For simple STED scanning microscopy we kept the two wavelengths merged along the whole optical path to preserve their intrinsic alignment. However, the STED beam is to display a dark center, while the excitation beam should remain Gaussian. Therefore, we used a beam shaping device which leaves the excitation beam unaffected and changes the polarization of the STED beam. This device is based on chromatic waveplates (λ/2 for 532 nm and λ for 355 nm) : four segments of such a waveplate are assembled in a star-like configuration, so that, with circularly polarized incident light, the polarization of the output beam is a combination of radial and azimuthal polarizations.
For simple STED-SPIM microscopy the device is simpler since only two segments are needed.
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Les masques de phase pour le STED simplifié
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