A method for detecting at least one reversibly photoswitchable fluorescent species including the following steps: (a) illuminating a sample containing said at least one reversibly photoswitchable fluorescent species with a first illuminating light beam, of wavelength λ 1, and periodically modulated according to a first function comprising one or more first illumination components modulated by pulsations ω i, said at least one pulsation ωi being associated with a reversibly photoswitchable fluorescent species, and with a second illuminating light beam of wavelength λ 2, different from λ 1, periodically modulated according to a second function comprising one or more second illumination components modulated by said at least one pulsation ω i, said at least one pulsation ω i being equal to said at least one pulsation ω i of the one or more first illuminating component; (b) detecting fluorescent radiation emitted by said sample thus illuminated; and (c) extracting the amplitude (I F out) from the one or more intensity component of said fluorescent radiation having the same pulsation ω i as each illumination component and in phase quadrature with respect to the one or more first illumination component; - for said at least one pulsation ω i, said one or more second illumination component modulated by the pulsation ω i being in antiphase with respect to said one or more first illumination component modulated by said pulsation ω i - and the average intensity of the first illuminating light beam, the average intensity of the second illuminating light beam, and said at least one pulsation being selected to approach a maximum of the at least one amplitude of the intensity component of the fluorescent radiation. (a) illuminating a sample containing said at least one reversibly photoswitchable fluorescent species with a first illuminating light beam, of wavelength λ 1, and periodically modulated according to a first function comprising one or more first illumination components modulated by pulsations ω i, said at least one pulsation ωi being associated with a reversibly photoswitchable fluorescent species, and with a second illuminating light beam of wavelength λ 2, different from λ 1, periodically modulated according to a second function comprising one or more second illumination components modulated by said at least one pulsation ω i, said at least one pulsation ω i being equal to said at least one pulsation ω i of the one or more first illuminating component; (b) detecting fluorescent radiation emitted by said sample thus illuminated; and (c) extracting the amplitude (I F out) from the one or more intensity component of said fluorescent radiation having the same pulsation ω i as each illumination component and in phase quadrature with respect to the one or more first illumination component; - for said at least one pulsation ω i, said one or more second illumination component modulated by the pulsation ω i being in antiphase with respect to said one or more first illumination component modulated by said pulsation ω i - and the average intensity of the first illuminating light beam, the average intensity of the second illuminating light beam, and said at least one pulsation being selected to approach a maximum of the at least one amplitude of the intensity component of the fluorescent radiation. - for said at least one pulsation ω i, said one or more second illumination component modulated by the pulsation ω i being in antiphase with respect to said one or more first illumination component modulated by said pulsation ω i - and the average intensity of the first illuminating light beam, the average intensity of the second illuminating light beam, and said at least one pulsation being selected to approach a maximum of the at least one amplitude of the intensity component of the fluorescent radiation.