Method and System for Characterizing Microorganisms by Digital Holographic Microscopy

A method for characterizing microorganisms present in a biological sample, comprising:

• a. acquiring a defocused digital holographic image by means of a microscopic imaging acquisition device with a coherent or partially coherent light source, said device being configured to form interference patterns on a matrix image sensor between the light source and the light diffracted by the sample;
• c. computer generating a focused image by applying a digital model for reconstructing a focused image to the defocused digital holographic image;
• d. characterizing the microorganisms as a function of the focused image,
• characterized in that:
• a.1. acquiring the defocused digital holographic image includes providing, in the field of view of the acquisition device corresponding to the defocused digital holographic image, a plurality of calibration objects distinct from the microorganisms present in the biological sample, the calibration objects
• being previously characterized in terms of size and in terms of refractive index;
• having dimensions that are selected so as to produce interference patterns on the matrix image sensor;
• having a shape that is selected such that said interference patterns can be computed using a wavefront propagation model integrating optical aberrations of the acquisition device;
• b. before computer generating the focused image, the method comprises:
• identifying calibration objects in the defocused digital holographic image;
• computing the interference patterns of the calibration objects by applying the wavefront propagation model, and quantifying the optical aberrations of the acquisition device as a function of the interference patterns of the calibration objects in the defocused holographic image and the computed interference patterns;
• c.1. the digital model for reconstructing the focused image integrates the quantified optical aberrations,
• wherein the computation of the aberrations is carried out computationally using a reverse parametric approach using the resolution of a problem according to the following relationships in order to obtain an aberration correction function p(x,y),
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• is a parametric propagator at the distance z6 between the surface of interest (6) and a plane of the sensor (2), like the Lorenz-Mie model, and p(x,y) is the correction function of the aberrations.