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Super resolution microscopy (STED) and scanning less microscopy

Imaging of living hippocampal neurons in primary culture in two contrast modes: one super-resolution STED channel and one label-free phase contrast channel [9]. Upper left: Spiral Phase Contrast (SPC) visualizes the neuronal network. Upper Right: Confocal fluorescence image. Lower left: SPC and confocal images are perfectly registered since they are recorded with the same scanner. They can therefore be easily overlayed. Lower right: STED microscopy resolves spine morphology in YFP-transfected living neurons in the region indicated by the white box in the SPC image.

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.

 
Achievements:
 
Several biological projects are currently running with our system in collaborations with various groups (J.-C. Poncer at Institut du Fer a Moulin, F. Darchen at University Paris Descartes, M. Groszer at Institut du Fer a Moulin, G. Nguyen at College de France, J. Boutet de Monvel and S. Safieddine at Institut Pasteur). In addition, an original implementation of spiral phase contrast was developed [9] and patented [11]. In confocal scanning mode, spiral phase contrast can measure quantitatively phase delays introduces by the sample [10], demonstrating sensitivities down to 10mrad ( lambda/1000).
 
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.
  9. M. A. Lauterbach, M. Guillon, A. Soltani, V. Emiliani. STED microscope with Spiral Phase Contrast, Scientific Reports vol. 3, art nr. 2050 (2013)
  10. Marc Guillon and Marcel A. Lauterbach. Quantitative confocal spiral phase contrast, J. Opt. Soc. Am. A, vol.31(6) pp. 1215-1225 (2014).
  11. Microscope for high spatial resolution imaging a strucutre of interest in a sample, EP12305509, submitted May 7th, 2012.