Abstract
Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.
Original language | English |
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Pages (from-to) | 1592-1603 |
Journal | ACS Photonics |
Volume | 11 |
Issue number | 4 |
DOIs | |
Publication status | Published - 2024 |
Keywords
- automated data acquisition
- data analysis
- fluorescence spectroscopy
- force spectroscopy
- optical tweezers
- protein−DNA interactions