Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

Area of Science:

• Biophotonics
• Microscopy
• Spectroscopy

Background:

• Fluorescence Lifetime Microscopy (FLIM) is crucial for analyzing biological samples.
• Current FLIM techniques have limitations in multiplexing capability.
• Advanced imaging methods are needed for real-time monitoring of multiple cellular processes.

Purpose of the Study:

• To introduce a novel excitation-emission multiplexed FLIM method with enhanced multiplexing capabilities.
• To enable simultaneous acquisition of multiple fluorescence lifetime images.
• To provide a cost-effective and accessible FLIM solution for biological research.

Main Methods:

• Utilized Fourier multiplexing for simultaneous acquisition of confocal fluorescence lifetime images.
• Employed multiple excitation wavelengths and emission color combinations.
• Achieved high-speed imaging at 44,000 pixels/sec using low-cost CW lasers and standard PMTs.

Main Results:

• Demonstrated a novel FLIM method surpassing current techniques in multiplexing.
• Successfully acquired multiplexed confocal fluorescence lifetime images at high speed.
• Developed a versatile spectral configuration compatible with commercial confocal microscopes.

Conclusions:

• The Fourier lifetime confocal method enables fast, multiplexed FLIM imaging.
• This technique facilitates monitoring of multiple biological processes in live cells.
• The low cost and compatibility offer broader accessibility of multiplexed FLIM to researchers.