Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

Area of Science:

• Biophysics
• Cell Biology
• Molecular Imaging

Background:

• Fluorescent proteins are crucial tools for visualizing molecular interactions in living cells.
• Existing fluorescent proteins often have limitations in brightness, spectral properties, or compatibility for multicolor applications.

Purpose of the Study:

• To report an improved monomeric red fluorescent protein, hmKeima8.5, with enhanced brightness and a large Stokes shift.
• To demonstrate its utility in multiphoton, multicolor imaging and molecular interaction studies in living cells.

Main Methods:

• Development and characterization of the hmKeima8.5 fluorescent protein.
• Utilizing hmKeima8.5 with mTFP1 for multiphoton excitation (MPE) at 850 nm.
• Application of multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS) to study protein-protein interactions.
• Detection of drug-protein interactions using hmKeima8.5 and Coumarin 343 (C343).

Main Results:

• hmKeima8.5 exhibits increased intracellular brightness and a large Stokes shift (∼180 nm).
• The mTFP1/hmKeima8.5 pair allows for well-separated emission peaks upon single MPE wavelength excitation.
• Robust and quantitative detection of homo- and hetero-oligomerization and drug-protein interactions in living cells was achieved.
• Demonstrated a practical toolbox for investigating molecular interactions in the cytoplasm.

Conclusions:

• hmKeima8.5 is a valuable red fluorescent protein for advanced cellular imaging.
• The mTFP1/hmKeima8.5 pair is effective for multiphoton, multicolor applications.
• The developed methods provide a versatile platform for studying molecular interactions in live cells.