Focus adjustment method for holographic imaging system

A focus adjustment method for acquiring an image of a surface of interest from a sample by an image sensor from an imaging system, comprising the following steps of:

• placing the sample in which the surface of interest is, in a field-of-view of an image sensor of the imaging system, the sample comprising at least one physical reference object having a known shape and described by characterising parameters associated with the reference object, these characterising parameters comprising at least position parameters of the reference object, the reference object being at a position in correspondence with a position of the surface of interest,
• illuminating the sample by an illumination light and acquiring a holographic image by the image sensor at an acquisition plane, of interference patterns caused by the reference object,
• determining the position of the reference object with respect to the acquisition plane, using a light diffraction model by the at least one reference object, the light diffraction model being based on the known shape of the reference object and involving the characterising parameters of the reference object, at least the position parameters of the reference object being estimated by adjustment in the light diffraction model to approximate the interference patterns caused by the reference object appearing in the holographic image,
• determining the position of the surface of interest with respect to the acquisition plane from a position of the reference object defined by the position parameters,
• performing focus adjustment of the image acquisition based on the position of the surface of interest with respect to the acquisition plane, focus adjustment consisting in making the positions of the surface of interest and of an object focal plane coincide, corresponding to the acquisition plane.