Method for analyzing a biological sample comprising determination of the spatial distribution of biomass along the optical axis
A method of analyzing a biological sample including biological agents and disposed in an analysis receptacle in a field of view of a holographic imaging system defining an acquisition focal plane, including, for each of a plurality of measurement times: acquiring a plurality of holographic images of the biological sample at different respective positions of the acquisition focal plane, and, from each acquired holographic image, determining a value of a biomass parameter representative of the quantity of biological agents at the position of the acquisition focal plane, the method including constructing a distribution indicator from values of the biomass parameter at the same measurement time for a plurality of positions of the acquisition focal plane, and providing, among the analysis results, a representation of the distribution of the biomass of biological agents, derived from at least one distribution indicator at a measurement time.
1 . A method for analyzing a biological sample by means of an analysis instrument, the biological sample comprising biological agents and being disposed in an analysis receptacle in a field of view of a holographic imaging system, the holographic imaging system defining an optical axis and an acquisition focal plane, the method comprising, for each measurement time from a plurality of measurement times of a measurement period:
the acquisition of a plurality of holographic images of the biological sample at various respective positions on the optical axis of the acquisition focal plane,
the determination, from each acquired holographic image, of a value of a biomass parameter representative of the quantity of biological agents at the position of the acquisition focal plane of said holographic image,
the method comprising the construction of a distribution indicator using values of the biomass parameter from the same measurement time for several positions of the acquisition focal plane, the distribution indicator being representative of the spatial distribution of the quantity of biological agents in the analysis receptacle along the optical axis at a measurement time, and the provision, from amongst the analysis results, of a representation of the distribution of the biomass of biological agents derived from at least one distribution indicator at one measurement time.
2 . The method as claimed in claim 1 , in which the distribution indicator is obtained by spatially organizing values of the biomass parameters according to the respective positions of the acquisition focal plane or the distribution indicator is obtained by a calculation applied to values of biomass parameters at various positions of the acquisition focal plane.
3 . The analysis method as claimed in claim 1 , in which the representation of the distribution of the biomass of biological agents is a representation of the time variation of the distribution of the biomass of biological agents which is derived from several distribution indicators at a plurality of measurement times.
4 . The analysis method as claimed in claim 3 , comprising the construction of the representation of the time variation of the distribution of the biomass of biological agents by spatially organizing distribution indicators according to their respective measurement times.
5 . The analysis method as claimed in claim 1 , in which the biomass parameter of a holographic image is derived from a statistic relating to the gray levels of the pixels of said holographic image and/or the biomass parameter is an average of the absolute values of the gray levels of each pixel of the holographic image.
6 . The analysis method as claimed in claim 1 , in which the determination of the biomass parameter of a holographic image comprises a prior normalization of the holographic image, comprising:
the determination of a background image by application of a smoothing filter to the acquired holographic image,
the division pixel to pixel of the gray levels of the acquired holographic image by the gray levels of the background image.
7 . The analysis method as claimed in claim 1 , in which the various respective positions on the optical axis of the acquisition focal planes number at least 10 in the analysis receptacle.
8 . The analysis method as claimed in claim 1 , in which the analysis receptacle comprises two opposing transparent faces situated at various positions on the optical axis, and the various respective positions on the optical axis of the acquisition focal planes extend from one transparent face to the other transparent face.
9 . The analysis method as claimed in claim 8 , in which at least one position of an acquisition focal plane on the optical axis is not situated between the two transparent faces, and/or a position of an acquisition focal plane on the optical axis corresponds to the position of a transparent face.
10 . The analysis method as claimed in claim 1 , in which the holographic imaging system defines a depth of field of at least 100 μm of depth in the direction of the optical axis around each acquisition focal plane.
11 . The analysis method as claimed in claim 1 , in which the plurality of holographic images are holograms or images reconstructed from holograms.
12 . An analysis instrument comprising a holographic system with a field of view configured for acquiring a holographic image and data processing means, the analysis instrument being configured for receiving a biological sample in an analysis receptacle in the field of view of the holographic system and for implementing the steps of the method as claimed in claim 1 .