Towards functional measurements of vision in the living macaque retina using two-photon fluorescence imaging

Abstract

Vision originates from the absorption of light by opsins in the retinal photoreceptors, requiring the regeneration of molecules through the visual cycle. Imaging in vivo the molecules involved in this process, such as retinol or NADH, will provide insight into the changes occurring with photon absorption and pigment regeneration in normal and diseased retina. In vivo two-photon fluorescence imaging through the pupil of the living macaque eye allows us to visualize fluorescent molecules that cannot be excited in vivo with single photon fluorescence due to the transmission of the ocular optics. The associated challenges of the low numerical aperture, the poor optical quality of the eye, the required high light levels, and eye motion were overcome by using an adaptive optics scanning laser ophthalmoscope (AOSLO). Based on the eye motion observed with simultaneously acquired high signal-to-noise ratio (SNR) near-infrared reflectance images of the cone mosaic, 16,500 low SNR two-photon images (12 minute acquisition) were registered and averaged to produce fluorescence images of the inner segments of the cone mosaic. In response to photopigment bleaching, the in vivo two-photon signal initially increases. This may be indicative of a functional increase in the concentration of retinol, NADH, FAD or an as yet unknown fluorophore.

Acknowledgments

Polgenix, Inc.; National Institute for Health, Bethesda, Maryland Grants P30-EY001319, R01-EY004367, BRP-EY014375, T32-EY007125, R01-EY009339, SBIR-EY020715; This work made use of STC shared experimental facilities supported by the NSF under Agreement No. AST-9876783; Research to Prevent Blindness; Alfredo Dubra-Suarez, Ph.D., holds a Career Award at the Scientific Interface from the Burroughs Welcome Fund; Klaus Tschira Foundation Heidelberg, Germany; European Life Scientist Organization.