Method of optical microscopy by scanning a sample containing an excitable species, said method comprising, for each predetermined scanning segment: - directing a first light beam having a first main wavelength λ 1, and a second light beam having a second main wavelength λ 2, onto respective, partially overlapped areas of said sample, wherein said first light beam is provided for exciting, in single- or multi-photon excitation mode, members of the excitable species to an excited state, and said second light beam is provided for reducing the number of excited members in said excited state; and - detecting an optical signal coming from the sample, said optical signal comprising a main component emitted by the members excited by said first light beam, and a spurious component emitted by other members of said excitable species undesirably excited by said second light beam, wherein said optical signal is detected during consecutive first and second time gates having respective durations T open and T close and defining a period T = T open + T close, said 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 said second time gate being provided for detecting the optical signal for a time interval during which the main component tends to or is zero while the spurious component remains present; said method further comprising: - processing the optical signal detected during said second time gate for extracting an estimate of said spurious component; and - subtracting said estimate of the spurious component from the optical signal detected during said first time gate; characterized in that said first light beam is operating during the first time gate T open and is interrupted during the second time gate T close and in that said second light beam is operating during both first time gate T open and second time gate T close, said time gates and said period T being such that T open » τ, T close » τ and T » τ, where τ is the average lifetime of said excited state, wherein said period T is such that ( T / τ) ≥ 10 2, wherein the durations T o-pen, T close of said first and second time gates are such that T open ≥ T close, and wherein 1 ≤ ( T o-pen / T close) ≤ 10. - directing a first light beam having a first main wavelength λ 1, and a second light beam having a second main wavelength λ 2, onto respective, partially overlapped areas of said sample, wherein said first light beam is provided for exciting, in single- or multi-photon excitation mode, members of the excitable species to an excited state, and said second light beam is provided for reducing the number of excited members in said excited state; and - detecting an optical signal coming from the sample, said optical signal comprising a main component emitted by the members excited by said first light beam, and a spurious component emitted by other members of said excitable species undesirably excited by said second light beam, wherein said optical signal is detected during consecutive first and second time gates having respective durations T open and T close and defining a period T = T open + T close, said 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 said second time gate being provided for detecting the optical signal for a time interval during which the main component tends to or is zero while the spurious component remains present; - processing the optical signal detected during said second time gate for extracting an estimate of said spurious component; and - subtracting said estimate of the spurious component from the optical signal detected during said first time gate;