Fig. 2
Characterization of miRFP670nano. a Fluorescence excitation and emission spectra of miRFP670nano. b Size-exclusion chromatography of miRFP670nano at concentration 10 mg ml−1 and indicated molecular weight standards. miRFP670nano with polyhistidine tag and linker runs as a monomer with the apparent molecular weight of 18.8 kDa. c pH dependencies of NIR fluorescence for miRFP670nano and miRFP670. d Kinetics of miRFP670nano and miRFP670 maturation. Time “0” corresponds to the beginning of the 1-h-long pulse-chase induction of the protein expression in bacteria. e Effective (cellular) brightness of miRFP670nano, miRFP703, and miRFP670 in mammalian cells. Live HeLa, U87, U-2 OS, PC6-3, and NIH3T3 cells were transiently transfected with miRFP670nano, miRFP703, or miRFP670. Fluorescence was analyzed by flow cytometry 72 h after transfection. NIR fluorescence intensity was normalized to that of co-transfected EGFP (to account for differences in transfection efficiency), to excitation efficiency of each NIR FP by 640 nm laser, and to emission spectrum of each FP in the emission filter. Effective brightness of miRFP670 was assumed to 100% for each cell line. Error bars, s.d. (n = 3; transfection experiments). f Photobleaching of miRFP670nano and miRFP670 in live HeLa cells. g Mean fluorescence intensity of HeLa cells transiently transfected with miRFP670nano, miRFP703, miRFP670, and EGFP before and after 4 h of incubation with 20 µg ml−1 cycloheximide. Error bars, s.d. (n = 5; transfection experiments). h Mean fluorescence intensity of HeLa cells transiently transfected with miRFP670nano, miRFP703, miRFP670, and EGFP before and after 4 h of incubation with 10 µM bortezomib. Error bars, s.d. (n = 5; transfection experiments). i Tolerance of miRFP670nano to fixation in paraformaldehyde. HeLa cells transfected with miRFP670nano, miRFP670, and miRFP703 were incubated with 4% paraformaldehyde for 10–60 min. The fluorescence of cells treated with paraformaldehyde was normalized to fluorescence of non-fixed cells. Error bars, s.d. (n = 3; transfection experiments)
