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      Optimized localization-analysis for single-molecule tracking and super-resolution microscopy

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          Abstract

          We optimally localize isolated fluorescent beads and molecules imaged as diffraction-limited spots, determine the orientation of molecules, and present reliable formulae for the precisions of various localization methods. For beads, theory and experimental data both show that unweighted least-squares fitting of a Gaussian squanders one third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, while maximum likelihood fitting is practically optimal.

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          Linear Statistical Inference and its Applications

          C. Rao (1973)
          Book 0 comments Cited 605 times – based on 0 reviews
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            Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.

            Ahmet Yildiz, Joseph Forkey, Sean McKinney … (2003)
            Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving approximately 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of <1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 + 2x nm and 37 - 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model.
            Article 0 comments Cited 347 times – based on 0 reviews     Review now
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              Localization accuracy in single-molecule microscopy.

              Raimund Ober, Sripad Ram, E. Ward (2004)
              One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101215604
                Journal ID (pubmed-jr-id): 32338
                Journal ID (nlm-ta): Nat Methods
                Title: Nature methods
                ISSN (Print): 1548-7091
                ISSN (Electronic): 1548-7105
                Publication date Nihms-submitted: 20 April 2010
                Publication date (Electronic): 4 April 2010
                Publication date (Print): May 2010
                Publication date PMC-release: 30 June 2011
                Volume: 7
                Issue: 5
                Pages: 377-381
                Affiliations
                [1 ]Department of Micro- and Nanotechnology, Technical University of Denmark - DTU, Building 345E, DK-2800 Kgs. Lyngby, Denmark
                [2 ]Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305
                [3 ]Department of Physics, Stanford University School of Medicine, Stanford, CA 94305
                Author notes
                [4]

                These authors contributed equally to this work

                Article
                Manuscript ID: nihpa186810
                DOI: 10.1038/nmeth.1447
                PMC ID: 3127582
                PubMed ID: 20364147
                SO-VID: 4dd71c8c-02ea-4a56-95d9-f3b61c07894c
                License:

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM033289-27 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM033289-21 ||GM
                Categories
                Subject: Article

                ScienceOpen disciplines: Life sciences
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                ScienceOpen disciplines: Life sciences

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