阅读说明：本技术 一种用于观察由物体反向散射的辐射的方法和装置 (Method and device for observing radiation backscattered by an object ) 是由 艾曼纽·舒尔茨 达米安·德克 米歇尔·洛克 于 2017-12-21 设计创作，主要内容包括：一种用于观察物体(3)、特别是生物物体的装置和方法。装置具有适于照射样本(2)的光源(10)。在照射的作用下,物体(3)发射传播到屏幕(20)的反向散射的辐射(14),屏幕的表面积大于100cm。反向散射的辐射(14)在屏幕(20)上的投射形成代表反向散射的辐射(14)的图像,由术语衍射图表示。使用图像传感器(30)来获取代表在屏幕(20)上形成的衍射图的图像。装置还包括反射元件(13)和连接支持件(17),反射元件设置在屏幕(20)和物体(3)之间,适于沿垂直于样本平面(XY)的入射轴(Z)反射入射光束(12)的一部分,反射元件与屏幕(20)的第一面(20i)刚性地固定；连接支持件(17)在反射元件(13)和屏幕(20)之间延伸,连接支持件(17)用于将反射元件(13)与到屏幕(20)固定,且反射元件(13)和/或连接支持件(17)被配置为吸收在物体(3)和屏幕(20)之间传播的反向散射的辐射(14)的至少50％。(An apparatus and a method for observing an object (3), in particular a biological object. The device has a light source (10) adapted to illuminate the sample (2). Under the influence of the illumination, the object (3) emits backscattered radiation (14) that propagates to a screen (20) having a surface area greater than 100 cm. The projection of the backscattered radiation (14) onto the screen (20) forms an image representative of the backscattered radiation (14), represented by the term diffraction diagram. An image sensor (30) is used to acquire an image representative of the diffraction pattern formed on the screen (20). The device further comprises a reflective element (13) and a connection support (17), the reflective element being arranged between the screen (20) and the object (3) and being adapted to reflect a portion of the incident light beam (12) along an incident axis (Z) perpendicular to the sample plane (XY), the reflective element being rigidly fixed to a first face (20i) of the screen (20); a connection support (17) extends between the reflective element (13) and the screen (20), the connection support (17) is for fixing the reflective element (13) to the screen (20), and the reflective element (13) and/or the connection support (17) is configured to absorb at least 50% of backscattered radiation (14) propagating between the object (3) and the screen (20).)

1. An apparatus for observing an object (3) present in a sample (2), comprising:

-a holder (6) capable of containing a sample (2), said holder defining a plane (XY), called sample plane, in which the sample extends when it is arranged on the holder;

-a light source (10) capable of emitting a light beam (12), called incident light beam, in order to illuminate the object;

-an image sensor (30) for acquiring an image representative of radiation (14) backscattered by the object under the effect of the incident light beam (12);

the apparatus is characterized by comprising:

-a screen (20) extending facing the holder (6) so as to be exposed to radiation backscattered by the object when the incident light beam illuminates the object, so as to form on the screen an image (I) representative of said backscattered radiation₂₀) Called diffractogram;

-the screen comprises a first face (20) exposed to backscattered radiation₁) The area of the first surface is more than 100cm²；

-the image sensor (30) is configured to acquire an image (I) of a diffraction pattern formed on a screen₃₀)，

The device also comprises a reflecting element (13) arranged between the screen (20) and the object (3) and capable of reflecting a portion of the incident light beam (12) along an incident axis (Z) perpendicular or substantially perpendicular to the sample plane (XY), said reflecting element being firmly fixed to the first face (20) of the screen₁)；

The device is characterized in that the device comprises a bonding medium (17) extending between the reflective element (13) and the screen (20), the bonding medium enabling the reflective element (13) to be fixed to the screen (20), the device enabling the reflective element (13) and/or the bonding medium (17) to be configured to absorb at least 50% of the backscattered radiation (14) propagating between the object (3) and the screen (20).

2. The device of claim 1, wherein the screen (20) is curved.

3. The apparatus of any preceding claim, wherein:

-the screen (20) comprises a second face (20)₂) So that on the first side (20)₁) The diffraction pattern formed above appears on the second face;

-the screen extends between the image sensor (30) and the holder (6) such that the image sensor (30) is coupled to the second face (20) by focusing optics (25)₂)。

4. Device according to any one of the preceding claims, wherein the distance (δ) between the reflecting element (13) and the screen (20) is less than 2 cm.

