Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization

Yıldız, Ahmet; Forkey, Joseph N.; McKinney, Sean A.; Ha, Taekjip; Goldman, Yale E.; Selvin, Paul R.

doi:10.1126/science.1084398

Cited by 1,735 publications

(1,610 citation statements)

References 30 publications

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“…As illustrated in Figure 3, in PALM/ STORM individual fluorophores are localized at resolutions well beyond the $200 nm diffraction limit by (i) stochastically photoactivating small percentages of the total population of fluorophores over time and (ii) determining the centroid position of these individual fluorophores from the Gaussian fits of their point spread functions (Figure 3). Given, that single-fluorophore localization precisions as low as $10 A have already been achieved in vitro [86,87], technical advances are expected to further improve the resolutions in live cells. SR-microscopy has so far done mostly in fixed cells.…”

Section: Chemical Tags For Super-resolution Imaging In Live Cellsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Chemical Tags For Super-resolution Imaging In Live Cellsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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“…Thus, this approach has the advantage of excellent S/N in a constantly illuminated plane near the surface. Spatial resolution can be as high as ∼1.5 nm, though the time resolution necessary to achieve this precision is relatively lengthy (∼500 ms) or requires highly fluorescent particles [26][27][28]. Disadvantages include the technical limitation that TIRF microscopy can only provide information about reactions close to a surface, and the spatial information obtained is typically in the plane of the surface.…”

Section: Motivationmentioning

confidence: 99%

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Yang

Musser

2006

Methods

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The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

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“…Alternatively, rapid molecular dynamics can be studied at high spatial resolution using single-particle tracking (SPT (11)(12)(13)(14)(15)(16)(17)(18)(19)). Whereas SPT has made important discoveries that change our view of plasma membrane organization (17,19) and molecular motor dynamics (20), the use of SPT in monitoring ''intracellular'' processes is rather limited because of the lack of three-dimensional (3D) tracking capacity that can follow a single particle inside a live cell for a long period of time. In the past decade, new SPT techniques have been developed to visualize molecular motion in the 3D space (termed 3D-SPT), including multiple imaging planes (21,22), orbital tracking (23)(24)(25), point spread function engineering (26,27), and confocal tracking (28,29).…”

Section: Introductionmentioning

confidence: 99%

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Liu

Perillo

Liu

et al. 2016

Biophysical Journal

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Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

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Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization

Cited by 1,735 publications

References 30 publications

Chemical tags: Applications in live cell fluorescence imaging

Chemical tags: Applications in live cell fluorescence imaging

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

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