Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155-157

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nature Methods (Impact Factor: 32.07). 03/2008; 5(2):155-7. DOI: 10.1038/nmeth.1176
Source: PubMed

ABSTRACT We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

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Available from: Suliana Manley, Jul 13, 2015
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    • "Recently, assays such as uPAINT [19] or sptPALM [20] have allowed the generation of hundreds of trajectories in a short amount of time and thereby opened the door to efficient and detailed mapping of interaction sites of membrane molecules with submembrane scaffolds and their functional characterization [5, 21–23]. "
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    ABSTRACT: Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity.
    06/2015; 3(2):1-8. DOI:10.1088/2050-6120/3/2/024001
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    • "This strictly limited the number of events that could be measured. This problem was eventually circumvented by tracking many temporally separated subsets of photoswitchable FP-tagged molecules in live-cell PALM experiments (sptPALM) (Hess et al., 2007; Manley et al., 2008). The high density of trajectories so obtained supplies enough statistical information to reconstruct high-resolution diffusion maps and infer distinct modes of diffusion. "
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    ABSTRACT: Observation of molecular processes inside living cells is fundamental to a quantitative understanding of how biological systems function. Specifically, decoding the complex behavior of single molecules enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble experiments. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new experiments to investigate the robustness and stochasticity of diverse molecular mechanisms with high spatiotemporal resolution. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 05/2015; 58(4):644-659. DOI:10.1016/j.molcel.2015.02.033 · 14.02 Impact Factor
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    • "In the past decade, various super-resolution techniques aiming at breaking the diffraction barrier have been proposed [14−18], such as stimulated emission depletion microscopy (STED) [19] [20], saturated structured illumination microscopy (SSIM) [21] [22], photoactivated localization microscopy (PALM) [23] [24], stochastic optical reconstruction microscopy (STORM) [25] [26], bleaching/blinking-assisted localization microscopy (BaLM) [27], and super-resolution optical fluctuation imaging (SOFI) [28, 29]. It should be noted that as PALM and STORM are based on the localization of a wide-field image, 3D super-resolution imaging has to be implemented with sophisticated point spread function (PSF) modulation following data postprocessing , such as astigmatism [26] and double helix [30]. "
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    ABSTRACT: Three-dimensional imaging cannot be achieved easily using previously developed localization super-resolution techniques. Here, we present a three-dimensional multimodal sub-diffraction imaging technique with spinning-disk (SD) confocal microscopy called 3D-MUSIC, which not only has all the advantages of SD confocal microscopy, such as fast imaging speed, high signal-to-noise ratio, and optical-sectioning capability, but also extends its spatial resolution limit along all three dimensions. Both axial and lateral resolution can be improved simultaneously by virtue of the blinking/fluctuating nature of modified fluorescent probes, exemplified with the quantum dots. Further, super-resolution images with dual modality can be obtained through super-resolution optical fluctuation imaging (SOFI) and bleaching/blinking-assisted localization microscopy (BaLM). Therefore, fast super-resolution imaging can be achieved with SD-SOFI by capturing only 100 frames while SD-BaLM yields high-resolution imaging.
    Nano Research 05/2015; DOI:10.1007/s12274-015-0736-8 · 7.01 Impact Factor
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