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Super-resolution Microscopy: 2-Color Stimulated Emission Depletion (STED)

Recent breakthroughs in microscopy techniques have led to the apparition of super-resolution microscopes.
These microscopes are able to break the conventional optical diffraction limit.
Amongst them, the STED is a fluorescent laser scanning microscopy technique where the resolution increase is purely physically-based. Such a microscope allows imaging of biological structures of acute slices or prepared cultures that confocal or 2-photon can not resolve.
STED relies on the quenching of fluorescence at the spatial extent of the excitation spot. This is achieved by aligning the excitation beam with a red-shifted continuous wave laser beam that provides a toroidal shaped depletion. Consequently the fluorescent radiation is preserved at the centre of the excitation spot while the stimulated emission is favored at the expense of the fluorescence process in the periphery of the spot. As a result, the fluorescence is emitted from a region which is smaller then the diffraction limited excitation spot. Thus STED microscopes can increase the spatial resolution up to ten fold compared to confocal.
The 2 colours Leica STED microscope installed at DBCM, was acquired through a joint financial support from the Swiss National Foundation program “R’Equip” and the Faculty of Biology and Medicine of UNIL and is the first one to be introduced in Switzerland. It will be utilized by groups of DBCM and other Departments of the Faculty to resolve the smallest cellular organelles, intercellular specialization domains or localization of membrane proteins that were previously done inaccurately or were simply impossible using confocal microscopy. See Volterra

 
Vesicles labeled with a fluorescent protein inside living PC12 cells.
The increased resolution given by the STED totally changes the vesicles perception (scale bar 1um)
 


 

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