PMC

Fluorescence excitation spectra of the retinal chromophore: comparison with absorption spectra and estimation of the efficiency of energy transfer. (A) Spectrum 1, absorption spectrum of xanthorhodopsin, pH 5.5. Spectrum 2, excitation spectrum for emission at 720 nm measured at magic angle (excitation with vertically polarized light (0°) and emission measured with polarizer set at 54.7°, which eliminates effects from preferential excitation). Excitation bandwidth 4 nm, emission bandwidth 32 nm. Spectrum 3, best fit of the excitation spectrum with retinal and carotenoid components in the absorption spectrum and with an efficiency for energy transfer of 0.49, determined as described in the text. (B) Spectra of the components of the excitation and absorption spectra: (1) carotenoid component in the absorption spectrum after subtraction of the retinal component and nonbound carotenoid; (2) carotenoid component in the excitation spectrum; (3) retinal component in the absorption and excitation spectra. (C) The ratio of the spectra of carotenoid components in the excitation and absorption spectra (spectrum 2 divided by spectrum 1 in Fig. 5 B). This ratio is the quantum efficiency of energy transfer, φ_m, from the carotenoid to the retinal.

Absorption, fluorescence, and excitation spectra of xanthorhodopsin. Effect of retinal removal with hydroxylamine. (A) Absorption spectrum of cell membranes of Salinibacter ruber containing xanthorhodopsin (at pH 5.5) before (spectrum 1) and after (spectrum 2) hydrolysis of the retinal Schiff base with hydroxylamine. The treatment converts the covalently bound retinal to the retinal oxime, whose absorption maximum is shifted from 563 nm to 367 nm, and the sharp carotenoid vibronic bands broaden. (B) Fluorescence emission of xanthorhodopsin, pH 5.5, excitation at 560 nm (spectrum 1) and 520 nm (spectrum 2). Spectrum 3, after hydroxylamine treatment and removal of the retinal oxime, excitation at 560 nm. Spectrum 4, spectrum 1 normalized to the amplitude for 520 nm excitation, by multiplying by 1.7. (C) Comparison of the xanthorhodopsin fluorescence bands at pH 8 (spectrum 1) and pH 5.5 (spectrum 2) with that of bacteriorhodopsin, pH 6 (spectrum 3). Excitation: 560 nm, 8 nm bandwidth. Absorbances of the samples at 560 nm were 0.29, 0.32, and 0.33, respectively. A small contribution from the Raman scattering of water was subtracted. (D) Fluorescence excitation spectra for the emission of xanthorhodopsin, sampled at 720 nm, at pH 8 (spectrum 1) and pH 5.5 (spectrum 2). Excitation beam bandwidth 4 nm.

Model of di-4-ANEPPS voltage sensitivity (A) Vm-dependent spectral shift of excitation spectrum from Vm = rest (solid) to ΔVm = +100 mV (dashed). The black circle shows the isosbestic wavelength at which excitation is the same during depolarization and rest. (B) Difference spectrum taken by subtracting the resting excitation spectrum from the shifted excitation spectrum (top). Normalized power density spectrum for blue and cyan LEDs (bottom). (C) Emission spectrum peak as a function of excitation wavelength. Published peaks (squares) as well as peaks estimated for 450 nm and 505 nm excitation (circles) are shown. (D) Blue and cyan excitation result in two resting emission spectra (solid gray and black lines, respectively). The emission spectra undergo a Vm-dependent shift and amplitude modulation resulting from the Vm-dependent excitation shift (dashed lines). The black circles show the emission isosbestic points. (E) Difference spectra taken by subtracting the resting emission spectra from their respective shifted and amplitude modulated emission spectra. The gray box is the emission passband used in this study. (F) When emitted fluorescence is filtered with the passband shown in (E), upright OAPs result from blue excitation and inverted OAPs result from cyan excitation.

a) An FFT spectrum while the excitation drive is off, the proton is on resonance with the resonator and the Be⁺ ions are off resonance. b) An FFT spectrum while the excitation drive is on, the proton is off resonance with the resonator and the Be⁺ ions are on resonance. c) An FFT spectrum while the excitation drive is on, the proton is on resonance with the resonator and the Be⁺ ions are on resonance. d) An FFT spectrum while the excitation drive is on, the proton is on resonance with the resonator and the Be⁺ ions are off resonance.