Rotational Mobility of Single Molecules Affects Localization Accuracy in Super-Resolution Fluorescence Microscopy

Lew, Matthew D.; Backlund, Mikael P.; Moerner, W. E.

doi:10.1021/nl304359p

Cited by 108 publications

(133 citation statements)

References 26 publications

(48 reference statements)

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“…Therefore, the algorithm depends on emitters having sufficient rotational mobility as emission profiles deviating from symmetrical PSFs can result in significant localization errors as has been discussed. [28][29][30][31] In summary, we believe that pSMLM-3D holds great promise to replace or complement commonly used localization algorithms, as the combination of high localization speeds and high localization accuracy has not been shown to this extent before.…”

Section: Discussionmentioning

confidence: 99%

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

Martens

Bader

Baas

et al. 2017

The Journal of Chemical Physics

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We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 10 6 localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function to two phase vectors (phasors) by calculating the first Fourier coefficients in both the x-and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

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Section: Discussionmentioning

confidence: 99%

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

Martens

Bader

Baas

et al. 2017

The Journal of Chemical Physics

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“…On the other hand, if r f τ τ > , then the polarization of the excitation illumination source relative to the mean orientation of the molecule will induce an asymmetric emission probablity distribution within the constraint cone [35]. In this situation, the cone angle α cannot be deduced from the eigenvalues of M alone.…”

Section: Example: Relationship Of M Matrix To the 'Rotation Within A mentioning

confidence: 99%

Determining the Rotational Mobility of a Single Molecule from a Single Image: A Numerical Study

Backer

Moerner

2015

Imaging and Applied Optics 2015

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Measurements of the orientational freedom with which a single molecule may rotate or 'wobble' about a fixed axis have provided researchers invaluable clues about the underlying behavior of a variety of biological systems. In this paper, we propose a measurement and data analysis procedure based on a widefield fluorescence microscope image for quantitatively distinguishing individual molecules that exhibit varying degrees of rotational mobility. Our proposed technique is especially applicable to cases in which the molecule undergoes rotational motions on a timescale much faster than the framerate of the camera used to record fluorescence images. Unlike currently available methods, sophisticated hardware for modulating the polarization of light illuminating the sample is not required. Additional polarization optics may be inserted in the microscope's imaging pathway to achieve superior measurement precision, but are not essential. We present a theoretical analysis, and benchmark our technique with numerical simulations using typical experimental parameters for single-molecule imaging. ©2015 Optical Society of AmericaOCIS codes: (170.2520) Fluorescence microscopy; (100.0100) Image processing. References and links1. T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77(19), 3979-3982 (1996). 2. T. Ha, J. Glass, T. Enderle, D. S. Chemla, and S. Weiss, "Hindered rotational diffusion and rotational jumps of single molecules," Phys. Rev. Lett. 80(10), 2093-2096 (1998). A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, "Video-rate nanoscopy using sCMOS camera-specific single-molecule localization

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“…First, molecular orientation can itself affect localization properties (23)(24)(25), and therefore the quality of image reconstruction. This effect has been shown, however, to be less dramatic when rotational mobility occurs in 2D in the sample plane, or in a large angular range (26). Second, the measurement of molecular orientation involves splitting the signal into polarized detection/excitation channels, which can decrease localization precision if not properly processed.…”

mentioning

confidence: 99%

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Valades-Cruz

Shaban

Kress

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

126

138

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Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.iological processes are inherently driven by the molecularscale organization of biomolecular assemblies, which arrange in precise structures that are essential for biological functions in cells and tissues. The extent to which the biological function depends on the underlying molecular-scale organization is particularly evident in filamentous assemblies, such as DNA filaments and cytoskeletal protein filaments. Changes in the local higher-order organization of DNA filaments is tightly linked to essential DNA-mediated processes including control of gene expression, DNA replication, and DNA repair. However, how specific DNA-binding proteins affect DNA filament architecture and thus DNA-mediated functions is poorly understood (1). Similarly, the spatial organization of cytoskeletal filaments in cells and tissues is also weakly explored, despite their central role in generating forces and driving cell motility, cell division, and tissue morphogenesis (2). Electron microscopy has been widely used to provide molecular-scale images of the structure of such filament assemblies; however, it typically involves several daylong sample preparation and ultrathin sectioning of the biological material, thus limiting investigations in whole cells and tissues.Polarized fluorescence imaging is a powerful approach for elucidating the structural organization of filament assemblies because it is compatible with a wide variety of microscopy techniques, thus enabling studies across multiple spatial and temporal scales. Polarized fluorescenc...

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Rotational Mobility of Single Molecules Affects Localization Accuracy in Super-Resolution Fluorescence Microscopy

Cited by 108 publications

References 26 publications

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

Determining the Rotational Mobility of a Single Molecule from a Single Image: A Numerical Study

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

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