Cookies Disclaimer

OK Our site saves small pieces of text information (cookies) on your device in order to deliver better content and for statistical purposes. You can disable the usage of cookies by changing the settings of your browser. By browsing our website without changing the browser settings you grant us permission to store that information on your device.

Publication

Bibliographic Data

  • Authors: Hein, B.,Willig, K.I.,Wurm, C.A.,Westphal, V.,Jakobs, S. and Hell, S.W.
  • Title: Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins
  • Journal: Biophys. J.
  • Volume: 98
  • Issue: 1
  • Volume: 158-163
  • DOI: 10.1016/j.bpj.2009.09.053

Abstract

We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzy1guanine-substituted organic dyes, for labeling. Tetramethy1rhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells.