Fig. 1: Mechanistic investigation of reduced photobleaching.
From: Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity
a, Fluorescence images of EGFP immobilized in a PAA gel and illuminated at 470 nm (32 W/cm2, whole field) and at 900 nm (2 kW/cm2, red dashed circle). Scale bars, 20 µm. Representative of n = 4 replicate experiments. b, Photobleaching kinetics of EGFP illuminated solely at 470 nm (blue) or at both 470 nm and 900 nm (red). Values are mean ± s.d. (n = 4 samples). c,d, Dependence of the reduced photobleaching (RP) effect on NIR light intensity I900 (c; I470 = 32 W/cm2) and on visible light intensity I488 (d; I900 = 2 kW/cm2). Values are mean ± s.d. (n = 3 samples). Lines are simulations based on the model shown in f. e, Dependence of the RP effect on the NIR wavelength λNIR. Black line: measurement performed with I470 = 32 W/cm2 and INIR = 20 W/cm2. Red dashed line: EGFP triplet absorption spectrum from ref. 15. mOD stands for 10−3 OD (optical density). f, Photophysical model. Triplet T1, a precursor of bleached states, forms from the bright S1 state under 470-nm illumination. NIR co-illumination promotes T1 into the higher triplet Tn from which the fluorophore returns to S1 by RISC. g, RP effect predicted by the model as a function of I470 (left axis) and I900 (bottom axis). h, RP effect for different green and yellow FPs expressed in E. coli. I470 = 32 W/cm2; I900 = 2 kW/cm2. Values are mean ± s.d. (n = 3 samples).
