Safety assessment in macaques of light exposures for functional two-photon ophthalmoscopy in humans

Abstract

Two-photon ophthalmoscopy has potential for in vivo assessment of function 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 well established. To test the feasibility of safe in vivo two-photon excitation fluorescence (TPEF) imaging of photoreceptors in humans, we examined the effects of ultrashort pulsed light and the required light levels with a variety of clinical and high resolution imaging methods in macaques. The only measure that revealed a significant effect due to exposure to pulsed light within existing safety standards was infrared autofluorescence (IRAF) intensity. No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptors and retinal pigment epithelium appeared normal in adaptive optics images. No effect of repeated exposures on TPEF time course was detected, suggesting that visual cycle function was maintained. If IRAF reduction is hazardous, it is the only hurdle to applying two-photon retinal imaging in humans. To date, no harmful effects of IRAF reduction have been detected.

Two-photon imaging of the mammalian retina with ultrafast pulsing laser

“…We calculated the maximum permissible average light power per single pulse limit and obtained a value of 680 mW (72), well above the average power used in this study. Furthermore, our pulse peak power, approximately 588 W, assuming a Gaussian pulse and 1 mW of average power, was well below the critical power thresholds for self-focusing (78,79) and plasma formation calculated for 20-fs pulses: 1.5 MW and 159 kW, respectively (76). Therefore, the work reported here provides a foundation for obtaining informative 2-photon microscopy images with laser exposures below damage thresholds per current safety standards.…”

“…Previously, 2-photon images of the retina were not achieved within the safety limits prescribed by the American National Standard for Safe Use of Lasers, ANSI Z136.1 -2014. Moreover, potentially deleterious changes occurred to the retina following exposure to pulsed IR light at just 75% of light levels permissible by current safety standards (76). In our present study, informative images of mouse RPE in vivo were obtained in 50 seconds with 1 mW of average power.…”

Cellular-scale evaluation of induced photoreceptor degeneration in the living primate eye

“…Furthermore, although TPEF imaging with AOSLO has not yet been implemented in humans, it has potential to be an objective measure to inform the functional status of photoreceptors in damaged or degenerating retina [38,39,45]. Since the aforementioned diseases exhibit very similar phenotypes to the macaque model used here (i.e.…”

“…Non-confocal imaging has successfully been employed to visualize photoreceptors in both healthy and diseased retinas [7,12,19,20]. Intrinsic twophoton imaging has provided contrast in both inner and outer segments of photoreceptors [12,38] and has also successfully interrogated photoreceptor function [38,39,45]. Therefore, the combination of these imaging methods can be particularly useful when applied in cases of photoreceptor disruption, in which it may not be apparent from confocal reflectance AOSLO imaging and/or OCT whether photoreceptor structure is intact and the cells are functioning.…”

“…Using MATLAB, the TPEF time course of the segmented data was fit with exponential functions of the form / the fitting parameter τ is the time constant of TPEF increase and the fitting parameter ΔF F is the relative increase in TPEF[45].…”