Time-resolved confocal fluorescence imaging and spectrocopy system with single molecule sensitivity and...

Area of Science:

• Optics and Photonics
• Biophysics
• Analytical Chemistry

Background:

• Advanced fluorescence microscopy is crucial for investigating molecular dynamics at the single-molecule level.
• Existing techniques often face limitations in resolution, speed, or multicolor capabilities.
• Precise characterization of fluorescence dynamics requires sophisticated instrumentation and data analysis.

Purpose of the Study:

• To present the technical innovations and performance of the MicroTime 200, a two-channel confocal fluorescence lifetime microscope.
• To demonstrate its capability for high-accuracy imaging and time-resolved fluorescence analysis.
• To showcase its utility for investigating complex fluorescence phenomena, including FRET and single-molecule antibunching.

Main Methods:

• Utilizes a two-channel confocal setup with picosecond diode lasers for multicolor excitation.
• Employs a compact Piezo scanner with a novel scanning algorithm for superior positioning accuracy.
• Features data acquisition based on time-correlated single photon counting (TCSPC) in Time-Tagged Time-Resolved (TTTR) mode.

Main Results:

• Achieved efficient investigation of fluorescence dynamics down to the single-molecule level.
• Demonstrated multicolor excitation and high-accuracy imaging and positioning.
• Enabled multiparameter data analysis, including intensity, polarization, spectral composition, FLIM, and fluorescence fluctuation correlation spectroscopy (FfCS).

Conclusions:

• The MicroTime 200 offers a powerful platform for advanced fluorescence analysis.
• Its technical features enable detailed investigation of fluorescence dynamics and molecular interactions.
• The demonstrated capabilities are applicable to diverse samples, from synthetic beads to biological systems and single molecules.