Stimulated emission-depletion (STED) microscopy based on time gating of excitation beam and synchronous detection of fluorescence emission

Method of optical microscopy by scanning a sample containing an excitable species, the method comprising: directing a first and a second light beam onto respective, partially overlapped areas of the sample, wherein the first light beam is provided for exciting members of the excitable species, and the second light beam is provided for reducing the number of excited members; detecting an optical signal coming from the sample, comprising a main component and a spurious component, during consecutive first and second time gates, the first time gate being provided for detecting the optical signal for a time interval during which the main component and the spurious component are both present, and the second time gate being provided for detecting the optical signal for a time interval during which the main component tends to or is zero; processing the detected optical signal to separate its main component.