Near-infrared fluorescent proteins for multicolor in vivo imaging

Abstract

Near-infrared fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-infrared FPs--iRFP670, iRFP682, iRFP702 and iRFP720--from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with standard approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.

Figure 1. Characterization of NIR FPs

(a,b) Normalized excitation (a) and emission (b) spectra of different iRFPs. (c) Normalized full absorbance spectra of iRFPs. (d) Schematic representation of directed molecular evolution that led to iRFPs with distinct spectral properties. Development of previously reported iRFP713 is shown in a dash line. (e) Brightness of HeLa cells transiently transfected with iRFPs, normalized to the value for iRFP713-expressing cells. For each type of cells, the mean NIR fluorescence intensity of cellular population was normalized to FP transfection efficiency (co-transfection with EGFP), excitation efficiency of each FP with the 635 nm laser, and fluorescence signal of each FP in the emission filter Error bars, s.d. (n = 3 transfection experiments). (f) Comparison of iRFPs with GFP-like far-red FPs as fluorescent probes in deep-tissue imaging. Equal amounts of purified FPs (upper row, FP) or buffer with no FPs (lower row, no FP) were imaged in epifluorescent mode inside a mouse phantom at 7.0 mm depth using several filter channels. Total radiant efficiencies of the indicated area with the FP sample (ROI1) and without (ROI2) were measured and signal-to-background ratios (ROI1-ROI2)/ROI2 were calculated for each channel. Images with the highest ratio among channels are shown for each FP. The color bar indicates the total fluorescence radiant efficiency ([photon/s/cm²/steradian]/[μW/cm²]). (g) Quantification of the signal-to-background ratios for the images shown in (f). (h,i) Same as in (f,g) with protein samples located at 18.1 mm depth. Error bars, s.d. (n = 4 (g,i)).

Figure 2. Multicolor imaging in vivo and in cells

(a-f) Two-color imaging. (a) Spectra of iRFP670 and iRFP720 were overlaid with the profiles of filter channels optimal for two-color imaging: 640/30 nm excitation and 680/20 nm emission, and 710/30 nm excitation and 760/20 nm emission. (b) Representative images of a living mouse with two co-injected tumors expressing iRFP670 (left tumor) and iRFP720 (right tumor). The images in different channels and the overlay of two optimal channels were obtained on day 24 after cell injections. (c) Representative images of a living mouse with a tumor expressing iRFP670 (lower left) and liver expressing iRFP713 (upper right). The images in different channels and the overlay of two optimal channels were obtained on day 29 after cell injection and day 6 after adenovirus infection. (c,d) The color bars indicate the fluorescent radiant efficiency ([photon/s/cm²/steradian]/[μW/cm²]). (d) Spectra of iRFP670 were overlaid with the profiles of the filter channels used in microscopy: 605/40 nm excitation and 667/30 nm emission for iRFP670, and 682/12 nm excitation and 721/42 nm emission for iRFP720. (e,f) Two-color microscopy of transiently transfected HeLa cells co-expressing iRFP670 and iRFP720 in nucleus and mitochondria. The overlays of two pseudocolor images, corresponding to iRFP670 channel (in green, left column) and iRFP720 channel (in magenta, middle column) are shown. Scale bar, 10 μm. (g-i) Multicolor spectral imaging. (g) Flow cytometry analysis of MTLn3 cells stably expressing iRFP670, iRFP682, iRFP702, and iRFP720. 10,000 events of each cell type were collected. A 635 nm excitation laser and a combination of two indicated emission filters were used. (h) Confocal microscopy with spectral detection and linear unmixing of four types of MTLn3 cells shown in (g). Emission spectra were detected from 660 nm to 790 nm with an excitation by 633 nm laser. Unmixed channels and the overlay are shown in pseudocolors. Scale bar, 20 μm. (i) Separate detection of five types of tumors expressing iRFPs in living mice. Mice were injected with two types of MTLn3 cells each, from left to right in each image: iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, and iRFP670. Images were acquired in 19 spectral channels on day 12 after cell injections, and unmixed using reference spectra. Representative images of mice in unmixed channels and the composite pseudocolored image are shown. The color bar indicates the fluorescent radiant efficiency ([photon/s/cm²/steradian]/[μW/cm²]).