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Direct optical nanoscopy with axially localized detection

Research Square

Research Square

  • First Author :
    N. Bourg
  • Co-authors :
    C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort & S. Lévêque-Fort
  • Journal Name :
    Nature Photonics
  • Read Full text :
  • DOI :
    10.1038/nphoton.2015.132

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Abstract

Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip

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