Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Single-molecule fluorescence and optical trapping are key techniques in molecular studies.
• Combining these methods presents significant technical challenges but offers enhanced capabilities.
• Previous combined instruments have limitations in design and functionality.

Purpose of the Study:

• To develop and characterize a novel instrument integrating dual-trap optical tweezers and single-molecule fluorescence microscopy.
• To overcome fundamental and technical challenges in combined single-molecule approaches.
• To demonstrate the instrument's capability in studying complex molecular dynamics.

Main Methods:

• Constructed a hybrid instrument with base-pair resolution dual-trap optical tweezers and single-molecule fluorescence microscopy.
• Operated optical tweezers in constant and variable force modes.
• Implemented single-molecule fluorescence detection in wide-field and confocal configurations.

Main Results:

• Successfully imaged a single stretched lambda DNA molecule.
• Investigated DNA hairpin dynamics by monitoring fluorescence and extension changes.
• Observed simultaneous fluorescence signal changes and extension pauses linked to oligonucleotide association/dissociation.

Conclusions:

• The developed versatile microscopy offers enhanced flexibility for single-molecule studies.
• This combined approach facilitates the investigation of molecular machines and assemblies.
• The instrument provides new avenues for understanding molecular interactions with high precision.