Figure 1. Properties of red-shifted miRFP718nano in comparison with parental miRFP670nano.

(a) Absorbance spectra. (b) Fluorescence excitation spectra (emission at 740 nm). (c, d) Fluorescence emission spectra (excitation at 580 nm for miRFP670nano and 650 nm for miRFP718nano). (e) pH dependencies of NIR fluorescence. Data are presented as mean values ± s.d. for n = 3 transfection experiments. In (a-e) spectroscopic measurements were performed in phosphate-buffered saline (PBS). (f) Photobleaching in live HeLa cells with 665/45 nm excitation filter. Light power density at the back aperture of the objective lens was 8.25 mW cm⁻², resulting in 4.3 W cm⁻² at the focal objective plane. Cells were incubated in a live cell imaging solution. (g) Cellular brightness of miRFP718nano, miRFP670nano and miRFP703 in live mammalian cells. Fluorescence was analyzed 72 h after transfection. The effective brightness of miRFP670nano was assumed to be 100% for each cell type. NIR fluorescence intensity was normalized to the excitation efficiency of each NIR FP by 640 nm laser, and to the emission spectrum of each FP in the emission filter. Gating was performed as shown in Supplementary Fig. 3. (h) Mean fluorescence intensity of HeLa cells transiently transfected with miRFP718nano, miRFP670nano and EGFP before and after 4 h of incubation with a protein synthesis inhibitor cycloheximide (20 μg/ml). Fluorescence was normalized to control conditions. (i) Mean fluorescence intensity of HeLa cells transiently transfected with miRFP718nano, miRFP670nano and EGFP before and after 4 h of incubation with an inhibitor of proteasome-dependent protein degradation bortezomib (10 μM). Fluorescence was normalized to control conditions. In (g, h, i) data are presented as mean values ± s.d. for n = 3 transfection experiments. (j) Mean fluorescence intensity of live HeLa cells transiently transfected with miRFP718nano miRFP670nano and EGFP 48 h and 120 h after transfection normalized to that at 48 h. (k) Number of FP expressing (fluorescent) HeLa cells transiently transfected with parental miRFP670nano, miRFP718nano or EGFP was calculated at 48 h and 120 h after transfection and normalized to that at 48 h. In (j, k) data are presented as mean values ± s.d. for n = 4 transfection experiments.