Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Fluorescent proteins (FPs) engineering aims to extend fluorescence to longer wavelengths, particularly the near-infrared (NIR) spectrum.
• Existing red-shifted FPs often require orange lasers not standard in flow cytometry.
• Bacterial phytochrome-based NIR fluorescent proteins (iRFPs) offer excitation with common red and NIR lasers.

Purpose of the Study:

• To introduce and characterize a series of novel NIR fluorescent proteins (iRFPs).
• To demonstrate the utility of iRFPs in combination with advanced flow cytometry for biological applications.

Main Methods:

• Development of five iRFP variants (iRFP670, iRFP682, iRFP702, iRFP713, iRFP720) with distinct excitation and emission profiles.
• Utilized standard red and NIR lasers for excitation.
• Employed spectral cytometry with spectral deconvolution for simultaneous multi-FP analysis.
• Applied iRFPs for deep tissue imaging and cell sorting in live animals.

Main Results:

• iRFP variants showed maximal excitation across visible red (590-610 nm) and NIR (685-730 nm) laser ranges.
• Simultaneous detection of multiple iRFPs was achieved using red and NIR lasers.
• Spectral cytometry enabled unmixed analysis of all five iRFPs.
• Demonstrated feasibility of deep tissue imaging and cell subpopulation sorting ex vivo.

Conclusions:

• The iRFP series provides versatile NIR fluorescent probes compatible with standard and advanced flow cytometry.
• This technology facilitates in vivo imaging and precise ex vivo cell analysis.
• Combines spectral cytometry and iRFPs for high-content analysis of biological systems.