5. A device according to any preceding claim, wherein the reflective element has an area of less than 4cm²Or less than 2cm²Or less than 1cm²。

6. The device according to any one of the preceding claims, wherein the screen (20) is translucent.

7. The device according to any one of claims 3 to 6, wherein the screen (20) comprises a first face (20) for displaying₁) And a second face (20)₂) A light guide for transmitting light therebetween.

8. The apparatus of claim 7, wherein the screen comprises a plurality of optical fibers extending between a first side and a second side.

9. The device according to any one of claims 1, 4 and 5, wherein the screen (20) is a light-sensitive part of an image sensor (30).

10. The apparatus of claim 3 or any one of claims 4 to 8 when dependent on claim 3, wherein the screen transmits less than 90% of the backscattered radiation (14) between the first and second faces.

11. Device according to any one of the preceding claims, wherein the screen (20) is movable relative to a holder (6), the distance (d) between holder and screen being adjustable.

12. The device according to any of the preceding claims, wherein the incident light beam (12) propagates between the reflecting element (13) and the object (10) along an axis, called the incidence axis, the device comprising a component, called a ring reflector (18), the ring reflector (18) extending around the incidence axis between the sample (2) and the screen (20), the reflector being capable of reflecting a portion of the backscattered radiation (14) to the screen (20).

13. For observing existence in sampleThe method of the object (3) in the present (2), said sample facing comprising a first face (20)₁) The screen (20) of (2) is extended, the method comprising the steps of:

a) illuminating an object (3) with an incident light beam (12) emitted by a light source (10), the incident light beam (12) propagating to a reflecting element (13) arranged between the screen and the object, the reflecting element directing all or part of the incident light beam towards the object, the reflecting element being connected to a first face (20) of the screen₁)；

b) Make the first side (20) of the screen₁) Is exposed to optical radiation (14) backscattered by the sample under the effect of the illumination so as to form, on said first face (20), an image representative of said backscattered radiation (14), called a diffractogram₁) Is larger than 100cm²；

c) Acquiring an image of a diffraction pattern formed on a screen using an image sensor (30);

the method is characterized in that incident radiation (12) propagates along an incident axis from the reflective element (13) to the object (10), and wherein backscattered radiation (14) propagating along the incident axis towards the screen is absorbed before reaching the screen so as to form a shadow in a diffraction pattern formed on the screen.

14. The method according to claim 13, wherein the area of the reflective element (13) is less than 5cm²Or less than 2cm²Or less than 1cm²。

15. The method according to any one of claims 13 or 14, wherein the screen (20) is curved.

16. A method according to any one of claims 13 to 15, wherein the distance (δ) between the reflecting element and the screen is less than 1 cm.

17. The method according to any one of claims 13 to 16, wherein the screen (20) comprises a second face (20)₂) The screen extending between the image sensor (30) and the specimen (2) such that the image is sensedThe device is optically coupled to the second face by focusing optics (25), the screen being such that on the first face (20)₁) The diffraction pattern formed on the second surface (20)₂) The above.

18. The method of claim 17, wherein:

-the screen (20) is translucent;

-or the screen comprises at least one light guide, in particular an optical fiber, extending between a first face and a second face;

or one of said faces of said screen is configured to form a lens.

19. The method of claim 17 or claim 18 when dependent on claim 17, wherein the screen transmits less than 90% of the backscattered radiation (14) from the first face to the second face.

20. The method of any one of claims 13 to 16, wherein the screen is a light sensitive portion of an image sensor.

21. Method according to any one of claims 13 to 20, comprising, after step c), a step of adjusting the distance (d) between the sample (2) and the screen (20) according to the image acquired by the image sensor (30), steps a) to c) being repeated after adjusting said distance.

22. Method according to any one of claims 13 to 21, comprising a step d) of characterizing the object (3) on the basis of the image acquired by the image sensor.

23. The method of claim 22, wherein step d) comprises:

-determining features of the image;

-identifying the object using said features and calibration features established by carrying out steps a) to c) of the method on a standard sample.

24. The method of any one of claims 13 to 23, wherein the object comprises a microorganism.

Technical Field

The technical field of the invention is the observation and identification of objects, in particular biological objects, in particular bacterial colonies, based on images of radiation backscattered by the object.

