Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to...

Area of Science:

• Biophysics
• Microscopy
• Spectroscopy

Background:

• Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for studying molecular environments.
• Accurate measurement of Förster Resonance Energy Transfer (FRET) requires high temporal resolution and detection efficiency.
• Existing methods may struggle with low excitation levels and photobleaching in living cells.

Purpose of the Study:

• To develop and validate a novel multi-dimensional time-correlated single photon counting (TCSPC) technique for FLIM.
• To apply this technique for measuring FRET efficiency and interchromophore distances.
• To demonstrate its utility in imaging FRET in living neurons.

Main Methods:

• Utilized a laser-scanning microscope with microsecond pixel dwell-time.
• Employed multi-dimensional TCSPC for high temporal accuracy and detection efficiency.
• Applied the technique to enhanced cyan fluorescent protein (ECFP)/enhanced yellow fluorescent protein (EYFP) constructs and styryl dyes (FM1-43/FM4-64).

Main Results:

• Successfully measured fluorescence lifetimes of ECFP and ECFP/EYFP.
• Quantified FRET efficiency and calculated interchromophore distances with <10% error.
• Demonstrated intermolecular FRET between FM1-43 and FM4-64 in living neurons, providing high-resolution lifetime images.

Conclusions:

• The novel multi-dimensional TCSPC technique offers high time resolution, accuracy, and counting efficiency for FLIM.
• This method enables precise FRET measurements and distance calculations, even at low excitation levels.
• The technique is suitable for studying intermolecular FRET dynamics in living biological systems, such as neuronal plasma membranes.