Process of acquiring and processing images in confocal microscopy, comprising the following step: A. acquiring (500, 510; 520, 530), by means of a sensor of image acquisition having its own resolution R x R, a first overall image A k -1 of an entire plane of a sample scanning the same with a first excitation light beam, obtained by making a basic light beam pass through one or more first pinholes (1255; 1255') having a shape and a first size (L 1; L a1, L b1) and transmitted to an optics of a confocal microscope that is focused on said plane of the sample and that has a PSF, and acquiring a second overall image A k of the entire plane of the sample scanning the same with a second excitation light beam, obtained by making said basic light beam pass through one or more second pinholes (1265; 1265') having a shape equal to the shape of said one or more first pinholes (1255; 1255') and a second size (L 2; L a2, L b2) larger than the first size (L 1; L a1, L b1), wherein said one or more first pinholes (1255; 1255') and said one or more second pinholes (1265; 1265') are arranged in a plane optically conjugated to said plane of the sample; characterized in that the process comprises the following further steps: B. calculating an estimate g of the point spread function, PSF, of the optics of the microscope, through an inverse Fourier transform of the Fourier transform ĝ calculated as follows: g ^ ω = Â k − 1 * ω Â k ω | Â k − 1 ω | 2 where Â k -1 ( ω) is the Fourier transform of the acquired first overall image A k -1 and Â k ( ω) is the Fourier transform of the acquired second overall image A k; and C. deconvolving the acquired second overall image A k using the estimate g of the PSF of the optics of the microscope through a main deconvolution algorithm, obtaining the resulting image I of the entire plane of the sample: I = A k • g. A. acquiring (500, 510; 520, 530), by means of a sensor of image acquisition having its own resolution R x R, a first overall image A k -1 of an entire plane of a sample scanning the same with a first excitation light beam, obtained by making a basic light beam pass through one or more first pinholes (1255; 1255') having a shape and a first size (L 1; L a1, L b1) and transmitted to an optics of a confocal microscope that is focused on said plane of the sample and that has a PSF, and acquiring a second overall image A k of the entire plane of the sample scanning the same with a second excitation light beam, obtained by making said basic light beam pass through one or more second pinholes (1265; 1265') having a shape equal to the shape of said one or more first pinholes (1255; 1255') and a second size (L 2; L a2, L b2) larger than the first size (L 1; L a1, L b1), wherein said one or more first pinholes (1255; 1255') and said one or more second pinholes (1265; 1265') are arranged in a plane optically conjugated to said plane of the sample; B. calculating an estimate g of the point spread function, PSF, of the optics of the microscope, through an inverse Fourier transform of the Fourier transform ĝ calculated as follows: g ^ ω = Â k − 1 * ω Â k ω | Â k − 1 ω | 2 where Â k -1 ( ω) is the Fourier transform of the acquired first overall image A k -1 and Â k ( ω) is the Fourier transform of the acquired second overall image A k; and C. deconvolving the acquired second overall image A k using the estimate g of the PSF of the optics of the microscope through a main deconvolution algorithm, obtaining the resulting image I of the entire plane of the sample: I = A k • g.