Abstract: A focus adjustment method for acquiring an image of a surface of interest of a sample by a holographic imager includes the steps of: placing the sample including at least one reference object having a known shape and described by characterising parameters having at least position parameters acquiring an image and determining the position of the reference object with respect to the acquisition plane, by applying a light diffraction model involving the spatial parameters of the reference object estimated by approximating the appearance of the reference object in the holographic image acquired, and determining the position of the surface of interest with respect to the acquisition plane from a position of the reference object and focus adjustment of the image acquisition.