Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
  • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
  • G01N21/453—Holographic interferometry
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1434—Optical arrangements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N2015/1006—Investigating individual particles for cytology
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1434—Optical arrangements
  • G01N2015/1454—Optical arrangements using phase shift or interference, e.g. for improving contrast
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N2015/1486—Counting the particles

Definitions

• the present invention relates to the field of the analysis of biological samples by imaging, and more particularly relates to the monitoring over time of the spatial distribution of biomass along the optical axis.
• a biological sample consists of a suspension of biological agents.
• Biological agents are, for example, microorganisms (bacteria, yeasts, molds, etc.).
• the analysis of a biological sample is an in vitro analysis of the biological sample.
• the analysis of a biological agent in the biological sample can comprise the identification of said biological agent or the determination of a characteristic of this biological agent, such as for example the minimum inhibitory concentration of an antibiotic which would be effective at against said biological agent or else the ability to form a biofilm.
• the biological sample called inoculum in its initial state, is placed in a receptacle, or well, at least partially transparent through which the analysis instrument can carry out measurements of optical properties of the biological sample.
• the well contains a nutrient medium and also one or more reagents, such as an enzymatic substrate or antibiotics, intended to interact with biological agents present in the biological sample.
• reagents such as an enzymatic substrate or antibiotics
• a plurality of wells are provided to each receive the inoculum, each of the wells being provided with different reagents or with the same reagent at different concentrations. Depending on the nature of the biological agents present in the inoculum, these react with certain reagents, and not with other reagents, or at certain concentrations and not at others.
• the reagents consist of different antibiotics at different concentrations, and the biological agents will multiply in the wells containing the antibiotics to which they are not sensitive or in which the concentration of antibiotics is insufficient, or on the contrary will see their development more or less hampered in the wells containing the antibiotics to which they are sensitive in sufficient concentrations.
• the biomass i.e. the quantity of biological material present in each well, directly influences the optical properties of the biological sample present in each well, since the biological agents themselves present different optical properties from the solution. in which they are suspended.
• the transmittance of the biological sample is affected by the evolution of the concentration of biological agents.
• methods for the analysis of biological samples had been developed based on the determination of the evolution over time, during an incubation phase, of the overall transmittance (or absorbance, which is equivalent) of a well filled with the biological sample, in order to determine a turbidity measurement, typically expressed in McFarland (McF).
• McFarland McFarland
• This turbidity measurement is directly representative of the biomass of biological agents in the biological sample.
• an emitting diode illuminates the sample with a light beam of known intensity, and a point photodiode arranged opposite the emitting diode with respect to the sample makes it possible to determine the light intensity received after the light beam has passed through the biological sample.
• Such a transmittance measurement has a fairly low sensitivity, such that it is not possible to measure a turbidity of less than 0.05 McF, or even less than 0.1 McF.
• the biomass does not always make it possible to determine the concentration of biological agents: in the event of an increase in the volume of the biological agents, for example by elongation for bacteria, the biomass increases for the same number of biological agents.
• the spatial distribution of the growth of the biomass of biological agents is then affected at least temporarily by the spatial heterogeneity of the reactants, but an overall measurement does not make it possible to account for this.
• heterogeneity can indicate that a biological agent is likely to form a biofilm, in which case measures Special measures can be taken, for example by adapting a medical treatment to the presence of a biofilm.
• the invention therefore aims to make it possible, during the analysis of a biological sample, to have available for the latter the evolution over time of the spatial distribution of the biomass of biological agents along an optical axis. , for example in order to highlight characteristics of biological agents such as the ability to form a biofilm.
• the invention proposes a method for analyzing a biological sample by means of an analysis instrument, the biological sample comprising biological agents and being placed in an analysis receptacle in a field of view of a holographic imaging system, said holographic imaging system defining an optical axis and an acquisition focal plane, the method comprising, for each measurement instant of a plurality of measurement instants of a duration of measure :
• the method comprising the construction of a distribution indicator based on values of the biomass parameter of a measurement instant for several positions of the acquisition focal plane, said distribution indicator being representative of the spatial distribution of the quantity of biological agents in the receptacle of analysis along the optical axis at said instant of measurement, and the provision, among the analysis results, of a representation of the distribution of the biomass of biological agents derived from at least one indicator of distribution at an instant of measurement.