Background

Identification of microorganisms (particularly bacteria) is desirable for various fields. For example, in the diagnostic field, the identification of bacteria allows to understand the nature of the pathogen causing the infection and to optimize the treatment of the patient. In addition, bacterial identification is a fundamental technology in epidemiology or against nosocomial infections. In addition to the health area, applications may include, but are not limited to, hygiene, safety and food processing areas.

There are currently a variety of effective instruments that allow such identification. The methods used are, in particular, mass spectrometry, Raman spectrometry, colorimetric tests, colony morphology analysis or nucleic acid amplification techniques. Methods using spectroscopic techniques (mass spectrometry or raman spectroscopy) require expensive equipment and qualified operators. Colorimetric methods are simpler, but generally slower. With regard to nucleic acid amplification, many steps need to be performed continuously while satisfying precise operating conditions.

Patent US74665560 describes a method for characterizing microorganisms based on the use of the scattering and diffraction of an incident laser beam by the microorganisms. The microorganism is disposed between the laser light source and the image sensor. Under the effect of the laser beam irradiation, an image is obtained in which a diffraction pattern is present, which pattern constitutes the characteristics of the observed microorganisms. Patent US8787633 describes a method for the same purpose. These documents describe a method for identifying bacteria which seems promising, but which becomes unsuitable if the medium provided with the bacteria is opaque, coloured or scattering. In particular, these methods use images formed in a so-called transmission configuration, in which the sample is disposed between a light source and an image sensor. The sample must be sufficiently transparent if a usable image is to be obtained. Thus, the method is incompatible with samples containing colored media, such as media known as Columbia Blood agar (COS) which contains Columbia Blood agar in sheep Blood. The method is also not applicable to scattering substrates such as Cystine Lactose Electrolyte Deficient (CLED) agar, or opaque substrates such as chocolate agar. However, such media are frequently used for clinical diagnosis.

Patent application WO2016/097063 partially solves this problem by proposing a method for observing microorganisms in which the image is not formed in a transmission configuration, but in a backscatter configuration. The sample is irradiated by a laser beam. The backscattered radiation is focused on the image sensor by the collection optics. Document WO2016/054408 describes a similar arrangement, the backscattered radiation being collected by a CMOS image sensor, the effective surface area of which can reach 17.28cm²。

Devices operating in a backscattering configuration are also described in the publication by Huisung Kim et al, "influenced scatterometry for non-invasive interpretation of bacteriology", International society for optical engineering, SPIE, vol.21, N10, october 2016 ("reflection scattering method for non-invasive interrogation of bacterial colonies", SPIE by the International society of optical engineering, vol.21, No.10,2016, month 10). In this configuration, a flat screen is disposed between the specimen and the image sensor. The screen allows for back projection of radiation backscattered by the sample. The sample is irradiated with a laser beam, which is reflected by a reflecting plate before reaching the sample. The area of the reflecting plate is 25cm². The use of a translucent screen is also described in US 5241369.

The inventors have carried out the process described in WO2016/097063 and have discovered certain limitations described below.

The object of the present invention is to overcome these limitations by proposing a method for observing and characterizing microorganisms in a backscattered configuration. The invention is particularly applicable to opaque samples while remaining naturally applicable to transparent samples. The method allows the observation and characterization of microbial colonies at various stages of growth, whether microcolonies or macrocells. Another advantage is that the method is easy to implement and stable and does not require expensive equipment. Moreover, the method implemented is non-destructive. The method can be applied to colonies in the culture medium of the colonies without the need for sampling. Finally, the analysis is fast, taking about one second.

Disclosure of Invention

The invention relates firstly to a device for observing an object present in a sample, comprising:

-a holder capable of holding a sample;

-a light source capable of emitting a light beam, called incident light beam, in order to illuminate the object;

-an image sensor for acquiring an image representative of radiation backscattered by the object under the influence of the incident beam illumination;

the device is characterized in that it comprises:

a screen extending facing the holder so as to be exposed to radiation backscattered by the object when the incident beam illuminates it, forming an image on the screen representative of the backscattered radiation, called a diffractogram;

-the screen comprises a first side exposed to the backscattered radiation;

the image sensor is configured to acquire an image of the diffraction pattern formed on the screen.

