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Improved optical slicing by Stimulated Emission Depletion light sheet microscopy.

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Optical microscopy, which uses fluorescent markers, is essential for imaging biological samples. Often, excitation and detection are done via a single objective, we speak of epifluorescence technique. When the sample is largely illuminated, the image is made on the eye via an eyepiece but now preferably on a two-dimensional matrix of photodetectors (CCD or CMOS), so-called full-field imaging is carried out. This method is very fast but only gives a two-dimensional image of the sample.

While maintaining full-field acquisition, lateral lighting in the form of a sheet, substituted for axial lighting, provides optical sectioning and therefore the third dimension. However, if you want to have very thin sheets, the laws of diffraction mean that its thickness will not be uniform
As part of the "Nanoscolas" project supported by the ANR, in collaboration with a team from the Grenoble Institute of Neuroscience (GIN), we have shown that a superesolutiv method could reconcile finesse and uniformity. It uses two collinear lasers, one for the formation of the fluorescent sheet and the other to selectively extinguish it thanks to the stimulated emission (STED effect) provided that the stimulating beam is shaped via a phase mask. We have developed simple and very efficient phase masks and demonstrated that highly resolved three-dimensional images of fluorescent microspheres and biological tissue can be obtained at high speed (with STED in image c and without in image b). This work resulted in a publication in a special edition of "biomedical optics express".

View online : Improved optical slicing by stimulated emission depletion light sheet microscopy José Martínez Hernández, Alain Buisson, Irène Wang, and Jean-Claude Vial, Biomed. Opt. Express 2020