Analysis method including the determination of a position of a biological particle

A method for analyzing a sample receiving biological particles, among which is a particle of interest, the sample being arranged between a first light source and an optical system, the optical system carrying out an optical conjugation between an object plane and an image plane wherein is located an image sensor, and the sample being located inside a fluidic chamber delimited on a side of the image sensor by an upper slide, the method comprising:

• illuminating a reference point located on a face of the upper slide by a laser beam focused by the optical system;
• using the image sensor to receive an image of a spot formed by of the laser beam, wherein the laser beam, the image sensor, and the optical system are arranged such that a focusing point of the laser beam is in the object plane;
• adjusting a distance, along an axis parallel to the optical axis of the optical system, between the sample located in the fluidic chamber and the optical system based upon the image and by detecting specular reflection of the laser beam on the upper slide, wherein the reference point is positioned in the object plane of the optical system when said specular reflection is detected;
• offsetting the object plane of the optical system relatively to the reference point, along an axis parallel to the optical axis of the optical system, by a known distance as a useful distance, and so as to place the particle of interest outside of the object plane of the optical system;
• using the first light source, illuminating of a region receiving the particle of interest, as an illuminated region;
• using the image sensor, acquiring a holographic image of the illuminated region, as a reference image or defocused image;
• using the reference image, digitally constructing a series of reconstructed images, each associated with a simulation of a predetermined offset of the object plane along the optical axis of the optical system;
• and
• using the series of reconstructed images, determining a distance along an axis parallel to the optical axis of the optical system, between the particle of interest and the object plane.