Near-infrared fluorescent proteins

Abstract

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

Figure 1

Cytotoxicity and spectral characteristics. (a) Katushka and Katushka-9-5 cytotoxicity in microinjected X. laevis embryos. Percentages of surviving embryos by the tailbud and tadpole stages are shown (mans ± s.d.; n = 5 experiments each performed on 60 embryos). Significance was analyzed by t-test. (b) Flow cytometry analysis of HeLa cells 48 h after transient transfection with vectors encoding E2-Crimson, mNeptune, Neptune and eqFP650. Fluoresence brightness was normalized to the relative efficiency of excitation by the 488 nm laser line used. Emission was collected at 660–700 nm. Means ± s.d. are shown (n ≥ 3 transfection experiments). (c) Normalized fluorescence excitation (solid lines) and emission (dashed lines) spectra for eqFP650 and eqFP670. (d) Normalized absorption spectra for eqFP650 and eqFP670. (e,f). Normalized photobleaching curves for eqFP650, eqFP670, mNeptune and E2-Crimson using widefield fluorescence microscopy under metal halide illumination (e) or laser scanning confocal microscopy (f). Error bars, s.d. (n = 4 experiments).

Figure 2

Whole-mouse imaging with IVIS Spectrum system (Caliper). (a) Representative fluorescence reflectance images (excitation filter, 605/30 nm and emission filter, 660/20 nm) of mice injected intramuscularly with HEK 293T cells expressing E2-Crimson, mNeptune or eqFP650. Asterisk denotes background fluorescence in mice injected with E2-Crimson cells. Scale bar shows pseudocolor scale for fluorescence. (b,c) Fluorescence efficiency from cell implants imaged with 570/30 nm (b) or 605/30 nm (c) excitation filters and a range of emission filters, normalized to photons from firefly luciferase to control for transfection efficiency and numbers of implanted cells. Means ± s.e.m. are shown (n = 6–10 per point). *P < 0.05 (t-test) for eqFP650 relative to other proteins.

Figure 3

Whole-mouse infrared imaging with the Biospec system. Infrared fluorescence of subcutaneous injections of equal amounts of protein samples into a living mouse after normalization using the extinction coefficient at the absorption maximum wavelength of each protein. Excitation at 635 nm with photodiodes was used, and emission was measured over the integrated 700–900 nm range. Scale bar, 1 cm.