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Fluorescence-enhancement microscopy at 10 nm resolution

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Abstract

The historical importance of optical microscopy to biological study is difficult to overstate. Optical techniques are relatively non-invasive and minimally perturbative both during sample preparation and measurement, enabling temporally resolved imaging of dynamically evolving samples in vivo or at near physiological conditions. Spectroscopic contrast provided by fluorescence and Raman imaging, in particular, provides superior molecular addressability and chemical differentiability. Although spectroscopic techniques are widely utilized in molecular and cellular biology, typically the spatial resolution is limited by light diffraction to 200 – 500 nm, well above the length scale where individual molecules or complexes interact (<10 nm). Alternate microscopy techniques, such as X-ray crystallography, high-resolution nuclear-magnetic-resonance tomography, electron microscopy, scanning-tunneling microscopy, and atomic-force microscopy (AFM), can provide exquisite, sub-nm spatial resolution but do so at the price of chemical differentiability and/or temporal resolution. This gap in imaging functionality has been addressed by scanning near-field optical techniques and recently 25-30 nm resolution for fluorescence [1] and Raman [2] microscopy has been demonstrated using so-called apertureless approaches which rely on enhancement of the optical signal near a sharp (metal) probe. Although this resolution is well below the diffraction limit, it is only incrementally better than what can be obtained with standard near-field techniques where an apertured illumination source (or detection region) is scanned in close proximity to the sample.

© 2003 Optical Society of America

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