Human infrared vision is triggered by two-photon chromophore isomerization

Abstract

Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400- to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experiments with rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by two-photon isomerization of visual pigments.

Keywords: multiscale modeling; rhodopsin; transretinal electrophysiology; two-photon absorption; visual pigment.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Color perception caused by IR laser radiation of the human eye in vivo. (A) Experimental optical setup. AM, adjustable monochromator. (B) Plot showing the results of matching frequency-doubled wavelength perception with the true visible radiation illuminating the retina for 30 healthy volunteers. (C) Reproducibility of results obtained from two healthy volunteers (subjects 1 and 2) for different beam powers at 1,040 nm. Points plotted on the graph represent means of matching wavelengths performed by all examined subjects. Error bars in B represent SDs of adjusted wavelengths. Error bars in C are SDs of the means.

Fig. 2.

A nonlinear process contributes to spectral sensitivity of human visual perception. Spectral sensitivity data obtained with 0.3- to 0.6-ns (long) laser pulses are shown in black and the spectral sensitivity data obtained with 0.5- to 1.25-ps (short) laser pulses are in red. Dashed vertical lines separate regions of wavelengths with different color perception. Measurements were taken on two healthy dark-adapted subjects (S1 and S2). Each point represents the average of 10 measurements.

Fig. 3.

WT mouse rods respond to IR light with much greater sensitivity than predicted by 1PO excitation theory. (A) Rod photoresponses to 10-ms stimulation with varying light energy at 730 (Left) and 1,000 nm (Right). (B) Amplitude data from responses in A are shown as a function of flash energy in photons per micrometer squared. Data were fitted with Eq. 2, with n = 1.4 and Q1/2 = 3.1 × 107 photons/μm2 (730 nm) and with n = 2.4 and Q1/2 = 4.6 × 108 photons/μm2 (1,000 nm). (C) Average (four mice) rod sensitivity (S = 1/Q1/2) normalized to the sensitivity at 730 nm (black square) plotted as a function of wavelength (λ) 748 nm (triangle), 768 nm (star), 800 nm (open diamond), 900 nm (open circle), and 1,000 nm (open square or red circle, see below). Small insets show the normalized logarithmic amplitude data from one representative retina [x scale 1 log10(Q) and y scale 0.5 log(R/Rmax)]. Eq. 2 was fitted to each dataset with Q1/2 (730 nm) = 1.6 × 108 photons/μm2 and Q1/2 (800 nm) = 9.4 × 109 photons/μm2. The response−energy function became steeper for 900- and 1,000-nm stimulation and could be described by an exponential factor n > 1 (Methods). Assuming the maximum amplitude to be the same as at 730 nm yielded n = 1.4 for this particular retina with Q1/2 of 4.9 × 109 photons/μm2 (900 nm) and 3.5 × 109 photons/μm2 (1,000 nm, no inset). The average sensitivity for two retinas where saturation became evident (A and B) at 1,000 nm is shown as a red circle. This sensitivity is higher because the maximum amplitude at 1,000 nm was smaller than that at 730 nm. Setting the maximum in this way also yielded a steeper response–energy function as shown in the Inset and B (red). (D) Effect of pulsed laser light dispersion on rod sensitivity at 730 (Left) and 1,000 nm (Right). Black traces show responses to 6.6 × 107 photons/μm2 (730 nm) and 1.6 × 109 photons/μm2 (1,000 nm) flashes without dispersion compensation (DC). Red traces show the corresponding responses with DC, providing evidence that rod sensitivity is significantly affected by DC with 1,000-nm but not with 730-nm laser light stimulation.

Fig. 4.

IR isomerization of native and model visual pigments. (A) Bleaching of detergent-solubilized bovine rhodopsin with 1,000-nm light. (Left) Structure of rhodopsin is shown. (Center) Absorption spectra from detergent-solibilized bovine rhodopsin are presented. The spectrum from the control unbleached sample is shown in black and the spectrum after bleaching with 1,000-nm laser light is in red; the indigo arrow indicates the loss in 500-nm absorbance. (Right) Absorption spectra are shown immediately after adding an excess of 11-cis-retinal (red) and after overnight incubation on ice (black). The indigo arrow indicates the recovery of 500-nm absorbance. (B) Bleaching of a rhodopsin crystal with 1,000-nm light. (Left) Image obtained with visible light by using a sectioning microscope immediately after IR bleaching. The yellow rectangle outlines the region that was bleached with 1,000-nm light and the red color of the unbleached lower portion of the crystal is clearly visible. (Right) Picture obtained after bleaching the entire crystal with white light. (C) Chemical formulas of the protonated Schiff base. (Upper) 11-cis-propylamine Schiff base. (Lower) All-trans-propylamine Schiff base. (D) The protonated 11-cis-retinal-propylamine Schiff base was isomerized by intense IR light at 880 nm. Shown are HPLC chromatograms of Schiff bases after conversion into their oximes. (Left) Unbleached control. (Right) Sample bleached with 880-nm light. Isomers indicated in each chromatogram are as follows: 11-cis, 11-cis-retinal oxime; 13-cis, 13-cis-retinal oxime; 9-cis, 9-cis-retinal oxime; all-trans, all-trans-retinal oxime.

Fig. 5.

Rhodopsin 2PO cross sections from hybrid QM/MM calculations. Computed 2PO cross section from quadratic response and TD-DFT at the CAM-B3LYP(66) level of theory (36). The thin dashed lines represent contributions from single thermally equilibrated configurations sampled during QM/MM dynamics. (Inset) QM region defined in the calculations (represented in thick licorice), comprising the protonated Schiff base formed by condensation of 11-cis-retinal with the Lys296 side-chain.