A practical implementation of multifrequency widefield frequency-domain fluorescence lifetime imaging microscopy

Area of Science:

• Biomedical Optics
• Microscopy Techniques
• Fluorescence Imaging

Background:

• Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is crucial for kinetic studies in biomedical research.
• Commercial FD-FLIM instruments have limitations due to a narrow range of available modulation frequencies.
• Existing modulation methods can reduce optical resolution.

Purpose of the Study:

• To develop and demonstrate a practical implementation of multifrequency widefield FD-FLIM.
• To overcome the limitations of conventional FD-FLIM systems.
• To provide a versatile tool for fluorescence imaging studies.

Main Methods:

• Utilized a pulsed supercontinuum laser and a direct digital synthesizer for multifrequency measurements.
• Employed pulse modulation of the image intensifier instead of sine-wave modulation.
• Implemented Fast Fourier Transform and cross-correlation techniques for parallel multifrequency analysis.
• Applied the phasor method for real-time, fit-free FLIM data analysis.

Main Results:

• Achieved precise and simultaneous isolation of individual frequencies.
• Restored optical resolution lost during sinusoidal modulation.
• Demonstrated the system's capability through measurements on standard fluorescent samples and a Föster resonance energy transfer pair.
• Enabled graphical analysis of FLIM data during acquisition.

Conclusions:

• The developed widefield multifrequency FD-FLIM system offers a valuable and simple solution for fluorescence imaging.
• The pulse modulation approach enhances resolution and analytical capabilities.
• This technique is well-suited for kinetic studies and various fluorescence imaging applications in biomedical research.