Effect of laser-beam diameter on the visibility of two-photon stimuli.

Two-photon vision enables perception of near-infrared light through nonlinear absorption in retinal photopigments, showing a quadratic dependency on laser intensity. We measured two- and one-photon visual thresholds for varying beam diameters (with effective NA = 0.02-0.09) and defocus levels under dark- and light-adapted conditions. Two-photon visual thresholds varied significantly with beam diameter for stimuli focused on the retina and showed greater sensitivity to defocus than one-photon thresholds, especially at larger NA. While defocus blurs visible stimuli, in two-photon vision, it mainly decreases retinal photon density, reducing brightness without image degradation. These results emphasize, for the first time, to the best of our knowledge, the importance of beam diameter, focus quality, and accurate defocus correction in optimizing two-photon--based visual displays.