A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging.

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Fluorescence Lifetime Imaging Microscopy (FLIM) is crucial for studying cellular processes.
• Existing FLIM techniques often face limitations in acquisition speed, hindering real-time cellular dynamics observation.

Purpose of the Study:

• To develop a novel, high-speed, diffraction-limited multiphoton microscope for FLIM.
• To achieve sub-50 picosecond temporal resolution and significantly enhance acquisition speed.

Main Methods:

• A massively parallel, fully addressable, time-resolved multi-beam multiphoton microscope was engineered.
• The system achieved a 64x improvement in acquisition speed compared to single-beam laser scanning FLIM.
• Demonstrated FLIM acquisition in live cells within 500 milliseconds.

Main Results:

• Successfully imaged live cells expressing green fluorescent protein with high temporal and spatial resolution.
• Observed time-dependent Förster Resonance Energy Transfer (FRET) between epidermal growth factor receptor (EGFR) and Grb2.
• Visualized ligand-dependent association and endosomal accumulation of HER2-HER3 receptor tyrosine kinases.

Conclusions:

• The novel FLIM technique offers a significant advancement in imaging speed without compromising resolution.
• This technology is broadly applicable for studying the spatio-temporal dynamics of protein-protein interactions in live cells.