Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

Area of Science:

• Biophysics
• Single-molecule imaging
• Fluorescence microscopy

Background:

• Single-molecule fluorescence imaging is crucial for studying biomolecular dynamics.
• Simultaneous multi-color excitation and detection are key but under continuous development.
• Existing methods often lack flexibility in wavelength selection and control.

Purpose of the Study:

• To design and construct a versatile, economical, and compact multi-color fluorescence instrument.
• To enable sophisticated single-molecule dynamics studies with enhanced excitation control.
• To apply acousto-optic tunable filters (AOTF) to wide-field total internal reflection fluorescence (TIRF) microscopy for the first time.

Main Methods:

• Integration of two inverted microscopes sharing a laser combiner with six laser sources (400-640 nm).
• Utilized acousto-optic tunable filters (AOTF) for flexible, microsecond-timescale control of excitation wavelength, intensity, and duration.
• Employed single-mode optic fibers for independent TIRF angle optimization per wavelength and spectral splitting for multi-channel detection.

Main Results:

• Demonstrated a novel, cost-effective, and compact multi-color fluorescence setup.
• Achieved high flexibility in selecting, sequencing, and controlling excitation wavelengths using AOTF.
• Successfully performed two-color alternating excitation single-molecule FRET and four-color FRET experiments.

Conclusions:

• The developed instrument offers unprecedented flexibility and control for multi-color single-molecule fluorescence studies.
• The application of AOTF to TIRF microscopy represents a significant advancement in the field.
• The setup is capable of handling complex multi-color FRET experiments on challenging biomolecular structures.