KIP-Veröffentlichungen

We present a novel technique of far-field localization
nanoscopy combining spectral precision distance microscopy
with widely used fluorochromes like the Green Fluorescent
Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent
Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and
Alexa 568, as well as fluoresceine derivates. Spectral precision
distance microscopy allows the surpassing of conventional
resolution limits in fluorescence far-field microscopy by
precise object localization after the optical isolation of
single signals in time. Based on the principles of this
technique, our novel nanoscopic method was realized for
laser optical precision localization and image reconstruction
with highly enhanced optical resolution in intact cells.
This allows for spatial assignment of individual fluorescent
molecules with nanometre precision. The technique is based
on excitation intensity dependent reversible photobleaching
of the molecules used combined with fast time sequential
imaging under appropriate focusing conditions. A meaningful
advantage of the technique is the simple applicability as a
universal tool for imaging and investigations to the major
part of already available preparations according to standard
protocols. Using the above mentioned fluorophores, the
positions of single molecules within cellular structures were
determined by visible light with an estimated localization
precision down to 3 nm; hence distances in the range of       
10–30 nm were resolved between individual fluorescent
molecules allowing to apply different quantitative structure
analysis tools.