Method for analysing a sample (111; 311) receiving biological particles, among which a particle of interest, wherein the sample is arranged between a first light source (120; 320) and an optical system (131; 331), the optical system (131; 331) carrying out an optical conjugation between an object plane (133; 333) and an image plane in which an image sensor (132; 332) is located, and wherein the sample is arranged inside a fluidic chamber delimited by an upper slide (114; 314) on the side of the image sensor, the method comprising the following steps: - defining a reference point (118; 318) located on a face (117; 317; 315) of said upper slide (114; 314), and illuminating the reference point (118; 318) with a laser beam which is focused by the optical system (131; 331) and arranged in such a way that the image sensor (132; 332) receives the image of a spot formed by specular reflexion of the laser beam on the upper slide (114; 314), wherein the laser beam, the image sensor and the optical system are arranged in such a way that the focalisation point of the beam is in the object plane; - adjusting a distance, along an axis parallel to the optical axis of the optical system, between the sample arranged in its fluidic chamber and the optical system, by identifying the specular reflexion and in such a way that the reference point is located in the object plane of the optical system; - offsetting the object plane (133; 333) of the optical system with respect to the reference point (118; 3185), along an axis parallel to the optical axis of the optical system, by a known distance named useful distance, and so as to place the particle of interest outside the object plane (133; 333) of the optical system; - using the first light source, illuminating a region receiving the particle of interest, referred to as illuminated region; - using the image sensor (132; 332), acquiring a holographic image of the illuminated region, referred to as reference image or defocused image; - using the reference image, digital construction of a series of reconstructed images, each one associated with a simulation of a predetermined offset (Di 1, Di 2, Di n, Di 1, Di -n) of the object plane along the optical axis of said optical system; - using the series of reconstructed images, determining the distance (D ref) along an axis parallel to the optical axis of the optical system, between the particle of interest and said object plane. - defining a reference point (118; 318) located on a face (117; 317; 315) of said upper slide (114; 314), and illuminating the reference point (118; 318) with a laser beam which is focused by the optical system (131; 331) and arranged in such a way that the image sensor (132; 332) receives the image of a spot formed by specular reflexion of the laser beam on the upper slide (114; 314), wherein the laser beam, the image sensor and the optical system are arranged in such a way that the focalisation point of the beam is in the object plane; - adjusting a distance, along an axis parallel to the optical axis of the optical system, between the sample arranged in its fluidic chamber and the optical system, by identifying the specular reflexion and in such a way that the reference point is located in the object plane of the optical system; - offsetting the object plane (133; 333) of the optical system with respect to the reference point (118; 3185), along an axis parallel to the optical axis of the optical system, by a known distance named useful distance, and so as to place the particle of interest outside the object plane (133; 333) of the optical system; - using the first light source, illuminating a region receiving the particle of interest, referred to as illuminated region; - using the image sensor (132; 332), acquiring a holographic image of the illuminated region, referred to as reference image or defocused image; - using the reference image, digital construction of a series of reconstructed images, each one associated with a simulation of a predetermined offset (Di 1, Di 2, Di n, Di 1, Di -n) of the object plane along the optical axis of said optical system; - using the series of reconstructed images, determining the distance (D ref) along an axis parallel to the optical axis of the optical system, between the particle of interest and said object plane.