrast, lifetime measurements of fixed macaque rods and cones showed no significant differences (p = 0.75).

Conclusions : AOFLIO using two-photon excitation makes possible measurements of the fluorescence lifetime of intrinsic retinal fluorophores at a cellular scale in the living macaque. AOFLIO revealed differences in fluorescence lifetime between rods and cones in vivo but not in fixed retina, emphasizing the importance of investigating retinal processes in their natural environment. AOFLIO may be a new method for distinguishing cell classes in the retina by their fluorophore composition or environment.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Fig. 1. Two-photon excited fluorescence images of in vivo macaque photoreceptors. Cones can be distinguished by their longer fluorescence lifetime.

Fig. 1. Two-photon excited fluorescence images of in vivo macaque photoreceptors. Cones can be distinguished by their longer fluorescence lifetime.

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