Hadamard-transform fluorescence-lifetime imaging.

Area of Science:

• Biophotonics and Imaging Science
• Microscopy Techniques
• Spectroscopy

Background:

• Fluorescence-lifetime-imaging microscopy (FLIM) is crucial for analyzing biological samples.
• Traditional FLIM techniques face challenges with speed and analyzing complex, multi-component samples.
• Limitations in detector frame rates can hinder real-time imaging and analysis.

Purpose of the Study:

• To introduce and evaluate a novel Hadamard-transform-based fluorescence-lifetime-imaging (HT-FLI) technique.
• To address the limitations of existing FLIM methods, particularly for multi-component sample analysis.
• To improve the speed and efficiency of fluorescence lifetime measurements in microscopy.

Main Methods:

• Utilized a Fourier-transform phase-modulation fluorometer (FT-PMF) for fluorescence lifetime measurements.
• Employed a linearly swept modulation frequency of excitation light from zero to a maximum.
• Derived fluorescence lifetimes via Fourier transforms of fluorescence and reference waveforms.
• Implemented electronic exchange of Hadamard transform (HT) illumination mask patterns for imaging.
• Incorporated a high-speed, high-sensitivity photomultiplier to overcome detector frame-rate issues.

Main Results:

• The FT-PMF successfully enabled simultaneous analysis of multi-component samples.
• The HT imaging approach effectively eliminated frame-rate limitations associated with 2D image detectors.
• The technique provides a robust method for fluorescence lifetime determination.

Conclusions:

• The developed HT-FLI technique offers a significant advancement in FLIM.
• This method allows for rapid and simultaneous analysis of complex biological samples.
• The HT-FLI technique enhances imaging speed and analytical capabilities in fluorescence microscopy.