Extended Data Fig. 4: Photophysical properties of nLightR2.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a, Simplified drawing of nLightR2 fluorescence photocycle and b, reversible positive and negative photoswitching. c, Experimental design. blue shapes correspond to 488 nm and green to 560 nm light, both at 30 mW/mm2. d, Exemplary images showing positive photoswitching of nLightR2-expressing HEK cells in its apo and NE-bound states (10 µM NE). Color bar represents nLightR2 fluorescence intensity in arbitrary units (a.u.). e, Relative positive photoswitching to ligand response. Bars show the mean +/- SEM of the 488 nm light effect (data are normalized to 10 µM NE response). Positive photoswitching effect apo vs. NE-bound (two-sided paired t test, ***p = 0.0002, n = 4 dishes). Data shown as mean +/- SEM (error bars). f, Positive photoswitching kinetics in apo and NE-bound state. Data presented as fold change from no 488 irradiation. Effect is larger on the NE-bound than the apo state (two-sided unpair t test, p = 7.39e-14, n = 18 trials, from two dishes) g, Mixed photobleaching and negative photoswitching behavior of NE-bound nLightR2 vs. jRGECO1a + Ionomycin. h, Zoom-in view from g to highlight negative photoswitching effect. i, Negative photoswitching: Exemplary NE-bound nLightR2 trace before (top), and after (bottom) photobleaching correction. Image blocks are represented by different colors. Imaging blocks were intercalated with 30 s of no 560 light (recovery phase). j, Normalized maximum to minimum fluorescence per image block after photobleaching correction (solid line, n = 4 different dishes) +/- SEM (error bars). Dashed lines represent the recovery period without 560 nm light.
