posted on 2024-03-18, 06:29authored byZhaowei Liu, Edo van Veen, Humberto Sánchez, Belén Solano, Francisco J. Palmero Moya, Kaley A. McCluskey, Daniel Ramírez Montero, Theo van Laar, Nynke H. Dekker
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.