• the distribution indicator is obtained by spatially organizing biomass parameter values according to the respective positions of the acquisition focal plane or the distribution indicator is obtained by a calculation relating to biomass parameter values at different positions the acquisition focal plane;
• the representation of the distribution of the biomass of biological agents is a representation of the temporal evolution of the distribution of the biomass of biological agents which is derived several distribution indicators at a plurality of measurement instants;
• the method comprises the construction of the representation of the temporal evolution of the distribution of the biomass of biological agents by spatially organizing distribution indicators according to their respective measurement instants;
• the biomass parameter of a holographic image is derived from a statistic relating to the gray levels of the pixels of said holographic image
• the biomass parameter is an average of the absolute values of the gray levels of each pixel of the holographic image
• the determination of the biomass parameter of a holographic image includes a prior normalization of the holographic image, comprising:
• the different respective positions on the optical axis of the acquisition focal planes are at least 10 in number in the analysis receptacle, and preferably at least 20;
• the analysis receptacle comprises two opposite transparent faces located at different positions on the optical axis, and the different respective positions on the optical axis of the acquisition focal planes extend from one transparent face to the other face transparent;
• At least one position of an acquisition focal plane on the optical axis is not located between the two transparent faces, and/or one position of an acquisition focal plane on the optical axis corresponds to the position of a transparent face;
• the holographic imaging system defines a depth of field of at least 100 ⁇ m in the direction of the optical axis around each acquisition focal plane;
• the holographic image is a hologram or an image reconstructed from a hologram.
• the invention also relates to an analysis instrument comprising a holographic system with a field of view configured to acquire a holographic image and data processing means, the analysis instrument being configured to receive a biological sample in a receptacle of analysis in the field of view of the holographic system and to implement the steps of the method according to the invention.
• FIG. 1 shows an example of an analysis card comprising a plurality of receptacles in the form of wells that can be used for the placement of a biological sample to be analyzed, according to a possible embodiment of the invention
• FIG. 2 schematically shows an example of a holographic imaging system that can be used in an analysis instrument according to a possible embodiment of the invention
• FIG. 3 is a diagram illustrating steps of the analysis method according to a possible embodiment of the invention.
• FIG. 4 is a schematic view of a section of an analysis receptacle along a plane containing the optical axis
• FIG. 5 is an example of a graphical representation of the evolution over time of the spatial distribution of biomass of biological agents along an optical axis.
• the method of analyzing a biological sample is carried out using an analysis instrument comprising a holographic imaging system with a field of view, the analysis instrument being configured to receive a biological sample in a receptacle analysis in the field of view of the holographic imaging system.
• Figure 1 shows an example of an analysis card 1 comprising a plurality of analysis receptacles 2 in the form of wells that can be used for placing a biological sample to be analyzed.
• the analysis receptacles 2 are here organized according to a two-dimensional network on a plane, each receptacle 2 being associated with different analysis conditions, typically by means of different reagents present in the analysis receptacles 2.
• the reagents are made up of different antibiotics at different concentrations.
• the use of an analysis card 1 is not required, but such an analysis card 1 makes it possible to proceed during the same analysis period to a plurality of tests in a standardized way.
• Each analysis receptacle 2 is at least partially transparent to at least one wavelength of light, visible or not, and preferably is at least partially transparent to the visible spectrum. This transparency allows the analysis of the sample biological material contained therein by optical means such as the holographic imaging system.
• an analysis receptacle 2 has at least two opposite transparent faces 2a, 2b, so as to have a transparent axis for the propagation of light. These two opposite transparent faces 2a, 2b are for example less than 10 mm apart, and preferably less than 5 mm.
• the opposite transparent faces 2a, 2b are separated, along the optical axis 16, by more than 0.1 mm, and preferably by more than 0.5 mm, and more preferably by more than 1 mm.
• the opposite transparent faces 2a, 2b are transparent films defining the analysis receptacle 2. It is common for the reagents to be attached to at least one of the transparent faces. The reagents can thus be introduced into the analysis receptacle 2 by placing them on the film intended to form a transparent face 2a, 2b, before the latter is applied to the analysis card 1.
• such an analysis card 1 may for example comprise a conduit 5 intended to be immersed in a volume 3 of inoculum 3 prepared in a tube 4.
