Simultaneous time- and wavelength-resolved fluorescence microscopy of single molecules

Area of Science:

• Spectroscopy
• Physical Chemistry
• Biophysics

Background:

• Single molecule spectroscopy provides detailed insights into molecular behavior.
• Current methods often lack simultaneous temporal and spectral resolution at the single molecule level.
• Fluorescence Resonance Energy Transfer (FRET) is a powerful tool for measuring molecular distances.

Purpose of the Study:

• To develop and demonstrate a new confocal fluorescence microscope capable of simultaneous time- and wavelength-resolved photon detection with single molecule sensitivity.
• To explore novel temporal and spectral correlation spectroscopies at the single molecule level.
• To present initial results on single fluorophore and single-pair FRET measurements.

Main Methods:

• Utilized a novel confocal fluorescence microscope with a dispersive optical system and a time/position-sensitive photon detector.
• Simultaneously recorded the wavelength, emission time relative to excitation pulse, and absolute emission time for each detected photon.
• Applied the technique to single molecules of rhodamine 6G (R6G), tetramethylrhodamine (TMR), and Cy3, as well as single-pair FRET between Alexa fluorophores.

Main Results:

• Achieved simultaneous time- and wavelength-resolved fluorescence measurements at the single molecule level.
• Demonstrated the capability to generate full fluorescence spectra and correlated decay plots for individual molecules.
• Successfully measured single-pair FRET efficiency between Alexa fluorophores.

Conclusions:

• The developed microscope enables unprecedented simultaneous temporal and spectral analysis of single molecules.
• This technology opens new avenues for advanced correlation spectroscopies.
• The findings validate the potential of this approach for detailed molecular investigations.