Do-It-Yourself Guide: How to Use the Modern Single-Molecule Toolkit

Department of Chemistry, Single Molecule Analysis Group, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, USA.
Nature Methods (Impact Factor: 32.07). 07/2008; 5(6):475-89. DOI: 10.1038/nmeth.1215
Source: PubMed


Single-molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state of the art in single-molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy. We also provide guidelines for choosing the right approach from the available single-molecule toolkit for applications as diverse as structural biology, enzymology, nanotechnology and systems biology.

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Available from: Nils Walter, Oct 06, 2014
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    • "From an image acquired using a CCD-based camera, parameters of interest can be estimated to obtain useful information about the imaged object. In single molecule microscopy (e.g., Walter et al. 2008; Moerner 2007), for example, a major topic of interest has been the accurate estimation of the location of a fluorescent molecule (e.g., Thompson et al. 2002; Ober et al. 2004; Andersson 2008; Pavani and Piestun 2008; Ram et al. 2008), which has important applications in, for instance, the study of intracellular processes (e.g., Ram et al. 2008). In this context, we have proposed a general framework (Ram et al. 2006) for calculating the Fisher information, and hence the Cramer-Rao lower bound (Rao 1965), for the estimation of parameters from an image produced by a microscope. "
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    ABSTRACT: The high quantum efficiency of the charge-coupled device (CCD) has rendered it the imaging technology of choice in diverse applications. However, under extremely low light conditions where few photons are detected from the imaged object, the CCD becomes unsuitable as its readout noise can easily overwhelm the weak signal. An intended solution to this problem is the electron-multiplying charge-coupled device (EMCCD), which stochastically amplifies the acquired signal to drown out the readout noise. Here, we develop the theory for calculating the Fisher information content of the amplified signal, which is modeled as the output of a branching process. Specifically, Fisher information expressions are obtained for a general and a geometric model of amplification, as well as for two approximations of the amplified signal. All expressions pertain to the important scenario of a Poisson-distributed initial signal, which is characteristic of physical processes such as photon detection. To facilitate the investigation of different data models, a "noise coefficient" is introduced which allows the analysis and comparison of Fisher information via a scalar quantity. We apply our results to the problem of estimating the location of a point source from its image, as observed through an optical microscope and detected by an EMCCD.
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    • "Michelotti discussed the imaging methods and experimental challenges of very low velocities (e.g., 3 nm/min) of nanowalkers [8]. The state of the art in single-molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy can be found in the survey by Walter et al. [54]. "
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    ABSTRACT: Motion analysis plays an important role in studing activities or behaviors of live objects in medicine, biotechnology, chemistry, physics, spectroscopy, nanotechnology, enzymology, and biological engineering. This paper briefly reviews the developments in this area mostly in the recent three years, especially for cellular analysis in fluorescence microscopy. The topic has received much attention with the increasing demands in biomedical applications. The tasks of motion analysis include detection and tracking of objects, as well as analysis of motion behavior, living activity, events, motion statistics, and so forth. In the last decades, hundreds of papers have been published in this research topic. They cover a wide area, such as investigation of cell, cancer, virus, sperm, microbe, karyogram, and so forth. These contributions are summarized in this review. Developed methods and practical examples are also introduced. The review is useful to people in the related field for easy referral of the state of the art.
    Full-text · Article · Jan 2012 · Computational and Mathematical Methods in Medicine
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    • "The evanescent wave decays exponentially from the interface and thus selectively illuminates fluorophores in the close proximity to the interface (100’s nm) [61,62]. The most common TIRF configurations use a prism [55,63], or a high NA objective [64,65]. The excitation intensity and the resulting fluorescent signal [61,66] are several times stronger in the TIRF condition than in epifluorescence (Figure 2). "
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    ABSTRACT: Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR) of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking.
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