The light source may in particular be a laser source. The apparatus may comprise collimating optics such that the light beam emitted by the light source is collimated. The apparatus may include beam expansion optics to adjust the diameter of the beam to the size and morphology of the analyte body.

The object may be a colony of microorganisms, for example bacteria, in which case the screen allows obtaining a diffraction pattern whose size is large enough to characterize the colony at a sufficiently advanced stage of growth.

According to one embodiment, the area of the first side of the screen is greater than 100cm²。

The apparatus may comprise a reflective element disposed between the screen and the object, the reflective element being capable of reflecting a portion of an incident light beam along an incident axis perpendicular or substantially perpendicular to the sample plane, the reflective element being fixedly secured to the first side of the screen. This makes it possible to avoid interference of the diffraction pattern formed on the screen by the arms supporting the reflective elements, which arms extend transversely to the backscattered radiation.

The device may comprise any of the following features, alone or in a technically feasible combination:

the screen comprises a second face, such that the diffraction pattern formed on the first face appears on the second face; the screen extends between the image sensor and the holder such that the image sensor is coupled to the second side through the focusing optics. The screen serves as a backlight screen, transmitting the diffraction pattern projected onto the first face to the second face.

The distance between the reflecting element and the screen is less than 2 cm.

-reflectionThe area of the element is less than 4cm²Or less than 2cm²Or less than 1cm²。

The device comprises a bonding medium extending between the reflective element and the screen, the bonding medium enabling the reflective element to be fixed to the screen, the device enabling the reflective element and/or the bonding medium to be configured to absorb at least 20%, or even at least 30%, or even at least 50% of the backscattered radiation propagating between the object and the screen.

The screen is translucent.

The screen comprises a light guide, such as an optical fiber, for transmitting light between the first and second faces. The screen may include a plurality of optical fibers extending between the first side and the second side.

The screen is a photosensitive part of the image sensor, which allows converting backscattered radiation into charge carriers.

The screen transmits less than 90% of the backscattered radiation from the first face to the second face.

The screen is movable relative to the holder, the distance between the holder and the screen being adjustable.

-the incident light beam propagates between the reflecting element and the object around an axis called the incidence axis, the device comprising a component called a toroidal reflector extending around the incidence axis between the sample and the screen, the toroidal reflector being capable of reflecting a portion of the backscattered radiation (14) towards the screen (20).

The screen is curved, in particular towards the sample (or object).

Another subject of the invention is a method for observing an object present in a specimen extending towards a screen comprising a first face, the method comprising the steps of:

a) illuminating an object with an incident light beam emitted by a light source, the incident light beam propagating to the object;

b) exposing a first face of a screen to optical radiation backscattered by a sample under the action of illumination so as to form on said first face an image representative of said backscattered radiation, called a diffractogram;

c) an image of the diffraction pattern formed on the screen is acquired using an image sensor.

According to one embodiment, the device comprises a reflective element arranged between the screen and the object, the reflective element directing all or part of an incident light beam emitted by the light source towards the object. The reflective element may in particular be connected to the first side of the screen.

The method may include any of the following features, alone or in a technically feasible combination:

the area of the reflective element is less than 5cm²Or less than 2cm²Or less than 1cm²。

The screen is curved, and in particular curved towards the sample.

The screen is translucent.

The screen comprises at least one light guide, in particular an optical fiber, extending between the first and the second face.

One side of the screen is configured to form a lens.

The screen transmits less than 95% or 90% of the backscattered radiation.

The screen comprises a second face, the screen extending between the image sensor and the sample such that the image sensor is optically coupled to the second face by the focusing optics, the screen causing a diffraction pattern formed on the first face to appear on the second face.

After step c), the method comprises a step of adjusting the distance between the sample and the screen according to the image acquired by the image sensor, steps a) to c) being repeated after adjusting said distance.

The method comprises a step d) of characterizing the object on the basis of the image acquired by the image sensor or on the basis of the resulting image. The characterization of the image may include:

■ determining the characteristics of the image;

■ use of said features and by carrying out steps a) to c) of the method on standard samples

And the established calibration features identify the object.

The object comprises a microorganism. The object may in particular comprise a plurality of micro-organisms forming colonies. The object may be a bacterial colony.

The method is carried out using the apparatus described herein.

