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

Zhaowei Liu, Edo van Veen, Humberto Sánchez, Belén Solano, Francisco J. Palmero Moya, Kaley A. McCluskey, Daniel Ramírez Montero, Theo van Laar, Nynke H. Dekker*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

33 Downloads (Pure)

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 languageEnglish
Pages (from-to)1592-1603
JournalACS Photonics
Volume11
Issue number4
DOIs
Publication statusPublished - 2024

Keywords

  • automated data acquisition
  • data analysis
  • fluorescence spectroscopy
  • force spectroscopy
  • optical tweezers
  • protein−DNA interactions

Fingerprint

Dive into the research topics of 'A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy'. Together they form a unique fingerprint.

Cite this