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Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Proceedings of the National Academy of Sciences - PNAS, 2011-12, Vol.108 (52), p.21081-21086 [Peer Reviewed Journal]

copyright © 1993—2008 National Academy of Sciences of the United States of America ;Copyright National Academy of Sciences Dec 27, 2011 ;ISSN: 0027-8424 ;EISSN: 1091-6490 ;DOI: 10.1073/pnas.1117430109 ;PMID: 22167805

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  • Title:
    Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules
  • Author: Burnette, Dylan T ; Sengupta, Prabuddha ; Dai, Yuhai ; Lippincott-Schwartz, Jennifer ; Kachar, Bechara
  • Subjects: Animals ; Biological Sciences ; Bleaching ; bleaching agents ; Cercopithecus aethiops ; COS Cells ; dyes ; Fluorescence ; Fluorescent Dyes ; Green Fluorescent Proteins - metabolism ; image analysis ; Image Interpretation, Computer-Assisted - methods ; Image reconstruction ; Image resolution ; Imaging ; Microscopy ; Microscopy, Fluorescence - methods ; Microtubules ; Molecules ; Photobleaching ; Proteins ; Software ; Wave diffraction
  • Is Part Of: Proceedings of the National Academy of Sciences - PNAS, 2011-12, Vol.108 (52), p.21081-21086
  • Description: Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.
  • Publisher: United States: National Academy of Sciences
  • Language: English
  • Identifier: ISSN: 0027-8424
    EISSN: 1091-6490
    DOI: 10.1073/pnas.1117430109
    PMID: 22167805
  • Source: Geneva Foundation Free Medical Journals at publisher websites
    MEDLINE
    PubMed Central
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