Method for high-resolution optical scanning of a sample, preferably using a laser scanning fluorescence microscope, wherein the sample comprises a substance which is able to be brought into different energy states - first state Z1 and second state Z2, wherein the first state Z1 and the second state Z2 differ from each other in at least one optical property, wherein the sample is illuminated with focused and pulsed light of a wavelength of the excitation spectrum of the substance to regionally produce the first state Z1 of the substance, wherein the sample is illuminated with pulsed light of a suitable deexcitation wavelength to produce the second state Z2 of the substance in a focus peripheral region of the excitation, and wherein emission light which emanates from the sample and is the result of the decay of remaining first states Z1 is detected by means of a detection device, wherein the sample comprises at least one further substance, which is able to be brought into a first state Z1' and into a second state Z2', wherein the substances (S1, S2) differ from each other in at least one property, specifically with respect to their excitation spectra, and that the substances (S1, S2) at least partially match in at least one other property, specifically with respect to their emission spectra, wherein the substances (S1, S2) are excited with pulsed light of different excitation wavelengths but are deexcited stimulated with pulsed light of the same deexcitation wavelength, characterized in that a pulse sequence is repeated multiple times which includes the succession • laser pulse of the wavelength (λ1) of the excitation spectrum of the substance, • laser pulse of the deexcitation wavelength (λ3), • laser pulse of the wavelength (λ2) of the excitation spectrum of the further substance, • laser pulse of the deexcitation wavelength (λ3), or being • laser pulse of the wavelength (λ1) of the excitation spectrum of the substance, • laser pulse of a wavelength (λ2) of the excitation spectrum of the further substance, • laser pulse of the deexcitation wavelength (λ3). wherein the sample comprises a substance which is able to be brought into different energy states - first state Z1 and second state Z2, wherein the first state Z1 and the second state Z2 differ from each other in at least one optical property, wherein the sample is illuminated with focused and pulsed light of a wavelength of the excitation spectrum of the substance to regionally produce the first state Z1 of the substance, wherein the sample is illuminated with pulsed light of a suitable deexcitation wavelength to produce the second state Z2 of the substance in a focus peripheral region of the excitation, and wherein emission light which emanates from the sample and is the result of the decay of remaining first states Z1 is detected by means of a detection device, wherein the sample comprises at least one further substance, which is able to be brought into a first state Z1' and into a second state Z2', wherein the substances (S1, S2) differ from each other in at least one property, specifically with respect to their excitation spectra, and that the substances (S1, S2) at least partially match in at least one other property, specifically with respect to their emission spectra, wherein the substances (S1, S2) are excited with pulsed light of different excitation wavelengths but are deexcited stimulated with pulsed light of the same deexcitation wavelength, characterized in that a pulse sequence is repeated multiple times which includes the succession • laser pulse of the wavelength (λ1) of the excitation spectrum of the substance, • laser pulse of the deexcitation wavelength (λ3), • laser pulse of the wavelength (λ2) of the excitation spectrum of the further substance, • laser pulse of the deexcitation wavelength (λ3), or being • laser pulse of the wavelength (λ1) of the excitation spectrum of the substance, • laser pulse of a wavelength (λ2) of the excitation spectrum of the further substance, • laser pulse of the deexcitation wavelength (λ3). • laser pulse of the wavelength (λ1) of the excitation spectrum of the substance, • laser pulse of the deexcitation wavelength (λ3), • laser pulse of the wavelength (λ2) of the excitation spectrum of the further substance, • laser pulse of the deexcitation wavelength (λ3), • laser pulse of the wavelength (λ1) of the excitation spectrum of the substance, • laser pulse of a wavelength (λ2) of the excitation spectrum of the further substance, • laser pulse of the deexcitation wavelength (λ3).