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A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy

Journal Article (2024)
Author(s)

Zhaowei Liu (TU Delft - Applied Sciences, Kavli institute of nanoscience Delft)

Edo van Veen (TU Delft - Applied Sciences, Kavli institute of nanoscience Delft)

Humberto Sánchez (TU Delft - Applied Sciences, Kavli institute of nanoscience Delft)

Belén Solano (Kavli institute of nanoscience Delft, TU Delft - Applied Sciences)

Francisco J. Palmero Moya (Kavli institute of nanoscience Delft, TU Delft - Applied Sciences)

Kaley A. McCluskey (TU Delft - Applied Sciences, Kavli institute of nanoscience Delft)

Daniel Ramírez Montero (Kavli institute of nanoscience Delft, TU Delft - Applied Sciences)

Theo van Laar (TU Delft - Applied Sciences, Kavli institute of nanoscience Delft)

Nynke H. Dekker (University of Oxford, TU Delft - Applied Sciences, Kavli Institute of Nanoscience Discovery)

Research Group
BN/Nynke Dekker Lab
DOI related publication
https://doi.org/10.1021/acsphotonics.3c01739 Final published version
More Info
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Publication Year
2024
Language
English
Research Group
BN/Nynke Dekker Lab
Issue number
4
Volume number
11
Pages (from-to)
1592-1603
Downloads counter
387
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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.