Guide to red fluorescent proteins and biosensors for flow cytometry

Abstract

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed.

Fig. 1

The fluorescence excitation and emission spectra of LSSmKate2 (solid lines) are shown. The wavelengths of the laser lines, 405, 458, and 488 nm, are shown by vertical lines with indication of excitation efficiency. The emission that passes through the 550 nm long pass (red dash line) and 640/50 nm band pass (blue dash line) filters is shown as cross-hatched region with square S₁ and S₂, respectively. The fluorescence intensity collected through the filters is proportional to the S₁ and S₂ squares. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

Fig. 2

The excitation (A) and emission (B) spectral profiles of the LSS red (LSSmKate2), orange (mOrange), red (mKate2), and far-red (TagRFP657) FPs are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

Fig. 3

(A) A cell expressing a fluorescent timer gradually changes fluorescence from blue to red with time. The respective time changes of the excitation and emission spectra for the blue and red forms of the fluorescent timer are shown. (B) A flow cytometry plot shows the same population of cells, which expresses the blue-to-red fluorescent timer, analyzed at different times after its expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)