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用于集成力-共聚焦荧光显微镜中可靠数据采集与处理的生物物理工具箱

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

作者信息

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

机构信息

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.

出版信息

ACS Photonics. 2024 Mar 18;11(4):1592-1603. doi: 10.1021/acsphotonics.3c01739. eCollection 2024 Apr 17.

DOI:10.1021/acsphotonics.3c01739
PMID:38645993
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11027178/
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.

摘要

集成单分子力荧光光谱装置能够同时对单个分子进行荧光成像以及机械力操控与测量，从而提供全面的动态和时空信息。双光束光镊（OT）与共聚焦扫描显微镜相结合，构成了一种广泛用于研究各种生物过程，特别是蛋白质与DNA相互作用的力荧光光谱仪器。此类实验通常包括对与DNA结合的荧光标记蛋白质进行成像，以及对捕获的单个DNA分子进行力谱测量。在此，我们展示了一个多功能的先进工具箱，包括蛋白质 - DNA复合物样品的制备、结合光镊的微流控流动池的设计、光镊 - 共聚焦扫描测量的自动化，以及用于力和荧光光谱数据处理的简化数据分析软件包的开发与实施。其组件可适配任何配备共聚焦扫描显微镜的商业化或自制双光束光镊装置，这将有助于对多种生物系统进行单分子力荧光光谱研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/3d8e0f754b09/ph3c01739_0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/e96ba6c36ce5/ph3c01739_0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/fab2582b89d7/ph3c01739_0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/c86e4b8dd1e3/ph3c01739_0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/4df208b65579/ph3c01739_0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/39aac84ed03f/ph3c01739_0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/3d8e0f754b09/ph3c01739_0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/e96ba6c36ce5/ph3c01739_0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/fab2582b89d7/ph3c01739_0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/c86e4b8dd1e3/ph3c01739_0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/4df208b65579/ph3c01739_0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/39aac84ed03f/ph3c01739_0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53b/11027178/3d8e0f754b09/ph3c01739_0006.jpg

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