(a) Absorbance in arbitrary units (a.u.) with absorbance at 280 nm set to 100%. (b) Fluorescence excitation and emission spectra normalized to 100% for both proteins. (c) Fitted curves of the maturation kinetics in hours (h) in bacteria at 37°C. (d) Equilibrium pH dependence of fluorescence. (e and f) FACS dot-plots representing NIR fluorescence of iRFP and IFP1.4 (x axis) and green fluorescence from co-expressed EGFP (y axis) of transiently transfected HeLa cells not treated (e) or treated (f) with 25 μM of BV for 2 hours before analysis. A 676 nm laser line for excitation and a 700 nm long pass filter to collect emission from iRFP and IFP1.4 were used. (g) Mean NIR fluorescence intensity of the double-positive cells from (a) and (b) normalized to transfection efficiency (EGFP signal), absorbance of the respective protein at 676 nm, and overlap of the fluorescence spectrum of the respective protein with the transmission of the emission filter. (h) Fluorescent images of the transiently transfected HeLa cells with and without addition of 25 μM BV for 2 hours before imaging. Scale bar is 20 μm. (i) Photobleaching in HeLa cells. The curves were normalized to absorbance spectra and extinction coefficients of the proteins (calculated based on BV absorbance), spectrum of an arc lamp and transmission of a photobleaching filter. Plot represents the data obtained with endogenous BV but both proteins demonstrated no change in photostability after addition of exogenous BV. (j) Degradation of the proteins in HEK293 cells after treatment with 1 mM puromycin. Cells were incubated with 25 μM BV to achieve a higher fluorescent signal. Protein concentration was assessed by measuring fluorescence intensity of crude cell lysates. (k) BV binding to iRFP and IFP1.4 proteins in HeLa cells. Cells were incubated with the respective amounts of BV during 2 hours before harvesting on the second day after adenovirus infection. Fluorescence intensity was measured in crude cell lysates and normalized to 100%. Lines are fitted based on the Scatchard equation. (l) Protein expression in HeLa cells 48 hours after adenovirus infection. Data for the cells without exogenous BV, with 25 μM of BV added 2 hours and 42 hours before the analysis are shown. Fluorescence intensities were normalized to the total cell number, excitation wavelength, emission collection bandwidth, and protein molecular brightness to represent the iRFP or IFP1.4 concentrations.

(a) Overlay of representative light and fluorescent images of iRFP or IFP1.4 adenovirus infected mice with and without injection of 250 nmol BV. A non-infected control mouse is shown on the right. The fluorescence images were acquired using IVIS Spectrum instrument equipped with 675/30 nm excitation and 720/20 nm emission filters. The color bar indicates the fluorescence radiant efficiency, multiplied by 10⁹. (b) Near infra-red fluorescence total radiant efficiency of the liver areas of the iRFP and IFP1.4 expressing mice in (a), normalized to the bandwidth of the excitation and emission filters. (c) Time course of the NIR fluorescence total radiant efficiency of the liver areas of the iRFP and IFP1.4 expressing mice in (a) after BV injection. (d) Overlay of the photograph and fluorescent image of the isolated livers from the BV-injected infected and non-infected (control) mice. (e) Time course of the NIR fluorescence total radiant efficiency of the liver areas of the mice not being injected with BV. The fluorescence signals were normalized to the bandwidth of the excitation and emission filters.

Samples consisting of the equal amounts of the purified proteins of the same concentration were placed inside of the phantom mouse in the bores located 7.0 mm (a) or 18.1 mm (c) deep from the mouse surface. Each protein sample was imaged using epifluorescence mode in several wavelength channels. A signal-to-background ratio in each channel was calculated as (ROI1 - ROI2) / ROI2, where ROI1 or ROI2 were total radiant efficiencies of the respective areas with and without the protein sample. Images for the highest signal-to-background ratio for each protein are shown. The color bar indicates the fluorescence radiant efficiency, multiplied by 10⁸. Panels (b) and (d) represent the highest signal-to-background ratio values, calculated for the respective images in (a) and (c).