Method of modulated stimulated emission depletion microscopy at high spatial resolution (STED microscopy) comprising the following steps: A. illuminating a sample including one or more fluorophores in an observation volume with an excitation light beam having a constant excitation intensity, to energize said one or more fluorophores in the sample; B. illuminating the sample with a donut-shaped light beam, to de-energize by stimulated emission said one or more excited fluorophores obtaining an actual observation volume, the depletion light beam having an intensity I STED modulated over time I STED = I STED (t) with a temporal pattern having a modulation period T ; C. acquiring one or more fluorescence signals emitted from the sample on an image plane (x, y) of pixels; D. obtaining an image modulation M(x,y) of the sample on the image plane (x, y) of pixels, resulting from the modulation of the intensity I STED (t) of the depletion beam for each pixel (x,y), on the basis of said one or more fluorescence signals acquired at step C; E. processing the image modulation M(x,y) to obtain a fraction f center (x,y) of fluorescence intensity on the image plane (x, y) of pixels, corresponding to a contribution to said one or more fluorescence signals, acquired at step C, originating from fluorophores present in a center of the actual observation volume of the sample; F. calculating a final image F M-STED (x,y) of the sample on the image plane (x, y) of pixels, from said one or more fluorescence signals acquired at step C and from the image modulation function M(x,y) obtained at step D, as follows: F m − STED x y = f center x y F ′ STED x y wherein F' STED (x,y) is selected between a fluorescence image <F t-STED (x,y,t)> averaged in time and any fluorescence image of a temporal series F t-STED (x,y,t), said fluorescence images being obtained from said one or more fluorescence signals acquired in step C. A. illuminating a sample including one or more fluorophores in an observation volume with an excitation light beam having a constant excitation intensity, to energize said one or more fluorophores in the sample; B. illuminating the sample with a donut-shaped light beam, to de-energize by stimulated emission said one or more excited fluorophores obtaining an actual observation volume, the depletion light beam having an intensity I STED modulated over time I STED = I STED (t) with a temporal pattern having a modulation period T ; C. acquiring one or more fluorescence signals emitted from the sample on an image plane (x, y) of pixels; D. obtaining an image modulation M(x,y) of the sample on the image plane (x, y) of pixels, resulting from the modulation of the intensity I STED (t) of the depletion beam for each pixel (x,y), on the basis of said one or more fluorescence signals acquired at step C; E. processing the image modulation M(x,y) to obtain a fraction f center (x,y) of fluorescence intensity on the image plane (x, y) of pixels, corresponding to a contribution to said one or more fluorescence signals, acquired at step C, originating from fluorophores present in a center of the actual observation volume of the sample; F. calculating a final image F M-STED (x,y) of the sample on the image plane (x, y) of pixels, from said one or more fluorescence signals acquired at step C and from the image modulation function M(x,y) obtained at step D, as follows: F m − STED x y = f center x y F ′ STED x y wherein F' STED (x,y) is selected between a fluorescence image <F t-STED (x,y,t)> averaged in time and any fluorescence image of a temporal series F t-STED (x,y,t), said fluorescence images being obtained from said one or more fluorescence signals acquired in step C. <EOS>