• the inoculum is prepared by an operator who introduces biological agents, for example taken from a culture in a Petri dish by means of a rod or a swab, suspended in a saline solution, with a dilution corresponding to a determined range of turbidity, for example between 0.5 and 0.63 McF for bacteria as biological agents or else between 1.8 and 2.2 McF for yeasts as biological agents, the range depending on the type of analysis carried out and the measuring instrument.
• This preliminary suspension is then further diluted, for example with a factor of 20, or even 100, to analyze Gram- bacteria or with a factor of 10, or even 100, to analyze Gram+ bacteria.
• This subsequent dilution can in particular be automated, and therefore be carried out by the measuring instrument after the tube 4 has been placed in the analysis instrument.
• Other determined ranges of turbidity can be used, depending on the protocols used.
• the desired dilution can be obtained all at once, or as in the example above, in several times.
• conduit 5 is then immersed in the volume 3 of inoculum resulting from the preparation in tube 4, and the assembly is introduced into the analysis instrument. Of course, all or part of these preparation steps can be automated.
• the inoculum travels through the conduit 5, then by a fluidic circulation circuit provided in the analysis card 1, is distributed between the analysis receptacles 5. This movement of the inoculum in the conduit 5 and the card d analysis 1 can be caused by capillarity and/or by depressurization of the air present at the open end of the tube 4.
• the air present in the analysis card 1 which is at atmospheric pressure, leaves the analysis card 1 by the tube 5 through the inoculum 3 and gives way to the inoculum 3 which thus goes up by the tube 5 in the analysis card 1.
• the biological sample constituted by the inoculum is then in place in an analysis receptacle 2.
• the analysis instrument includes a holographic imaging system with a field of view configured to acquire a holographic image of that field of view.
• the acquisition of a holographic image allows a significant depth of field, and therefore a very good detection sensitivity of biological agents.
• the holographic imaging system is placed facing an analysis receptacle 2.
• FIG. 2 schematically represents a holographic imaging system 10 in line arranged so that the field of view 11 of said holographic imaging system 10 is contained in the volume of biological sample contained in an analysis receptacle 2.
• the analysis card 1, and therefore the analysis receptacles 2 that it comprises, is placed in an object plane of the holographic imaging system 10.
• the holographic imaging system 10 defines an imaging axis 16, simplified here by a straight line corresponding to the optical axis but which can consist of a set of successive lines defining the light path, depending on the configuration of the optical components of the holographic imaging system 10.
• a light source 4 configured to illuminate the analysis receptacle 2 in the field of view (or "field-of-view") of the holographic imaging system 10 by means of an illumination beam of sufficiently coherent light.
• the light source 14 can produce the illumination light, or simply be the termination of an optical fiber conveying this illumination light, optionally provided with a diaphragm or iris.
• the illumination beam has the conventional characteristics for holographic imaging, without particular additional constraints.
• the illumination beam can thus be monochromatic (for example with a wavelength around 640-670 nm) or possibly be composed of several wavelengths, for example used one after the other.
• an image sensor 12 On the other side of the analysis receptacle 2, here on the optical axis 16, is an image sensor 12, which is a digital sensor such as for example a CMOS or CCD sensor.
• the image sensor 12 is placed on an image plane of the holographic imaging system 10, and is configured to acquire a hologram, that is to say a spatial distribution of intensity of interference caused by interactions between the inoculum placed in the field of view 11 and the illumination beam.
• the holographic imaging system 10 is here provided with a set of optical components 18 arranged between the analysis receptacle 2 and the digital image sensor 12 such as for example a microscope objective 18a and a tube lens 18b in the example shown.
• An optical component such as the microscope objective 18a is however optional, the invention not being limited to holographic microscopy with lens.
• the arrangement described here is of course a non-limiting example. Any holographic imaging system 10 can be used, with different optical elements (with or without a microscope objective, etc.). Thus, when a holographic imaging system 10 can acquire an image in which the interference patterns generated by the biological sample appear, this holographic imaging system is suitable for implementing the method.
• the holographic imaging system 10 is configured to define a depth of field of at least 100 ⁇ m in depth in the direction of the optical axis 16 around each acquisition focal plane 20, and preferably at least 150 ⁇ m, and more preferably at least 250 ⁇ m.
