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

doi:10.1021/acsphotonics.3c01739

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

Research output: Contribution to Journal › Article › Academic › peer-review

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
Pages (from-to)	1592-1603
Journal	ACS Photonics
Volume	11
Issue number	4
DOIs	https://doi.org/10.1021/acsphotonics.3c01739
Publication status	Published - 17 Apr 2024
Externally published	Yes

Funding

Netherlands Organization for Scientific Research (NWO) Top grant 714.017.002 Netherlands Organization for Scientific Research - Spinoza Prize Dutch Foundation on Fundamental Research on Matter grant 16PR1047 ‘BaSyC–Building a Synthetic Cell’ Gravitation grant 024.003.019 European Research Council Advanced Grant (REPLICHROMA; grant number 789267) EMBO Postdoctoral Fellowship ALTF 484-2022 Boehringer Ingelheim Fonds PhD fellowship European Union Marie Curie-Skłodowska Actions program (TOPOREF; grant number REP-800488). N.D. acknowledges funding from The Netherlands Organization for Scientific Research (NWO) through Top grant 714.017.002 and a Spinoza Prize, from the Dutch Foundation on Fundamental Research on Matter (part of NWO) through grant 16PR1047, from ‘BaSyC–Building a Synthetic Cell’ Gravitation grant (024.003.019) of The Netherlands Ministry of Education, Culture and Science (OCW), and from the European Research Council through an Advanced Grant (REPLICHROMA; grant number 789267). Z.L. acknowledges funding from EMBO Postdoctoral Fellowship (grant number ALTF 484-2022). D.R.M. acknowledges funding from a Boehringer Ingelheim Fonds PhD fellowship. K.M. acknowledges funding from the Marie Curie-Skłodowska Actions program of the European Union (TOPOREF; grant number REP-800488). The authors thank Andrea Candelli for help in designing the microfluidic flow cell, Aafke van den Berg for help in experiment automation, John Diffley for discussions of biochemical experiments, Carlas Smith for discussions of localization precision, and Pang Yen Wang for discussions and assistance in dual-beam OT-confocal scanning microscope experiments.

Funders	Funder number
European Union Marie Curie-Skłodowska Actions
H2020 Marie Skłodowska-Curie Actions
Boehringer Ingelheim Fonds
714.017.002 Netherlands Organization for Scientific Research - Spinoza Prize Dutch Foundation on Fundamental Research on Matter
European Commission
PhD fellowship
Ministerie van Onderwijs, Cultuur en Wetenschap
Dutch Foundation on Fundamental Research on Matter	16PR1047, 024.003.019
TOPOREF	REP-800488
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	714.017.002
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
European Research Council	789267
European Research Council
European Molecular Biology Organization	ALTF 484-2022
European Molecular Biology Organization

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

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.",

author = "Zhaowei Liu and {van Veen}, Edo and Humberto S{\'a}nchez and Bel{\'e}n Solano and {Palmero Moya}, {Francisco J.} and McCluskey, {Kaley A.} and {Ram{\'i}rez Montero}, Daniel and {van Laar}, Theo and Dekker, {Nynke H.}",

year = "2024",

month = apr,

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doi = "10.1021/acsphotonics.3c01739",

language = "English",

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AU - Liu, Zhaowei

AU - van Veen, Edo

AU - Sánchez, Humberto

AU - Solano, Belén

AU - Palmero Moya, Francisco J.

AU - McCluskey, Kaley A.

AU - Ramírez Montero, Daniel

AU - van Laar, Theo

AU - Dekker, Nynke H.

PY - 2024/4/17

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AB - 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.

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

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