Time-resolved fluorescent proteins expand fluorescent microscopy in temporal and spectral domains

Area of Science:

• Biophysics
• Cell Biology
• Molecular Imaging

Background:

• Fluorescence microscopy is a key tool in life sciences.
• Steady-state fluorescence intensity is commonly used, but time-resolved (tr) signals, specifically fluorescence lifetime, are less explored.

Purpose of the Study:

• To develop a novel set of time-resolved fluorescent proteins (tr-FPs) with tunable lifetimes.
• To enable advanced imaging techniques by controlling fluorescence lifetime without altering spectral properties.

Main Methods:

• Designed and synthesized a family of tr-FPs with rationally controlled lifetimes across the visible spectrum.
• Utilized temporal-spectral resolved microscopy for simultaneous multi-protein imaging in live cells.
• Applied tr-FPs in multiplexing super-resolution microscopy.

Main Results:

• Developed tr-FPs with a wide range of lifetimes, covering the visible spectrum.
• Achieved simultaneous imaging of 9 different proteins in live cells using temporal-spectral resolved microscopy.
• Enabled concurrent visualization of 4 proteins using lifetime signals in super-resolution microscopy and quantified cellular protein stoichiometry.

Conclusions:

• Introduced tr-FPs as a transformative toolset for biological research.
• Demonstrated the potential of tr-FPs for integrating system complexity and quantitative accuracy.
• Opened new avenues for advanced cellular analysis and understanding biological mechanisms.