• the analysis receptacle 2 comprises two opposite transparent faces 2a, 2b organized along the optical axis 16, and the depth of field extends over at least 100 ⁇ m between the two opposite transparent faces of the analysis receptacle. , and preferably over at least 150 ⁇ m, and more preferably over at least 250 ⁇ m, or even at least 300 ⁇ m.
• the field of view 11 is understood as being the space in which the presence of biological agents can be determined from a hologram imaging said field of view 11.
• the measuring instrument also includes components for processing data, such as a processor, memory, communication buses, etc. Insofar as these other components are specific only by the process that they implement and by the instructions that they contain, they will not be detailed below.
• FIG. 3 is a diagram illustrating the steps of the analysis method, which follow a prior placement (step S1) of the biological sample in an analysis receptacle 2 in the field of view 11 of a system holographic imaging 10, detailed above.
• the method comprises a plurality of cycles (steps S02) consisting of steps implemented repeatedly for a plurality of measurement instants of a measurement duration:
• These cycles are typically repeated over a period ranging from one minute to 30 minutes, depending on the speed of the analysis instrument, the number of biological samples processed in parallel, and for example depending on the number of receptacles of analysis 2 in an analysis map 1.
• the measurement duration extends over several hours, and typically more than 10 hours, resulting in several tens or even several hundreds of measurement instants.
• the acquisition of a plurality of holographic images of the biological sample at different respective positions on the optical axis 16 of the acquisition focal plane involves a displacement of the acquisition focal plane (step S02c) prior to each acquisition of a holographic image by the image sensor 12 (step S02a).
• These different positions of the acquisition focal plane can be obtained for example by moving the assembly formed by the image sensor 12 and the optical elements 18a, 18b, for example via a motorized rail or a motorized stage. It is also possible to obtain these different positions of the acquisition focal plane 20 by modifying an optical component of the holographic imaging system 10 moving the acquisition focal plane, by modifying a focusing of light rays incident on the sensor image 12 so as to move the acquisition focal plane of the image sensor 12.
• the various respective positions on the optical axis 16 of the acquisition focal planes are at least 2 in number in the analysis receptacle, preferably at least 5, and more preferably at least 15, although only six between them are illustrated in Figure 4, for the purpose of simplification.
• a greater number of acquisition planes makes it possible to refine the analysis of the heterogeneity of the spatial distribution of biological agents along the optical axis 16.
• the focal acquisition planes extend preferably substantially perpendicular to the optical axis 16, so that the different respective positions on the optical axis 16 correspond to as many different depths in the analysis receptacle 2.
• the analysis receptacle 2 typically comprises two opposite transparent faces 2a, 2b located at different positions on the optical axis 16, and different respective positions 20a, 20b, 20c, 20d, 20e, 20f on the optical axis 16 of the acquisition focal planes 20 extend from one transparent face 2a, 2b to the other transparent face 2a, 2b.
• different respective positions 20a, 20b, 20c, 20d, 20e, 20f are regularly spaced along the optical axis 16 between the two transparent faces 2a, 2b opposites.
• the various respective positions 20a, 20b, 20c, 20d, 20e, 20f of the acquisition focal plane are spaced apart so that each image acquired reveals different biological agents.
• the different respective positions 20a, 20b, 20c, 20d, 20e, 20f of the acquisition focal plane on the optical axis 16 extend over a depth, along the optical axis 16, greater than 0.1 mm, and preferably greater than or equal to 0.5 mm, and preferably greater than or equal to 0.8 mm.
• consecutive positions 20a, 20b, 20c, 20d, 20e, 20f of the acquisition focal plane on the optical axis 16 are spaced apart by at least 50 ⁇ m, preferably by at least 100 ⁇ m, and preferably still at least 150 iim.
• the zones close to the two transparent faces 2a, 2b constitute privileged zones for analyzing the spatial distribution of the biological agents.
• these faces also constitute mechanical supports on which the biological agents can develop a biofilm, a particularly interesting characteristic to detect.
• a position of an acquisition focal plane on the optical axis 16 corresponds to the position of a transparent face 2a, 2b.
• the holographic imager 10 acquires a hologram, which has the advantage of offering a great depth of field, and therefore a great sensitivity for detecting biological agents in the biological sample.
• the holographic imaging system acquires a hologram.
• the light source 14 emits a reference illumination beam, which can be translated into a reference plane wave propagating in the Z direction along the imaging axis 16.
• the biological agents present in the field of view 11 inside the analysis receptacle 2 by their diffraction properties, scatter the incident reference light.
• the wave scattered by the biological agents and the reference background interfere on the image sensor 12 to form the hologram.
• the hologram corresponds to the spatial intensity distribution of the total field corresponding to the addition of scattered background and reference background. , which is translated into a gray level value for each pixel.
• the holographic image used may be the hologram or may be an image reconstructed by backpropagation calculation from the hologram, using an algorithm of propagation for example based on Rayleigh Sommerfeld's diffraction theory.
• Using the hologram without reconstruction makes it possible to benefit from a high detection sensitivity, because each biological agent appears in the hologram surrounded by rings corresponding to the interference figures caused by the presence of said biological agents, thus facilitating detection. the presence of these biological agents.
• the non-reconstruction saves time and computational resources.
• using a reconstructed image has other advantages, such as making it possible to precisely locate, possibly in three dimensions, the biological agents appearing in the reconstructed image.
• a reconstructed image can be defined by gray levels for each pixel.
• 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 more preferably, the biomass parameter is an average of the absolute values of the gray levels of each pixel of the holographic image.
• Other biomass parameters can however be used, such as for example a count of biological agents appearing in the holographic image, or even the proportion of the holographic image in which biological agents appear or not.
• the determination of the biomass parameter of a holographic image comprises a prior normalization of the holographic image, comprising:
• a smoothing filter preferably a Gaussian filter
• the smoothing filter parameters for example the standard deviation for a Gaussian filter, are chosen to create a low-pass filter with a cutoff frequency low enough that the filtered image is representative only of the background. plane of the holographic image, hence the name background image, without for example the biological agents being discernible in this filtered image.
• the normalization of the image may further comprise a subtraction of a constant operated on the gray levels of the holographic image resulting from the division. It is then possible to obtain a normalized holographic image with negative gray level values. It is therefore then the absolute values of the gray levels which are taken into account to determine the values of the biomass parameter.
• a distribution indicator is constructed (step S03) from values of the biomass parameter of the same measurement instant for several positions of the acquisition focal plane.
• the distribution indicator is representative of the spatial distribution of the quantity of biological agents along the optical axis at this instant of measurement.
• the distribution indicator can be obtained by spatially organizing the biomass parameters according to the respective positions of their acquisition focal planes.
• the distribution indicator takes the form of a concatenation of the values of the biomass parameter, and preferably takes the form of a spatial concatenation, in which the values of the biomass parameter are spatially concatenated.
• the distribution indicator can therefore be interpreted as a vector whose components account for the quantity of biological agents at different positions.
• the different values of the biomass parameter are organized according to the same spatial organization as the respective positions of the acquisition focal planes of the holographic images from which they come.
• the distribution indicator can correspond to a single value, which accounts for the homogeneity or heterogeneity of the spatial distribution of the quantity of biological agents along the optical axis at a time of measure.
• the distribution indicator can thus be obtained by a calculation relating to values of biomass parameters at different acquisition focal planes, and in particular by differences between these values.
• a distribution indicator can correspond to a difference between at least one value of the biomass parameter with an acquisition focal plane close to the middle of the well (on the optical axis), and at least one value of the biomass parameter with an acquisition focal plane close to a transparent face 2a, 2b.
• Other calculations are possible, such as for example a standard deviation, a variance, or other.
• a first holographic image is acquired with an acquisition focal plane 20 at a first position 20a
• a second holographic image is acquired with an acquisition focal plane 20 at a second position 20b
• a third holographic image is acquired with an acquisition focal plane 20 at a third position 20c, etc., until a holographic image has been acquired for each position 20a, 20b, 20c, 20d, 20e, 20f of the acquisition focal plane 20 along the optical axis.
• a value of the biomass parameter is determined for each holographic image, and therefore is associated with each position 20a, 20b, 20c, 20d, 20e, 20f: a first value for the first position 20a, a second value for the second position 20b, a third value for the third position 20c, etc.
• the first value is therefore placed adjacent to the second value, itself placed adjacent to the third value, itself placed adjacent to the fourth value, etc.
• the second value is therefore between the first value and the third value, just as the second position 20b is between the first position 20a and the third position 20c.
• the spatial organization of the respective positions of the acquisition focal planes is therefore repeated.
