Imaging intracellular fluorescent proteins at nanometer resolution.
Eric Betzig,George H. Patterson,Rachid Sougrat,O. Wolf Lindwasser,Scott G. Olenych,Juan S. Bonifacino,Michael W. Davidson,Jennifer Lippincott-Schwartz,Harald F. Hess +8 more
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TLDR
This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.Abstract:
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 approximately 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 fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.read more
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Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy
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Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy
TL;DR: 3D stochastic optical reconstruction microscopy (STORM) is demonstrated by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy, allowing the 3D morphology of nanoscopic cellular structures to be resolved.
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References
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Book ChapterDOI
Total internal reflection fluorescence microscopy.
TL;DR: Total internal reflection fluorescence (TIRF) microscopy provides a means to selectively excite fluorophores in an aqueous or cellular environment that is very near a solid surface (within ≤100 nm: less than one-fifth the thickness of a confocal microscopy section) without exciting fluorescence from regions further from the surface as discussed by the authors.
Journal ArticleDOI
Superresolution by localization of quantum dots using blinking statistics.
TL;DR: It is shown that the intermittent fluorescence or 'blinking' of quantum dots can be analyzed by an Independent Component Analysis so as to identify the light emitted by each individual nanoparticle, localize it precisely, and thereby resolve groups of closely spaced (< lambda / 30) quantum dots.
Journal ArticleDOI
Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time
TL;DR: 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.
Journal ArticleDOI
Site-specific labeling of proteins with small molecules in live cells.
Irwin Chen,Alice Y. Ting +1 more
TL;DR: The principal bottleneck for the utilization of small-molecule probes in live cells is the shortage of methodologies for targeting them with very high specificity to biological molecules or compartments of interest.
Journal ArticleDOI
Semi-rational engineering of a coral fluorescent protein into an efficient highlighter
TL;DR: The molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green‐to‐red photoconvertibility are reported, leading to the generation of an efficient highlighter, KikGR.
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