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  • Breaking the diffraction barrier in fluorescence microscopy by optical shelving.

Breaking the diffraction barrier in fluorescence microscopy by optical shelving.

Physical review letters (2007-08-07)
Stefan Bretschneider, Christian Eggeling, Stefan W Hell
ABSTRACT

We report the breaking of the diffraction resolution barrier in far-field fluorescence microscopy by transiently shelving the fluorophore in a metastable dark state. Using a relatively modest light intensity of several kW/cm(2) in a focal distribution featuring a local zero, we confine the fluorescence emission to a spot whose diameter is a fraction of the wavelength of light. Nanoscale far-field optical resolution down to 50 nm is demonstrated by imaging microtubules in a mammalian cell and proteins on the plasma membrane of a neuron. The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 532 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Sigma-Aldrich
Atto 532, BioReagent, suitable for fluorescence, ≥90% (HPCE)