Two-Photon Stimulation Efficiency with ns-Pulsed infrared Lasers

Abstract

Purpose : Two-photon ophthalmoscopy (2PO) is a promising technique for noninvasive functional imaging of the retina where endogenous fluorophores are excited via two-photon (2P) absorption with near-infrared pulsed lasers. When projected onto the retina, these lasers also produce 2P vision. Here we performed color matching experiments with a ns-pulsed laser to estimate the 2P stimulation of photoreceptors for typical exposures during 2PO with a fs-laser.

Methods : A retinal projection display was used for color matching experiments. Two 4x4 arcmin squares were projected side-by-side onto the fovea of 5 subjects. One square was produced by a visible-light OLED display to cause standard single-photon (1P) perception, whereas the other square was produced by a scanned ~1 ns-pulse laser in the near-infrared with a repetition rate of 3 or 15 kHz to produce 2P perception. Power settings of 10, 20 and 30 µW at the cornea at four wavelength bands of the pulsed laser towards the higher end of the excitation spectrum employed in 2PO (850, 880, 900, 920 nm; FWHM: 10-40 nm) were selected. Subjects matched the perceived color (hue, saturation, brightness) of the display to that of the laser. The color percepts measured were compared to the theoretical 2P benefit.

Results : All color percepts were located at the purple line. As settings changed to benefit 2P absorption (increased laser power/ reduced repetition rate), a blue shift in perceived color was measured. Increasing the laser power by a factor of 3 (9x 2P efficiency), resulted in a relative gain of 13% (±6) of blue vs red. Decreasing the repetition rate by a factor of 5 (5x 2P efficiency) showed a 11% (±6) increase. Combining both effects (45x 2P efficiency) resulted in a shift of 23% (±8) towards blue. Extrapolating data for the most efficient settings shows that 2P perception might already be visible at 780 nm with this setup. For 2PO at comparable wavelengths, 2P absorption should increase 10-fold when using a fs-lasers and even up to 10^5-fold when using AOSLO devices.

Conclusions : S cones can be stimulated substantially with a near infrared ns-pulsed laser. 2P stimulation of photoreceptors during 2PO can be expected to be much more efficient and might cause intense stimuli. This effect must be considered to define appropriate stimulation paradigms for functional imaging.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.