A method for detecting at least one reversibly photoswitchable fluorescent species (P) including the following steps: (a) illuminating a sample containing said at least one reversibly photoswitchable fluorescent species with a first illuminating light beam (FEX1), 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 (P), and with a second illuminating light beam (FEX2) 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 (FLU) 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 (FLU) 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 (FEX1), the average intensity of the second illuminating light beam (FEX2), 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 (FEX1), 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 (P), and with a second illuminating light beam (FEX2) 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 (FLU) 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 (FLU) 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 (FEX1), the average intensity of the second illuminating light beam (FEX2), 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 (FEX1), the average intensity of the second illuminating light beam (FEX2), 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. <EOS>