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

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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.

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          Robust single particle tracking in live cell time-lapse sequences

          Khuloud Jaqaman, Dinah Loerke, Marcel Mettlen … (2008)
          Single particle tracking (SPT) is often the rate-limiting step in live cell imaging studies of sub-cellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout the movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long and short-lived endocytic structures, and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.
          Article 0 comments Cited 314 times – based on 0 reviews     Review now
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            Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.

            Keir C. Neuman, Attila Nagy (2008)
            Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.
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              CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

              Wulan Deng, Xinghua Shi, Robert Tjian … (2015)
              Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.
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                Author and article information

                Journal
                Journal ID (nlm-ta): ACS Photonics
                Journal ID (iso-abbrev): ACS Photonics
                Journal ID (publisher-id): ph
                Journal ID (coden): apchd5
                Title: ACS Photonics
                Publisher: American Chemical Society
                ISSN (Electronic): 2330-4022
                Publication date (Electronic): 18 March 2024
                Publication date Collection: 17 April 2024
                Volume: 11
                Issue: 4
                Pages: 1592-1603
                Affiliations
                [† ]Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , 2629 HZ Delft, The Netherlands
                [‡ ]Clarendon Laboratory, Department of Physics, University of Oxford , Oxford OX1 3PU, U.K.
                [§ ]Kavli Institute of Nanoscience Discovery, University of Oxford , Dorothy Crowfoot Hodgkin Building, Oxford OX1 3QU, U.K.
                Author notes
                Author information
                https://orcid.org/0000-0001-8214-8882
                Article
                DOI: 10.1021/acsphotonics.3c01739
                PMC ID: 11027178
                PubMed ID: 38645993
                SO-VID: 733c6edb-f06b-4cd7-9ac2-40e9e3912aa2
                Copyright © © 2024 The Authors. Published by American Chemical Society
                License:

                Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained ( https://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 29 November 2023
                Date accepted : 01 March 2024
                Date revision received : 01 March 2024
                Funding
                Funded by: European Molecular Biology Organization, doi 10.13039/100004410;
                Award ID: Postdoctoral Fellowship grant number ALTF 484-2022
                Funded by: Marie Sklodowska-Curie Actions (MSCA), doi NA;
                Award ID: TOPOREF; grant number REP-800488
                Funded by: Netherlands Organization for Scientific Research (NWO), doi NA;
                Award ID: Top grant 714.017.002
                Funded by: Netherlands Organization for Scientific Research (NWO), doi NA;
                Award ID: Spinoza Prize
                Funded by: Dutch Foundation on Fundamental Research on Matter, doi NA;
                Award ID: 16PR1047
                Funded by: Netherlands Ministry of Education, Culture and Science (OCW), doi NA;
                Award ID: 024.003.019
                Funded by: Boehringer Ingelheim Fonds, doi 10.13039/501100001645;
                Award ID: PhD fellowship
                Funded by: European Research Council, doi 10.13039/501100000781;
                Award ID: Advanced Grant (REPLICHROMA; grant number 789267)
                Categories
                Subject: Article
                Custom metadata
                document-id-old-9 ph3c01739
                document-id-new-14 ph3c01739
                ccc-price

                Keywords: optical tweezers,force spectroscopy,fluorescence spectroscopy,data analysis,automated data acquisition,protein−dna interactions
                Data availability:
                Keywords: optical tweezers, force spectroscopy, fluorescence spectroscopy, data analysis, automated data acquisition, protein−dna interactions

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