Breaking the diffraction barrier in fluorescence microscopy by optical shelving.
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.
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37070 Göttingen, Germany.
Stefan
S
Bretschneider
Christian
C
Eggeling
Stefan W
SW
Hell
D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D055095:Optics and Photonics; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry
D005456:Fluorescent Dyes; D050825:Synaptosomal-Associated Protein 25