Fig. 2. Spectral properties of purified rhodopsin domains from the R. globosum RGCs.

a Normalized absorption spectra of the purified recombinant rhodopsin domains from RGC1, RGC2, and NeoR (red and UV-form); picture of the purified NeoR sample, inset. b Reversible bleaching of NeoR₆₉₀ with 680 nm light. c Reversible bleaching of NeoR₃₆₇ with 375 nm light. d Kinetic traces of NeoR bleaching with 680 nm light and subsequent recovery by 375 nm illumination. e Absorption spectra of purified NeoR expressed in media supplemented with A1-retinal or 3,4-dihydro-retinal (A2), as compared to those from biliverdin (BV)-containing iRFP⁵⁰. f Fluorescence spectra of NeoR₆₉₀: excitation (dots) overlaid with the absorbance (red) and emission (green). g Evolution-associated difference spectra (EADS) of fs–ns transient absorption spectra derived from pump-probe experiments, time trace at 708 nm, inset. h Representative confocal images of NeoR fluorescence (red) and the C-terminal eGFP tag (green) in ND7/23 cells. NeoR is reversibly bleached by illumination with 640- and 405 nm light. The reversible bleaching of NeoR fluorescence in single cells was repeated in three independent experiments using five fields of view each. Scale bar, 100 µm.