High-density mapping of single-molecule trajectories with photoactivated localization microscopy.

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nature Methods (Impact Factor: 25.95). 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.

  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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 · 14.46 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 6.96 Impact Factor


Available from