Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy

Abstract

Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or near-red fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.

Figure 1

TagRFP657 properties. (A) Excitation (dashed line) and emission (solid line) spectra. (B) Equilibrium pH dependence for TagRFP657 (solid line) and mKate2 (dashed line). (C) Photobleaching kinetics of TagRFP657 (solid line) and mKate2 (dashed line) at 47.1 mW/cm². (D) Photobleaching half-times for TagRFP657 (open columns) and mKate2 (dashed columns) at different light intensities. Intensities were measured at the back-focal plane of a 100× oil objective. Photobleaching data were normalized to spectral output of the lamp, transmission profiles of the filter and dichroic mirror, and FP absorbance spectra. (E) Maturation kinetics of TagRFP657 (solid line) and mKate2 (dashed line) at 37°C. (F) Residual green fluorescence of TagRFP657 (solid line), mKate2 (dashed line), and mNeptune (long dashed line) when excited at 460 nm. Relative areas under the emission spectra in the 470–570-nm range were 100% (mKate2), 83% (mNeptune), and 8% (TagRFP657).

Figure 2

Simultaneous detection of several red FPs expressed in live mammalian cells. (A) Using flow cytometry: sorting of a mixture of TagRFP657, mKate, and TagRFP expressing as well as nontransfected cells. (B) Using wide-field fluorescence microscopy: coexpression of histone-2B (H2B) tagged TagRFP657 and TurboRFP targeted to mitochondria. (Note that all colors used in this figure are pseudocolors.)

Figure 3

Imaging of EB3-TagRFP657 fusion in mammalian cells using (A) standard confocal and (B) superresolution STED microscopy. Magnified views of the square areas in panels A and B are shown for confocal (C) and STED (D) images. Profiles displayed in panels E and F were generated from identical regions of interest shown in panels C and D. Uncolored arrows indicate details only resolvable in STED (D). The profiles were measured along the longer side of the regions of interest, averaging over the shorter side. Solid red lines in panels E and F indicate the Gaussian (confocal) and Lorentzian (STED) fits used to determine the displayed full width at half-maximum values (illustrated with red arrows). Image color-maps were scaled to minimum and maximum values in panels A and B.