Retinal safety evaluation of two-photon laser scanning in rats

Abstract

Safe use of retinal imaging with two-photon excitation in human eyes is crucial, as the effects of ultrashort pulsed lasers on the retina are relatively unknown. At the time of the study, the laser safety standards were inadequate due to the lack of biological data. This article addresses the feasibility of two-photon retinal imaging with respect to laser safety. In this study, rat retinas were evaluated at various laser exposure levels and with different laser parameters to determine the effects of laser-induced optical damage. The results were experimentally verified using confocal reflectance imaging, two-photon fluorescein angiography, immunohistochemistry, and correlated to the IEC 60825-1 laser safety standard.

Fig. 1

Scanning laser beam exposure for 300 seconds with a 30° scan angle at 160 mW laser power in brown Norway rats (a-c) and albino rats (d-f). The confocal reflectance image (a, d) of the laser-exposed retina. Early phase (b, e - 5 to 15 seconds after injection) and late phase (c, f - 7 to 10 minutes after injection) of two-photon fluorescein angiography of the exposed retina

Fig. 2

Fluorescence microscopic (TUNEL assay) images of brown Norway and albino retinal sections exposed to the scanning laser beam. The fluorescence image of the control (a, 20 × ) and the experimental eye (b, 10 × ) of the brown Norway rat retina; and the control (c, 10 × ) and the experimental eye (d, 20 × ) of the albino rat retina. The images shown here are the merged images of DAPI and green fluorescence and cropped for better visualization of the retinal cells. GCL - ganglion cell layer; INL - inner nuclear layer; ONL - outer nuclear layer; RPE - retinal pigment epithelium

Fig. 3

The stationary laser beam exposure in the brown Norway rat at 13 mW laser power. (a) confocal reflectance image before exposure, (b) confocal reflectance image after exposure to the laser at 13 mW for 60 seconds (arrow 1), 100 seconds (arrow 2), 300 seconds (arrow 3) and 600 seconds (arrow 4). The early phase (c) and late phase (d) two-photon fluorescein angiography, the hyperfluorescence (d, arrow) seen in the laser-exposed area. (e) Confocal reflectance image of the laser-exposed retina on day 4. Early phase (f) two-photon fluorescein angiography of the exposed retina on day 4

Fig. 4

Light microscopic and fluorescence images of the retinal sections of the brown Norway rat exposed to a stationary laser beam at 13 mW laser power. H&E stained retinal sections of the exposed retina at different sections from the periphery to the laser- exposed area. Retinal section (a, 4 × ) at the periphery; retinal section (b, 4 × ) approximately 50 microns from the periphery towards the optic nerve; and the retinal section of the exposed retina (c, 4 × ) close to the optic nerve. Close-up view of the retinal light-induced damages (d, e, f (from a, b, c)) acquired using a 40 × objective lens. Fluorescence microscopic images of retinal sections (g, h, i of d, e, f) acquired using a 20 × objective lens

Fig. 5

Stationary laser beam exposure in brown Norway rat at 80 mW laser power. (a) Confocal image of the retina immediately after laser exposure. Early-phase (b) and late-phase (c) two-photon fluorescein angiography. H&E stained retinal sections (d, e) from the exposed area (black box). (f) Fluorescence image of the exposed area (yellow box)

Fig. 6

Stationary laser beam exposure in the albino rat at 160 mW laser power. Confocal reflectance image (a) before laser exposure, (b) confocal reflectance image after laser exposure, (c) the early phase and (d) late phase two-photon fluorescein angiography. The hyperfluorescence (arrow) noticed in the laser-exposed area. (e) Confocal reflectance and early phase (f) two-photon fluorescein angiography of the exposed retina on day 4.

Fig. 7

The fluorescence image of the control (a) and experimental (b) albino rat’s retinal sections exposed to a stationary laser beam for 300 seconds at 160 mW laser power.

Fig. 8

The confocal reflectance image (a) of the brown Norway retina exposed to the stationary laser beam of 13 mW for 300 seconds. (b) Confocal reflectance image on day 4 after laser exposure. The fluorescence image (c, 10 × ) of the exposed retina.

Fig. 9

Fluorescence images of the control (a) and experimental (b) retina of brown Norway rat exposed to the two-photon scanning laser beam. The positive control test on the experimental eye (c). The bright green fluorescent cells are the positive (apoptosis) cells, which is evident in the positive control retinal sections. Blue fluorescence is the DAPI nuclei staining, and the green fluorescence is the TUNEL-positive nuclei staining