Super resolution microscopy (STED) and scanning less microscopy


Confocal and STED images of immuno-labeled TI-VAMP vesicles obtained with our setup (Collaboration with A. Burgo, T. Galli, Université Paris 7, INSERM U950). The region marked in green is shown enlarged. The STED image resolves individual vesicles with a resolution of about 40 nm, whereas the confocal image just shows blurred accumulations of vesicles.

 

In recent years, light microscopy has largely contributed to the understanding of many biological processes. Moreover, light microscopy is advantageous compared with some higher resolving microscopy techniques (e. g. electron microscopy) because it is compatible with observations under physiological conditions. Dynamics can be studied and sample preparation is relatively easy. However, conventional light microscopy is fundamentally limited in resolution by diffraction whereas many cellular processes occur on a nanometer scale and are therefore not accessible.

Stimulated Emission Depletion (STED) microscopy [1, 2, 3] allows imaging beyond the diffraction limit. Nevertheless it works under physiological conditions since it uses not-harming visible light. Keeping the advantages of conventional microscopes, it enables thus imaging with unseen resolution, down to 6nm [4], which is equivalent to a higher useful magnification. Samples with conventional immonostainings or fluorescent proteins [5] can be observed. Fast scanning enables studying dynamics [6, 7], also in living cells [8].

Embedded in a neuroscience department, our STED microscope aims:

  • first at enhancing the versatility of STED microscopy especially in the scope of neuroscience research;
  • second, at tackling open questions in neuroscience with superior resolution (∼50nm) that is provided by STED microscopy.

References
  1. Hell, S. W. and Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optics Letters, 19(11):780–782, 1994.
  2. Klar, T. A. and Hell, S. W. Subdiffraction resolution in far-field fluorescence microscopy. Optics Letters, 24(14):954–956, 1999.
  3. Hell, S. W. Far-field optical nanoscopy. Science, 316(5828):1153–1158, 2007.
  4. Rittweger, E., Han, K. Y., Irvine, S. E., Eggeling, C., and Hell, S. W. STED microscopy reveals crystal colourcentres with nanometricresolution. Nature Photonics, 3(3):144–147, 2009.
  5. Willig, K. I., Kellner, R. R., Medda, R., Hein, B., Jakobs, S., and Hell, S. W. Nanoscaleresolution in GFP-based microscopy. Nature Methods,3(9):721–723, 2006.
  6. Westphal, V., Lauterbach, M. A., Di Nicola, A., and Hell, S. W. Dynamic far-field fluorescence nanoscopy. New Journal of Physics, 9:435, 2007.
  7. M. A. Lauterbach, C. K. Ullal, V.Westphal, and S.W. Hell.Dynamic imaging of colloidal-crystalnanostructurs at 200 frames per second. Langmuir, 26(18):14400–14404, 2010.
  8. Westphal, V., Rizzoli, S. O., Lauterbach, M. A., Kamin, D., Jahn, R., and Hell, S. W. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science, 320(5873):246–249, 2008.