Can in vivo two-photon retinal imaging be safely applied in humans?

Abstract

Purpose : In vivo two-photon imaging has the potential to be a powerful tool for functional assessment of normal and diseased retina. However, light safety of the sub-100 fs laser typically used is a major concern and safety standards are not yet established. To test the feasibility of safe two-photon autofluorescence (TPAF) imaging in humans, we examined the effects of exposure to ultrashort pulsed light in macaque.

Methods : The photoreceptor layer of 2 macaques (M1 and M2) was exposed at 8 retinal locations in the near-periphery with a pulsed laser (730 nm, ~55 fs, 80 MHz) on days 1, 8 and 15 of the study. Reflectance videos of photoreceptor structure and TPAF (400-550 nm) videos that assessed the kinetics of the retinoid cycle were simultaneously acquired with a two-photon adaptive optics scanning light ophthalmoscope (AOSLO). Following 15 min of dark adaptation, TPAF was recorded for either 40 s at 0.5 mW or 80 s at 1 mW. Rate constants and relative signal increases were extracted from exponential fits to the time course. Pre- and 2 days post-exposure, conventional fundus photography, fluorescein angiography, fundus autofluorescence imaging and OCT were performed (Heidelberg Spectralis). Several weeks after the last exposure, we imaged single-photon fluorescence from lipofuscin in the RPE cells underlying the exposed locations with an AOSLO.

Results : The only measure that revealed a significant effect due to exposure to pulsed light was infrared autofluorescence intensity which was reduced by 25%. IRAF reduction, presumably due to photobleaching, did not result in a detectable loss of visual function in a previous study (Masella et al., 2014). No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptor and RPE cells appeared normal and cone density did not change. No significant difference was measured before and after exposures in the rate constants (M1: 0.15±0.08/s, p>0.37; M2: 0.22±0.07/s, p>0.08) or relative signal increase (M1: 0.65±0.28, p>0.11; M2: 0.46±0.10, p>0.08) of photoreceptor TPAF.

Conclusions : Apart from IRAF reduction, no other significant changes were observed after repeated TPAF measurements with the minimal exposures required for two-photon imaging in our present instrument. While we cannot exclude the possibility of changes too subtle to detect, this study suggests that safe two-photon retinal imaging in humans is possible.

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