Using light sheet microscopy(SPIM) we plan to shown that the STED effect using our DPSS-lasers will drastically improve the axial resolution. Indeed, as shown on the schematics of the LSFM the axial resolution is determined by the thickness of the light sheet usually obtained by focusing through a cylindrical lens or by scanning the excitation beam The field of observation (imaged on a CCD camera) of a surface of quasi constant thickness is limited by the Rayleigh length of the illumination and consequently have a minimum thickness determined by the focusing law of a Gaussian beam. For example, if a field of observation of 1 square millimeter is whished the minimum thickness is 30µm which is prohibitive for cellular 3D imaging.
To show the improvement we use the laser optimized for "simple STED" and use an oscillating mirror to scan the excitation in order to create a sheet, the focusing optics being an achromatic lens . Modifications of phase masks", optimized for light sheets, are considered.
We first show that STED-SPIM improves axial resolution and expands the field of view