Adaptive optics fluorescence lifetime ophthalmoscopy of iGlucoSnFR-TS suggests increased glucose in RPE of rho^-/- compared to healthy mice

Abstract

Purpose : A potential consequence of outer retinal degeneration is an accumulation of glucose in the retinal pigment epithelium (RPE). iGlucoSnFR-TS (GS), a fluorescence lifetime-based sensor for cellular glucose, presents a powerful new tool to study disease mechanisms in the living eye. We developed adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO) to image GS in healthy mice and those with outer retinal degeneration.

Methods : A custom adaptive optics scanning light ophthalmoscope (4-6° square field of view, 25 Hz frame rate, 180 s exposure) was used for two-photon excitation of fluorescence (790 nm, ~55 fs pulse width, 80 MHz repetition rate, 7 mW mean power). We performed AOFLIO of RPE at multiple locations in one healthy C57BL/6J (B6/J) mouse and 3 GS-labeled mice (2 B6/J, 1 rho^-/-) injected subretinally at birth with an adeno-associated viral vector encoding GS driven by the Best1 promoter. The fluorescence lifetime decay histograms were measured using time-correlated single-photon counting (SPC-160, Becker & Hickl GmbH) across emission bandwidth 380-550 nm. A two-component exponential function was fit to each decay curve from which the mean fluorescence lifetime (τ_m) was calculated.

Results : AOFLIO can measure the lifetimes of both intrinsic fluorophores and extrinsic GS at cellular resolution. In GS-injected mice, we observed patches of RPE cells with high and low fluorescence intensity, which we interpret as high (GS+) and low expression. Additionally, some RPE appeared to have no expression (GS-). In GS-injected B6/J mice, there was a significant (p<.0001) difference in τ_m between GS+ (162±16 ps) and GS- (96±16 ps) RPE. τ_m of GS- B6/J RPE was not significantly (p=0.11) different from the uninjected mouse (79±3 ps). Similar trends were observed for τ_m in rho^-/- mice (GS+ 331±61 ps; GS- 194±105 ps). The τ_m of GS+ RPE in rho^-/- mice was significantly longer than in B6/J mice (p<.0001).

Conclusions : The longer lifetimes observed in labeled cells of rho^-/- mice suggest an increase in cellular glucose of RPE compared to the labeled B6/J. However, we have not determined the optical impact on fluorescence lifetime due to blur or disease-related photoreceptor loss. Two-photon AOFLIO will allow longitudinal assessment of fluorescence lifetime markers, such as GS, in vivo to track metabolism at cellular-scale in RPE of healthy and diseased mice.

This is a 2021 ARVO Annual Meeting abstract.