Widefield multifrequency fluorescence lifetime imaging using a two-tap complementary metal-oxide semiconductor camera...

Area of Science:

• Biomedical Optics
• Microscopy
• Fluorescence Imaging

Background:

• Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) offers efficient image-wide lifetime measurements.
• Existing systems face limitations in speed and multifrequency capabilities.

Purpose of the Study:

• To develop and demonstrate a novel widefield FD-FLIM system.
• To enable parallel multifrequency FLIM measurements for enhanced cellular analysis.

Main Methods:

• Utilized a complementary metal-oxide semiconductor camera with specialized lock-in pixels and charge modulators.
• Implemented parallel multifrequency FLIM via fast Fourier transform at a fundamental frequency of 20 MHz.
• Measured up to 31 harmonics with 64 phase images per laser repetition period.

Main Results:

• Successfully demonstrated parallel multifrequency FLIM in a single measurement.
• Analyzed Förster Resonance Energy Transfer (FRET) in cells expressing Cerulean and Cerulean-Venus constructs.
• Tracked intracellular temperature changes using Rhodamine B fluorescence lifetime.

Conclusions:

• The developed widefield multifrequency FD-FLIM system provides a fast and efficient imaging solution.
• This technology is a valuable tool for advanced biomedical research and diagnostics.
• The system enables detailed analysis of cellular dynamics and microenvironments.