Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

  title={Imaging Intracellular Fluorescent Proteins at Nanometer Resolution},
  author={Eric Betzig and George H. Patterson and Rachid Sougrat and O. Wolf Lindwasser and Scott G. Olenych and Juan S. Bonifacino and Michael W. Davidson and Jennifer Lippincott-Schwartz and Harald F. Hess},
  pages={1642 - 1645}
We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to ∼2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method—termed photoactivated localization microscopy—to image specific target proteins in thin sections of lysosomes and mitochondria; in… 
Localization-based super-resolution imaging of cellular structures.
Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction
Photoactivated Localization Microscopy for Cellular Imaging
The technical aspects of multicolor and three-dimensional imaging, and the software packages that are available, are summarized and several biological applications with an emphasis on neuroscience are highlighted.
Fluorescence Imaging Microscopy
The number of specific parameter-indicating fluorophores useful for nondestructive live cell imaging continues to increase, including markers of cell shape and volume, organelles, membrane potentials, ions, and other chemical constitutents.
Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo
The results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.
Photoactivatable fluorescent proteins for super-resolution microscopy.
This work illustrates how two fluorescent proteins with different photoactivation mechanisms can be utilized in high resolution dual color PALM imaging to obtain insights into a cellular process that otherwise would not be accessible.
A New Approach to Fluorescence Microscopy
Methods for tagging specific biomolecules with fluorescent labels, such as green fluorescent protein (GFP), provide a toolbox for the observation of protein organization, interactions, and dynamics that enables the visualization of a host of biological phenomena.
Photoswitchable fluorescent proteins for superresolution fluorescence microscopy circumventing the diffraction limit of light.
This work introduces a basic protocol of PALM through the visualization of actin bundles with superresolution and shows the applications of the fluorescent proteins to techniques such as molecular tracking and highlighting on a microscope.
Single-Molecule Localization Microscopy in Eukaryotes.
How SMLM has contributed new knowledge in eukaryotic biology is described, and the potential to pave the way toward a better understanding of how cells function at the molecular level is described.


Multicolor and Electron Microscopic Imaging of Connexin Trafficking
This approach was used to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicle of widely varying sizes.
A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells
We report a photoactivatable variant of theAequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by
Development and Use of Fluorescent Protein Markers in Living Cells
The development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells are traced.
Photoactivatable fluorescent proteins
The properties of the available photoactivatable fluorescent proteins and their potential applications are discussed.
Single Molecules Observed by Near-Field Scanning Optical Microscopy
Individual carbocyanine dye molecules in a sub-monolayer spread have been imaged with near-field scanning optical microscopy and the orientation of each molecular dipole can be determined to map the electric field distribution in the near- field aperture with molecular spatial resolution.
Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.
This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy, and the instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances.
Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.
The surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller is demonstrated, underscoring the potential to finally achieve molecular resolution in fluorescent microscopy by technical optimization.
Nanometer-localized multiple single-molecule fluorescence microscopy.
This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores to validate the NALMS microscopy approach.
Single-molecule high-resolution imaging with photobleaching.
  • M. Gordon, T. Ha, P. Selvin
  • Physics, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 2004
This work circumvented the Rayleigh limit and achieved nanometer-scale resolution by measuring the distance between single fluorophores separated by 10-20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface.