• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    The invention relates to an object observation system (1) comprising: - a light source (3), - a support (12) adapted to receive a translucent or opaque substrate, - a detector (7) capable of harvesting the backscattered light resulting from the interaction between the light emitted by the light source (3) and the objects, - a polarization separator (9), and - a quarter-wave plate (10), the separator (9) and the quarter-wave plate (10) being arranged so that the separator (9) directs the light emitted by the light source (3) towards the solid substrate and directs the backscattered light resulting from the interaction between the emitted light by the light source (3) and the objects towards the detector (7).
    公开号:FR3030748A1
    申请号:FR1462645
    申请日:2014-12-17
    公开日:2016-06-24
    发明作者:Valentin Genuer；Pierre Marcoux；Emmanuelle Schultz
    申请人:Commissariat a lEnergie Atomique CEA；Commissariat a lEnergie Atomique et aux Energies Alternatives CEA；
    IPC主号:
    专利说明:

    [0001] The present invention relates to an object observation system. It is known documents US 2008/0310692 Ai, US 7 465 560 B2 and EP 2 122 326 B1 to determine a biological species of a bacterium by studying the transmission figure obtained by the scattering of incident photons by the bacterium, the bacterium being on a substrate. However, such a transmission figure can not be obtained when the substrate comprises blood because the low transmission coefficient of blood prevents the obtaining of the diffraction pattern. Indeed, in the aforementioned documents, obtaining transmission image does not work when the substrate and its support are opaque. There is therefore a need for an object observation system for observing objects on a non-transparent substrate.
    [0002] For this purpose, it is proposed an object observation system comprising a light source adapted to emit light polarized rectilinearly in a first direction, a support adapted to receive a substrate having a surface comprising objects, at least one one of the support and the substrate being translucent or opaque, a detector capable of harvesting backscattered light resulting from the interaction between the light emitted by the light source and the objects, a polarization separator capable of reflecting polarized light rectilinearly according to a second direction of polarization and adapted to transmit polarized light rectilinearly in a third direction, the second polarization direction being perpendicular to the third direction, and a quarter-wave plate.
    [0003] The separator and the quarter-wave plate being arranged so that the separator directs the light emitted by the light source towards the substrate and directs the backscattered light resulting from the interaction between the light emitted by the light source and the objects towards the detector. By the expression "to direct the light beam", it is understood to transmit or reflect the light beam. Thus, according to a first embodiment, the separator and the quarter-wave plate are arranged so that the separator reflects the incident light with respect to the separator towards the substrate and transmits the backscattered light resulting from the interaction between the light emitted by the light source and the objects towards the detector. According to another embodiment, the separator and the quarter-wave plate are arranged so that the separator transmits the incident light with respect to the separator towards the substrate and reflects the backscattered light resulting from the interaction between the light emitted by the light source and the objects towards the detector. According to particular embodiments, the system comprises one or more of the following characteristics, taken in isolation or in any technically possible combination: the objects are microorganisms, the substrate being a solid substrate, in particular an agar substrate, adapted to the growth of said microorganisms. - the first direction and the second direction are identical. the first direction is different from the second direction in which case the system comprises a polarization direction adjusting element, the polarization direction adjusting element being arranged between the light source and the separator, so that the light incident on the separator is polarized in the second direction. the substrate has a surface intended to interact with the light source light, the surface being smooth. - The system further comprises a sensor adapted to acquire an image of the objects. the light coming from the light source forms a beam, the system comprising an optical system capable of modifying the size of the beam. - which objects are objects whose size is less than one millimeter. - the objects are microorganisms. - The observation system further comprises a half wave plate adapted to change the polarization of light emitted by the light source. - The observation system further comprises a computer adapted to analyze the backscattered light detect by the detector to derive at least one feature of the objects. the observation system is devoid of optics interposed between the separator and the quarter-wave plate and between the quarter-wave plate and the substrate. the objects are part of a petri dish having a cylindrical body whose base surface is flat and a cylindrical cover whose base surface is flat and forms an angle with the base surface of the body between 0.1 degree and 15 degrees. Other features and advantages of the invention will appear on reading the following description of embodiments of the invention, given by way of example only and with reference to the drawings which are: FIG. 1, a diagrammatic view a first example of an observation system; - Figure 2, a schematic view of a part of the system of Figure 1 in operation; FIG. 3, a diffraction pattern obtained using the system of FIG. 1 for a 6-hour growth of a micro-colony of bacteria (Escherichia elbow number ATCC 11775) in a petri dish; FIG. 4, a diffraction pattern obtained using an observation system devoid of cube and quarter-wave plate for a 6-hour growth of a micro-colony of bacteria (Escherichia coli number ATCC 35421) in a Petri dish; FIG. 5, a schematic view of a second example of an observation system; Figure 6, a direct-space image obtained using the system of Figure 5 for a 6-hour growth of a micro-colony of bacteria (Escherichia elbow number ATCC 25922) in a Petri dish; FIGS. 7 to 12, a set of images illustrating a procedure for aligning and adjusting the size of the probe beam on a colony of bacteria for a 6-hour growth of a micro-colony of bacteria (Escherichia coli number ATCC 11775) in a petri dish; FIG. 13, a schematic view of a third example of an observation system; Figure 14, a diffraction pattern obtained for a 6-hour growth of a micro-colony of bacteria (Escherichia coli number ATCC 25922) in a Petri dish when the lid of the Petri dish is laid flat, and Figure 15, a diffraction pattern obtained for a 6 hour growth of a micro-colony of bacteria (Escherichia coli number ATCC 25922) in a petri dish when the petri dish cover is inclined by 1 degree. An object observation system, referred to as system 1, is shown in FIG.
    [0004] The system 1 makes it possible to observe a sample 11. The sample 11 comprises a set of particles and a substrate supporting the set of particles. By particles, it is understood micrometric particles, for example cells, microorganisms. In particular, the microorganisms may be contiguous to one another so as to form bacterial colonies, spores, fungi or yeasts. It is considered, for the rest, that the set of particles is located on the surface of a substrate. Each particle has a maximum extension in one direction. By maximal extension, it is understood a characteristic size of the set of organisms.
    [0005] This is for example the diameter of the circle in which the set of organisms is inscribed. In general, the invention applies to organisms or groups of organisms whose maximum extension is between 100 nanometers (nm) and 1 millimeter (mm), preferably between 1 micrometer (1m) and According to a particular embodiment, the system 1 is suitable for allowing the observation of bacterial colonies of micrometric size, the characteristic size of which is less than 1 mm, for example between 100 μm and 1 mm, or even between 50 μm and 500 lm. This allows observation at an early stage of colony development. It is then time-saving compared to methods based on observation of bacterial colonies of millimeter size.
    [0006] Moreover, the system 1 also makes it possible to analyze a diffraction pattern. The analysis of the diffraction pattern makes it possible to count, identify, sort or monitor (also referred to as "monitoring") the particles observed. In particular, the identification of the particles can be done on the basis of a comparison of each detected diffraction pattern with a previously established library of reference diffraction patterns. The substrate may be able to grow at least a portion of the body of organisms. Thus, the substrate is itself a culture medium or is placed in contact with a culture medium. According to a particular embodiment, the substrate is a solid substrate.
    [0007] The solid substrate is, for example, an agar medium. In the example of Figure 1, the sample is in the form of a petri dish. A petri dish has a cylindrical body whose base surface is a circle. The base surface is flat. In this case, the base area is normal to the vertical of the place.
    [0008] In the case of Figure 1, the object to be observed is on the surface of a blood agar thickness of about 4-5 mm contained in a petri dish of diameter 86 mm. The system 1 comprises a light source 3, a support 12 of the sample 11, a detector 7, a lens 8, a polarization separator 9 and a quarter-wave plate 10.
    [0009] The light source 3 is capable of emitting spatially coherent light.
    [0010] The light that the light source 3 is able to emit is also polarized rectilinearly in a first direction D1. For example, the light source 3 is a laser source capable of emitting a laser beam. According to a variant, the light source 3 is a light emitting diode (often designated by the acronym of LED for "Light Emitting Diode"). The culture medium is placed in an enclosure, the enclosure being itself disposed on a support 12. The support 12 is adapted to receive a substrate having a surface adapted for the growth of objects. The relative position of the support 12 and the light source 3 can be adjustable so that light emitted by the light source 3 has an interaction with objects having grown on the substrate. At least one of the support 12 and the substrate is opaque or translucent.
    [0011] For example, the substrate may be opaque, translucent or transparent. When the substrate is transparent, the support 12 is absorbent or diffusing to prevent reflection of light on the support 12. The substrate may consist of a transparent culture medium to which an opacifying agent has been added. The opacifying agent may in particular be a mineral powder, of the Kaolin (white clay) or titanium dioxide type, or an organic dye (Methylene Blue, Phenol Red, Bromophenol Blue, etc.). The detector 7 is able to collect backscattered light resulting from the interaction between the light emitted by the light source and the objects. The lens 8 is focused at infinity. This means that the detector 7 is positioned on its image focus. By the term "polarization separator" is meant a device capable of reflecting a polarization in a given direction and transmitting a polarization in a perpendicular direction. In the example of FIG. 1, the polarization separator 9 is able to reflect polarized light rectilinearly in a second direction D2 and suitable for transmitting polarized light rectilinearly in a third direction D3. The second direction D2 is perpendicular to the third direction D3. According to the particular example of FIG. 1, the polarization splitter 9 is a blade.
    [0012] In this example, the function of the separator 9 is to transmit only one direction of polarization and to reflect the other directions. The light incident on the beam is reflected by the separator 9, while the light reflected by the sample 11 is transmitted. According to another variant not shown, the polarization splitter 9 is a cube.
    [0013] The quarter-wave plate 10 is a type 214 phase-delay plate. The quarter-wave plate 10 has the property of transforming a rectilinear polarization into circular polarization and vice versa, a circular polarization in rectilinear polarization. The cube 9 and the quarter-wave plate 10 are arranged so that the cube 9 reflects the light emitted by the light source 3 towards the sample 11 and transmits the backscattered light resulting from the interaction between the light emitted by the light source 3 and the objects to the detector 7. The operation of the system 1 is now described. The light source 3 emits light towards the separator 9.
    [0014] The light emitted by the light source 3 is incident on the separator 9 with a polarization s. The whole of the separator 9 and the quarter-wave plate 10 form a polarization selection device whose operation is explained with reference to FIG. 2. The separator 9 separates the polarizations. More precisely, according to the example of FIG. 1, the separator 9 transmits the light whose direction of polarization is parallel to the plane of incidence, the plane of incidence being the plane containing the incident ray and the normal to the face considered. . The direction of polarization parallel to the plane of incidence is usually called polarization p. The separator 9 also reflects light whose direction of polarization is perpendicular to the plane of incidence. The direction of polarization perpendicular to the plane of incidence is usually called s polarization. When the light incident on the separator 9 is polarized rectilinearly according to a polarization s (part A in FIG. 2), the light emitted by the light source 3 is reflected towards the sample 11.
    [0015] The light then propagates toward the quarter-wave plate 10. The quarter-wave plate 10 converts the linear polarization of the light into a left circular polarization. The left circular polarized light then propagates to the sample 11 (see Part B in Figure 2).
    [0016] The light is then backscattered by the sample 11.
    [0017] A change in polarization direction then takes place due to a property of circularly polarized light reflecting on an interface. Indeed, on reflection on an interface, the direction of polarization of a circularly polarized light changes direction. Thus, a right circular polarization light beam is, after reflection, left circular polarization. The backscattered light is thus polarized with a circular polarization in the opposite direction to the incident circular polarization, namely circular right polarized (see part C in FIG. 2). The quarter wave plate 10 then converts the right circular polarization of the light into a p-type straight polarization. The p-type straight polarized light then propagates to the separator 9 (see Part D in Figure 2). The separator 9 transmits the p-polarized light so that light propagates to the lens 8.
    [0018] The lens 8 serves to form on the detector 7 an image of the interferences which are located at infinity. The light then propagates towards the detector 7. The detector 7 then records a diffraction pattern corresponding to the elastic scattering of the photons by the objects.
    [0019] According to one variant, the separator 9 transmits the light emitted by the light source towards the sample 11 and reflects the backscattered light, the polarization direction of which is D3, towards the detector 7. In general, the separator 9 is capable of directing the incident light from the light source towards the sample 11. It is also able to direct the backscattered light by the sample 11, whose direction of polarization is perpendicular to that of said incident light The term "direct" then refers to the fact of transmitting or reflecting light. An example of a diffraction pattern obtained for a 6-hour growth of bacteria in a Petri dish is illustrated in FIG. 3. In FIG. In this case, the substrate is COS sheep blood agar. The acronym COS refers to the English phrase "Columbia Blood Sheep" which means "Columbia sheep blood". For comparison, the same diffraction pattern is shown in Figure 4 for a system devoid of cube and quarter-wave blade. An unexploitable diffraction pattern is observed.
    [0020] The differences observed between FIGS. 3 and 4 are explained by the fact that the polarization management proposed for the system 1 makes it possible to eliminate all the parasitic reflections caused by the optical surfaces in the path of the beam emitted by the light source. 3 and retroreflected beam. It is observed that the diffraction pattern obtained with the system 1 is more easily exploitable. As a result, the system 1 makes it possible to observe diffraction patterns formed by localized particles on an opaque substrate or on a substrate placed on an opaque support. According to this example, rapid bacterial micro-colony identifications, on the surface of an opaque medium, in particular in only 6 hours of culture are possible using the system 1.
    [0021] The system 1 can also be used for macro-colonies and, more generally, for any type of particles as previously defined. In addition, the system 1 makes it possible to measure directly on the substrate on which the objects rest, particularly when the substrate is a culture medium placed in a Petri dish. This is simpler than microscopy techniques involving the transfer of objects on a substrate compatible with a measurement in transmission. Another application is the detection of mold on an opaque substrate. Preferably, the substrate is a sufficiently smooth culture medium, to avoid the multiplication of reflections or backscattering on the surface of the substrate, which generates, in the image, a noise detrimental to the quality of the measurement. By smooth, it is understood a roughness of preferably less than 100 lm rms (root mean square for square root of the average squares), better 50 lm at least locally, in the vicinity of a particle to be detected . According to one embodiment, the only component between the separator 9 and the sample 11 is the quarter-wave plate 10. This means that no other optical component is interposed between the separator 9 and the sample 11. This makes it possible to increase the quality of the images by avoiding the generation of spurious reflections that would be caused by optical systems, for example, lenses or lenses, arranged between the separator 9 and the sample 11. According to one embodiment, the components The terms "upstream" and "downstream" are defined in relation to the direction of light propagation. Thus, the beam shaping components are positioned upstream of the separator 9 when the components are positioned between the light source 3 and the first face of the separator 9.
    [0022] This makes it possible to improve the quality of the diffraction images obtained. According to one embodiment, the quarter-wave plate 10 is anti-reflection treated. This makes it possible to avoid spurious reflections on the quarter-wave plate 10. Advantageously, the first direction D1 and the second direction D2 are identical. To modify the first direction D1, it may be envisaged to turn the light source 3 around its optical axis or to insert a half-wave plate rotatable about its axis of revolution. The half-wave plate then has the function of adjusting the polarization of the beam emitted by the light source 3, so that the incident beam at the separator 9 is polarized rectilinearly in the second direction D2. Naturally, such a blade, acting as a polarization direction adjustment element, is not necessary if the beam directly emitted by the light source is directly linearly polarized along the second direction D2. According to the embodiments, the adjustment half-wave plate is or is not part of the light source 3. A half-wave plate forming part of the light source 3 has the advantage of being compact and already positioned on the light source. mounting. According to one embodiment, the system 1 further comprises a computer. The computer is, for example, adapted to compare diffraction patterns acquired by the detector 7 and to determine at least one characteristic relating to all the particles from the result of the comparison. According to one embodiment, the sample 11 is part of the system 1. Alternatively, the substrate is adapted to vary the optical index of at least a portion of the particles of all the particles. This makes it possible to increase the differences between the different diffraction figures.
    [0023] Thus, for example, the substrate comprises precipitating chromogenic substrates. A second example of system 1 is shown in Figure 5. For the following, it is defined a vertical direction and two transverse directions. Each of these directions is symbolized by the axes shown in Figure 5, namely the Z axis for the vertical direction, the X axis for the first transverse direction and the Y axis for the second transverse direction. As for the system 1 according to the first example, the system 1 illustrated in FIG. 5 comprises the light source 3, the support 12, the detector 7, the polarization separator cube 9 and the quarter-wave plate 10.
    [0024] The system 1 of FIG. 5 also comprises an optical density 2, a half-wave plate 4, a first lens 5, a splitter plate 15, a second lens 6, a third lens 8, a fourth lens 14, a sensor 13 and a first translation 16. The optical density 2 makes it possible to attenuate the optical power at the output of the light source 3.
    [0025] The optical density 2 is, according to the example of FIG. 5, a neutral density filter of optical density 4. According to the particular example, the light source 3 is a monochromatic laser source emitting at a wavelength of 532, 2 nanometers (nm). Preferably, the light source 3 emits in a wavelength range of between 250 nm and 1200 nm. In general, the wavelength must be less than the maximum extension of the object observed, while allowing the use of usual detection means. Wavelengths in the visible or near-infrared range are then preferred. In a variant, the wavelength of the laser beam is in a band of different wavelengths. The wavelength depends in particular on the organism to be observed and its sensitivity to illumination by a laser beam. The laser beam obtained has a diameter of 0.334 millimeters and a divergence of 2.07 mrad. The laser beam is single mode (TEM00) and linearly polarized.
    [0026] The laser beam has a power of 20.1 milliWatts (mW). The combination of the first lens 5 and the second lens 6 makes it possible to shape the beam of the light source 3 to obtain a range of beam sizes of between 30 microns and 250 microns (that is to say greater than or equal to equal to 30 microns and less than or equal to 250 microns).
    [0027] The first lens 5 is, according to the example of Figure 5, a biconcave lens focal length -20 mm. According to the example of FIG. 5, the second lens 6 is a biconvex focal lens +75.0 mm having undergone antireflection treatment for the wavelength range 400 nm - 700 nm.
    [0028] The separating blade 15 makes it possible to separate an incident beam into two beams. In this case, the separating blade 15 sends a portion of the light to the separator 9 and another portion to the sensor 13. The detector 7 is, according to the example of Figure 5, a CMOS type camera (CMOS being the acronym for "Complementary Metal Oxide Semiconductor" for "Complementary Metal Oxide Semiconductor").
    [0029] According to another variant, the detector 7 is a CCD type camera (acronym for the English expression "Charged Coupled Device"). The third lens 8 makes it possible to collect the backscattered light and to focus it towards the detector 7, as previously described. According to the second example, the third lens 8 is a biconvex zoom lens +40.0 mm having undergone antireflection treatment for the wavelength range 400 nm - 700 nm. According to the illustrated example, the separator 9 is able to interact with waves whose wavelength is between 420 nm and 680 nm. The fourth lens 14 makes it possible to collect the light coming from the substrate in the direct space and to focus it towards the sensor 13. According to the example of FIG. 5, the fourth lens 14 is a plano-convex lens of focal length +150, 0 mm having undergone antireflection treatment for the 350 nm -700 nm wavelength range. The support 12 comprises second translation means allowing the sample to move in two directions, namely the first longitudinal direction X and the second longitudinal direction Y. The sensor 13 makes it possible to image the sample 11 in direct space at the same time. The sensor 13 is, for example, a CMOS-type camera (CMOS being the acronym for "Complementary Metal Oxide Semiconductor" for "complementary metal oxide semiconductor"). The first translation 16 allows a displacement of all the elements of the light source 3, the optical density 2 and the first and second lenses 5 and 6 along the first transverse direction X. The displacement of the first translation 16 contributes to vary the size of the beam on the object between 30 microns and 1 millimeter. For example, the maximum stroke of the first translation 16 is between 150 mm and 200 mm with an accuracy of 1 micron. The operation of the system 1 according to the second example is similar to the operation of the system 1 according to the first example. The system 1 also makes it possible to obtain images in direct space of the sample 11 via the sensor 13.
    [0030] The sensor 13 makes it possible to have an indication of the size of the micro-colonies as well as their shape.
    [0031] As an illustration, it is assumed that a micro-colony is to be imaged. At first, the micro-colony is localized on a large field image. In the proposed context, a wide field image corresponds to a position remote from the waist of the beam. An example of such a figure is shown in Figure 6. In this figure, there are several micro-colonies in the dashed circle. A microcolony is surrounded by a first contour, circular in shape. A second oval contour surrounds two overlapping colonies. The micro-colonies appear as shadows on the sensor 13 because the light beam enters the agar and is backscattered. The dark spots correspond to the absorption of all or part of the backscattered light, by the particles present on the surface of the substrate. The diffusion in the substrate has the effect of depolarizing the light. Thus, a portion of the light backscattered by the substrate is depolarized and is not transmitted through the separator 9 to the detector 7. This portion of the backscattered light is reflected to the splitter plate 15, the splitter blade 15 directing it to the sensor 13. The image provided by the sensor 13 is a control image, allowing an overview of the particles examined. Such a control image makes it possible to ensure that particles are present in the observed field. The control image also makes it possible to detect the presence of overlapping particles, particularly when the particles are micro-colonies. Such overlaps lead to the observation of deformed diffraction patterns, which may be detrimental in applications where each diffraction pattern is used for identification purposes. The control image may also allow a coarse centering of the incident beam relative to the particles to be examined. Following this step, the image produced by the detector 7 is observed. The image produced has one or a plurality of elementary diffraction patterns, each elemental diffraction pattern corresponding to a particle or an agglomerate of particles.
    [0032] The beam is then progressively aligned with the micro-colony until an optimal interference figure is obtained in terms of the number of fringes and contrast of the fringes. By the expression "alignment", it is understood a displacement of the sample 11, relative to the incident beam, in at least one of the three directions, that is to say the first transverse direction X, the second transverse Y and the Z axis.
    [0033] FIGS. 7 to 12 are a set of images obtained by the detector 7 illustrating such a procedure for aligning and adjusting the size of the probe beam on a colony of bacteria for a 6 hour growth of the bacteria in a box of Petri.
    [0034] In Figure 7, it is a large field image in which it is possible to see three micro-colonies. In FIG. 8, it is observed that the beam is slightly offset with respect to the micro-colony to be imaged. Figure 9 corresponds to the case where the beam is centered with respect to the micro-colony to be imaged. For the implementation, by way of example, the two translations 16 and 12 are used by controlling the symmetry of the diffraction pattern acquired on the detector 7. Such a control is either manual or automatic, for example by implementing mathematical tools such as a Hough transform for the detection of circles. Figure 10 illustrates the case where the beam has a size in the plane of the sample 11 which is smaller than the micro-colony. It is observed that the interference pattern is off. As a result, the beam size is gradually increased (see Figure 11). The increase is, for example, implemented by moving the first translation in steps of 100 microns to 500 microns. The progressive increase is implemented until an optimum pattern is achieved, that is to say a pattern having a maximum of contrasting rings. Figure 12 illustrates such a pattern. The determination of the optimal pattern is done by the eye of the operator, or by image processing. The proposed procedure makes it possible to adapt the size of the beam to the size of the micro-colonies. For example, System 1 is able to perform measurement on objects of different sizes, from 1 micron to 500 microns, thanks to a unique and simple architecture giving access to a range of suitable beam sizes. The system 1 thus makes it possible to detect and locate the micrometric objects over a large field of view. This location is performed upstream of the measurement of the interference image in reflection; this makes the measurement easier and faster. A third example of system 1 is shown in FIG. 13.
    [0035] The third example of system 1 is similar to the second example of system 1. The remarks applying to the common elements are thus also valid for the third example of system 1. These remarks are not repeated. Only the differences are highlighted. Unlike the system 1 according to FIG. 5, the third example of system 1 does not include a separator plate, a fourth lens and a sensor.
    [0036] Sample 11 of the preceding examples is included in a Petri dish without lid. On the other hand, the chamber of the sample 11 of FIG. 13 has a cover 18. This makes it possible to avoid cross-contamination (that is to say contamination of the external environment crossed with contamination of the substrate, which means leads to the contamination of boxes by previously used boxes). This also avoids contamination of the operators. In such a situation, the cover 18 is preferably transparent. The cover 18 has an angle with respect to the horizontal. According to the example of Figure 13, this angle is 1 degree. The comparison of FIGS. 14 and 15 makes it possible to show the interest of such a configuration. Indeed, FIG. 14 is a diffraction pattern obtained for a 6-hour growth of a bacterium in a Petri dish when the cover of the petri dish is laid flat whereas FIG. 15 is a diffraction pattern obtained. for a 6-hour growth of a bacteria in a Petri dish when the Petri dish lid is inclined by 1 degree. The diffraction pattern is of better quality in terms of contrast of the fringes in the case of FIG. 15. More generally, the parasitic reflections from the lid of the box can be eliminated by tilting the lid 18 by a few degrees with respect to the horizontal, for example between 0.1 ° and 15 °, preferably between 10 and 5 °. Otherwise formulated, the base surface of the lid 18 which is of cylindrical shape forms an angle with the base surface of the body of the petri dish. This angle is between 0.5 ° and 50. In all the embodiments presented, the system 1 allows the identification of bacterial colonies on opaque nutrient media by elastic diffusion in reflection geometry. In particular, it becomes possible to use a substrate which is a blood agar. Otherwise formulated, the system 1 allows the acquisition of interference figures resulting from the reflection of a light beam on micro-organisms (single bacteria, micro-colonies, macro-colonies) and on micrometric objects resting on substrates. opaque. These substrates do not allow measurement in transmission as it is performed by the current devices. System 1 therefore makes it possible to extend the field of application to opaque substrates by proposing a non-destructive, automated and fast method.
    [0037] It has also been shown that the system 1 makes it possible to solve several difficulties. In particular, the system 1 makes it possible to quickly locate all the micro-colonies present on the petri dish (whose diameter is 86 mm). The system 1 also makes it possible to adapt the size of the beam of the light source 3 to the size of the micro-colonies, which sizes may vary according to the bacterial species between 30 microns and 250 microns. The size can also vary from several tens of microns for the same species and the same culture. The adaptation can be done within a series of measurements made on the same box. The system 1 also makes it possible to center the beam coming from the light source 3 on the micro-colony or colonies considered. The system 1 is also adapted to acquire the interference patterns resulting from the reflection of the probe beam on the micro-colony or colonies studied. The system 1 also makes it possible to calculate parameters characterizing the interference patterns and to compare the parameters with an existing base. Such a possibility opens the way to learning techniques and especially supervised techniques of SVM type (acronym for "Support Vector Machine" translated into French by support vector machines or wide margin separators).
    [0038] Other non-illustrated variants of the system 1 are also conceivable provided that the characteristics are compatible. Typically, according to a non-illustrated embodiment, the system 1 is according to the third example and comprises a sensor 13. To illustrate the capabilities of the system 1, it is detailed in the following three particular experiments conducted with the previous system 1. In these experiments, the bacterial strains are ATCC (American Type Culture Collection) commercial strains. From a culture in a liquid medium after 24 hours of incubation at 37 ° C., a suspension of 5 milliliters (ml) comprising water and a quantity of cells of the strain such as the turbidity of the solution are removed. equal to 0.5 McF (McFarland standard). The suspension is diluted 1/1000 to 1/100 depending on the strain. Then, a volume of 10 μl of this suspension is then inoculated onto the COS culture medium contained in a Petri dish chamber. The box thus inoculated is incubated at 37 ° C. The dish was removed from the incubator 6 hours after inoculation.
    [0039] Following the method described above, it is acquired interference images in reflection on the colonies. Preferably, each image has only one interference figure, which corresponds to a mircoorganism to be characterized. Each image is then projected on a basis of Zernike moments from a family of orthogonal Zernike polynomials defined in polar coordinates on the unit disk. Such a base offers the advantage of a rotation invariance as well as a redundancy limitation of the information. Such a projection is known per se. This step makes it possible to have scalar indicators relating to the interference figure included in the image. Other methods of quantitative analysis can be used, which consist of projecting an image into a database, in order to obtain coordinates of the image analyzed in this database.
    [0040] A vector, gathering said indicators, called descriptor, is obtained for each image. Such a vector consists of 120 components corresponding to the modules of the components of the projection on the first 120 polynomials of the base. Once this database is constituted, it is used a support vector machine (SVM) type classification algorithm and more particularly the minimal sequential optimization algorithm (SMO). Indeed, the training of a support vector machine requires solving a large quadratic optimization problem and the algorithm SMO proposes to reduce the computation time by splitting it into quadratic optimization problems of sizes. smaller possible that will be solved analytically. This classification is then evaluated by a cross-validation step (also known as "10-fold cross-validation"). The results are then combined in the form of a confusion matrix. For a first experiment, 400 equidistant samples in 4 strains of the same Escherichia coli species are studied. The confusion matrix obtained is as follows: Strain Classified as EC10 Classified as EC21 Classified as Escherichia EC28 EC11 Coli EC10 - 96 1 1 2 ATCC 25922 EC21 - 7 80 4 9 ATCC 35421 EC28 - 6 6 80 8 ATCC11775 EC 11 - 5 4 2 89 ATCC 8739 Table 1 Confusion matrix for the first experiment The confusion matrix in Table 1 reads as follows: taking the line in front of the EC10 strain, out of 100 EC10 descriptors, 96 were recognized as EC10, 1 as EC21, 1 as EC28 and 2 as EC11 and so on. Applying this to each species, out of 400 descriptors, 345 were well recognized and 55 were confused with the descriptors of another strain. This corresponds to an overall classification rate of over 86%.
    [0041] Table 1 corresponds to a classification on 4 strains of the same species: Escherichia coli. This corresponds to a much finer level of identification than an interspecies identification. A very satisfactory overall classification rate of more than 80% (86.25% exactly) is obtained. This rate is of the same order of magnitude as the results obtained with diffraction patterns in transmission. For a second experiment, 500 equidistant samples in 5 species of bacteria are studied. The confusion matrix obtained is as follows: Strain Classed Rated Rated Rated Rated (Species) AS AS AS AS LIKE AS AS EC21 EC8 AB30 CF7 5E9 EC21 - ATCC 35421 (Escherichia colt) 89 8 0 1 2 EC10 - ATCC 25922 2 70 6 0 22 (Enterobacter cloacae) AB30 - ATCC 23220 2 4 94 0 0 (Acinetobacter baumanit) CF7 - ATCC 8090 1 0 0 98 1 (Citrobacter freundii) SE9 - ATCC 14990 0 14 0 0 86 (Staphylococcus epidermidis) Table 2: Confusion matrix for the second experience In the second experiment, the overall classification rate is over 87%. This rate is of the same order of magnitude as the results obtained with diffraction patterns in transmission.
    [0042] For a third experiment, 300 equidistant samples in 3 strains of the same genus of Candida fungus are studied. The resulting confusion matrix is as follows: Strain Classified as Classified (Species) EC10 EC21 as EC28 CA36 - ATCC 14053 88 0 10 (Candida albicans) CG38-ATCC 2001 0 100 0 (Candida glabrata) 0137 - ATCC 13803 6 0 94 (Candida tropicalis). Table 3: Confusion matrix for the third experiment In the third experiment, the overall classification rate is 94%. The method used makes it possible to obtain diffraction patterns exploitable by analysis and classification algorithms, in order to establish an aid for the identification of microorganisms.
    权利要求:
    Claims (12)
    [0001]
    CLAIMS1.- An object observation system (1) comprising: - a light source (3) capable of emitting polarized light rectilinearly in a first direction (D1), - a support (12) adapted to receive a substrate having a surface comprising objects, at least one of the support (12) and the substrate being translucent or opaque, - a detector (7) capable of harvesting backscattered light resulting from the interaction between the light emitted by the light source (3) and the objects, - a polarization separator (9) adapted to reflect polarized light rectilinearly in a second direction of polarization (D2) and suitable for transmitting polarized light rectilinearly in a third direction (D3), the second polarization direction (D2) being perpendicular to the third direction (D3), and - a quarter-wave plate (10), the separator (9) and the quarter-wave plate (10) being arranged so that , the separator (9) directs the light emitted by the light source (3) to the substrate and directs the backscattered light resulting from the interaction between the light emitted by the light source (3) and the objects towards the detector (7).
    [0002]
    2. Object observation system according to claim 1, wherein the objects are microorganisms, the substrate being a solid substrate, in particular an agar substrate, adapted to the growth of said microorganisms.
    [0003]
    3.- object observation system according to claim 1 or 2, wherein: - either the first direction (D1) and the second direction (D2) are identical; or the first direction (D1) is different from the second direction (D2) in which case the system (1) comprises a polarization direction adjustment element, the polarization direction adjustment element being disposed between the light source (3) and the separator (9), so that the light incident on the separator (9) is polarized in the second direction (D2).
    [0004]
    An object viewing system according to any one of claims 1 to 3, wherein the substrate has a surface for interacting with the light source light (3), the surface being smooth.
    [0005]
    5. Object observation system according to any one of claims 1 to 4, wherein the system further comprises a sensor (15) adapted to acquire an image of the objects.
    [0006]
    6. Object observation system according to any one of claims 1 to 5, wherein the light from the light source (3) forms a beam, the system (1) comprising an optical system (5, 6). ) fit to change the beam size.
    [0007]
    7. Object observation system according to any one of claims 1 to 6, wherein the objects are objects whose size is less than one millimeter.
    [0008]
    8. Object observation system according to any one of claims 1 to 7, wherein the observation system (1) is adapted to observe objects which are microorganisms.
    [0009]
    9. Object observation system according to any one of claims 1 to 8, wherein the observation system (1) further comprises a half-wave plate adapted to change the polarization of light emitted by the light source (3).
    [0010]
    10. An object observation system according to any one of claims 1 to 9, wherein the observation system (1) further comprises a computer adapted to analyze the backscattered light detected by the detector (7). ) to derive at least one characteristic of the objects.
    [0011]
    11. Object observation system according to any one of claims 1 to 10, wherein the observation system (1) is devoid of optics interposed between the separator (9) and the blade quarter-d ' wave (10) and between the quarter wave plate (10) and the substrate.
    [0012]
    12. Object observation system according to any one of claims 1 to 11, wherein the objects are part of a petri dish having a cylindrical body whose base surface is flat and a cover (18). cylindrical whose base surface is flat and forms an angle with the base surface of the body between 0.1 degree and 15 degrees.
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    同族专利:
    公开号 | 公开日
    US10247668B2|2019-04-02|
    WO2016097063A1|2016-06-23|
    FR3030748B1|2017-01-27|
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    法律状态:
    2015-12-08| PLFP| Fee payment|Year of fee payment: 2 |
    2016-06-24| PLSC| Publication of the preliminary search report|Effective date: 20160624 |
    2016-12-06| PLFP| Fee payment|Year of fee payment: 3 |
    2017-11-23| PLFP| Fee payment|Year of fee payment: 4 |
    2019-11-20| PLFP| Fee payment|Year of fee payment: 6 |
    2020-12-28| PLFP| Fee payment|Year of fee payment: 7 |
    2021-12-31| PLFP| Fee payment|Year of fee payment: 8 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1462645A|FR3030748B1|2014-12-17|2014-12-17|OBJECT OBSERVATION SYSTEM|FR1462645A| FR3030748B1|2014-12-17|2014-12-17|OBJECT OBSERVATION SYSTEM|
    EP15820467.7A| EP3234555B1|2014-12-17|2015-12-16|System for observing objects|
    PCT/EP2015/080106| WO2016097063A1|2014-12-17|2015-12-16|System for observing objects|
    US15/536,526| US10247668B2|2014-12-17|2015-12-16|System for observing objects|
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