Abstract: Disclosed herein is a pulse-shaping method for stimulated emission depletion (STED) microscopy. The method comprises generating an optical excitation/depletion pulse with a depletion wavelength ?d; splitting the excitation/depletion pulse in time into an excitation part and a depletion part such that the excitation part and the depletion part propagate along an optical axis and are separated by a time delay ?t; creating an effective phase difference ?? between the excitation part and the depletion part; and focusing the excitation part and the depletion part of the excitation/depletion pulse onto a focus point, wherein the time delay ?t and the effective phase difference ?? are chosen such that an intensity distribution of the excitation/depletion pulse has a local maximum at the focus point at a first time and a local minimum at the focus point at a second time.

Abstract: Disclosed herein are a method and a device for two-color confocal colocalization microscopy of a sample. The sample is labeled by a confocal imaging marker and a stimulated emission depletion (STED) imaging marker. The method comprises generating a first confocal excitation pulse with a first wavelength ?1 and a second confocal excitation pulse with a second wavelength ?2 different from the first wavelength; focusing the first and second confocal excitation pulses onto a confocal focus point; generating a STED excitation pulse with the first wavelength and a STED depletion pulse with the second wavelength; focusing the STED excitation pulse and the STED depletion pulse onto a STED focus point; and detecting light emitted from the sample at an emission wavelength ?b of the STED imaging marker and at an emission wavelength ?a of the confocal imaging marker.