• Each measurement cycle therefore makes it possible to obtain a distribution indicator representative of the spatial distribution of the quantity of biological agents along the optical axis 16 at the measurement instant in which the cycle takes place.
• the measurement cycles are typically repeated over a period ranging from one minute to 30 minutes, depending on the speed of the analysis instrument, the number of biological samples processed in parallel, and for example depending on the number of analysis receptacles 2 in an analysis card 1, but also depending on the speed of the interactions between the biological agents and the reagents.
• the analysis time typically extends over several hours, and the method thus typically comprises more than 10 cycles, and preferably more than 20 measurement cycles during this measurement time.
• the method then preferably comprises the construction (S04) of a representation of the distribution of the biomass of biological agents, which is derived from at least one distribution indicator at a measurement instant.
• the representation of the distribution of the biomass of biological agents is a representation of the temporal evolution of the distribution of the biomass of biological agents which is derived from several distribution indicators at a plurality of measurement instants.
• This representation of the distribution of the biomass of biological agents can be constructed by spatially organizing distribution indicators according to their respective measurement instants. Indeed, each distribution indicator corresponds to a particular measurement instant.
• the combination of the distribution indicators therefore makes it possible to follow the temporal evolution of the spatial distribution of the biomass of biological agents along the optical axis 16.
• it is a concatenation of distribution indicators , organized chronologically.
• Figure 5 shows an example of representation of this temporal evolution.
• the biological sample imaged is a saline suspension of Pseudomonas aeruginosa as biological agents.
• 27 holographic images were acquired at 27 acquisition focal plane positions along the optical axis, corresponding to 27 different depths.
• the measurement times are spaced 15 minutes apart.
• the ordinate axis is that of the positions in Z along the optical axis, corresponding to the depth
• the abscissa axis is that of the time, in minutes.
• the bottom of the figure therefore corresponds to a transparent face 2a, 2b of the analysis receptacle 2, and the top of the figure corresponds to the other transparent face 2a, 2b, while the left of the figure corresponds to the start of the duration of measurement and the right of the figure corresponds to the end of the measurement duration.
• This figure corresponds to a representation of the distribution of the biomass of biological agents in graphical form, derived from the distribution indicators at several measurement instants, which was obtained by translating the values of the biomass parameter by different shades of gray.
• the Pseudomonas aeruginosa bacteria develop in the form of structured aggregates called biofilms, which first extend over the surfaces of the analysis receptacle 2.
• the spatial distribution of the biological agents in the analysis receptacle is important information, which can for example constitute an additional indicator for identifying the biological agents of the sample, or for evaluating the interactions with reagents arranged on one face of the analysis receptacle 2, or even to reveal the ability of the biological agent to form a biofilm.
• the method comprises the supply, among the analysis results (step S05), of the representation of the distribution of the biomass of biological agents derived from at least one distribution indicator at a measurement instant, for example to provide information on the spatial distribution of biological agents at a measurement instant.
• This representation of the distribution of the biomass of biological agents can for example be provided in graphical form, such as an image or a curve or a table. It can be provided in the form of numerical values, for example corresponding to one or more distribution indicators. It is also possible that the representation of the distribution of the biomass of biological agents is simply one of the distribution indicators, in which case no construction step may be necessary, except for example if it is desired to provide this graphically. representation of the distribution of the biomass of biological agents. Thus, even with a single distribution indicator consisting of values of the biomass parameter, it is possible to construct a curve showing the distribution of the biomass of biological agents along the optical axis.
• a representation of the temporal evolution of the distribution of the biomass of biological agents can for example be obtained by spatially organizing indicators of distribution according to their respective measurement instants.
• the image in Figure 5 is an example of such a representation in graphical form.
• this representation of the distribution of the biomass of biological agents can take another form, such as for example a curve or a table or even a numerical value, as mentioned previously.
• the representation can be a table or a list chronologically organizing said numerical values of the distribution indicators, or representing it in the form of curves.
• the representation of the distribution of the biomass of biological agents can also result from calculation relating to values of distribution indicators, such as for example differences between these values.
• the representation of the distribution of the biomass of biological agents is understood here as allowing its communication to an operator, and its interpretation by the latter.
• the representation of the distribution of the biomass of biological agents can for example be displayed by a display screen, put in a format allowing its display, or can be transmitted to a printer to be printed.

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