Further advantages and features will become more apparent from the following description of a particular embodiment of the invention, given by way of non-limiting example and illustrated in the accompanying drawings listed below.

Drawings

Fig. 1A shows a device for observing microorganisms according to the prior art.

Fig. 1B and 1C show the spatial distribution of backscattered radiation emitted from two different objects, respectively.

Fig. 2A and 2B show a first embodiment of the present invention. Fig. 2C shows an example of a screen that can be implemented in the first, second, or third embodiment. Fig. 2D is a detail of fig. 2A. Fig. 2E and 2F show the distance variation between the screen and the sample. Fig. 2G is an example of an optical system for shaping a laser beam.

Fig. 3A and 3B show a second embodiment and a third embodiment, respectively. Fig. 3C and 3D show variants applicable to all embodiments. Fig. 3E shows a variant in which the screen is curved.

Fig. 4A and 4B show diffraction patterns of bacterial colonies according to two different arrangements of the device.

Fig. 5A, 5B and 5C illustrate a method for moving an observed bacterial colony to center it with respect to the incident beam and with respect to the screen. Fig. 5A is a diffraction pattern slightly off-center. Fig. 5B is the result of applying a filter to the diffraction pattern shown in fig. 5A. FIG. 5C shows the diffraction pattern of FIG. 5A after re-centering.

Fig. 6 shows an embodiment of a so-called high dynamic range, in which various images are combined.

FIGS. 7A, 7B, 7C, 7D, 7E and 7F show diffractograms of various bacterial colonies.

Fig. 8A and 8B show a micrograph and diffractogram of bacterial colonies. Fig. 8C and 8D show a micrograph and diffractogram of another bacterial colony.

FIGS. 9A, 9B and 9C are diffractograms of bacterial colonies formed on various media.

Fig. 9D shows a diffraction pattern of a bacterial colony formed on the bacterial layer, different from the observed bacterial colony formed.

FIG. 10 shows the change in diffraction pattern over time for a given bacterial colony.

Fig. 11A shows an experimental setup combining a flat screen and a curved screen.

FIG. 11B is an image of bacterial colonies observed using the apparatus shown schematically in FIG. 11A.

Detailed Description

Figure 1A shows a device for observing microorganisms, such as described in patent application WO 2016/097063. The laser light source 10 emits a linearly polarized light beam 102, the linearly polarized light beam 102 propagating to an object 3 to be characterized, for example a bacterial colony disposed on the surface of a culture medium 4. Before reaching bacterial colony 3, polarized light beam 102 is deflected by half-silvered mirror 103 so as to propagate in a direction called the direction of incidence, which is substantially perpendicular to the surface of culture medium 4. Light beam 102 passes through quarter wave plate 104 before reaching bacterial colony 3. The light beam 102 interacts with the bacterial colony 3, which results in the formation of backscattered radiation 14, which propagates in a direction substantially opposite to the direction of incidence. The backscattered radiation 14 is formed by multiple interactions of the beam 102 with the colonies 3, combining the effects of diffraction and elastic scattering in the colonies. The backscattered radiation 14 passes through a quarter-wave plate 104 and then through a half-silvered mirror 103 before being focused by an optical system 107 towards the image sensor 30. The image formed on the image sensor, called the diffraction pattern, represents the backscattered radiation 14. The diffractogram may be considered a representation of the bacterial colony to allow identification of the bacteria forming the colony. The device representing the prior art has been implemented by the inventors. The inventors have shown that this device is not able to satisfactorily observe certain bacterial colonies.

In particular, the backscattered radiation 14 is emitted in an angular range that varies according to the type of microorganism observed. Certain bacterial colonies (e.g. staphylococcal colonies) grow by the gradual formation of an outer surface 3s, said outer surface 3s having a shape close to a hemisphere delimited by an ambient medium 7 (e.g. air). This situation is shown in fig. 1B. In this type of configuration, the backscattered radiation 14 is divergent and forms a cone 15 covering a high angular range Ω. High is understood to include angles greater than 65 ° or even 85 °. This is caused in particular by the refraction of the backscattered radiation when it passes through the surface 3s in order to be refracted in the ambient medium 7. In contrast, as shown in fig. 1C, other bacterial colonies grow by gradually forming a flat surface 3s, which has a large radius of curvature. The backscattered radiation 14 is thus refracted in the ambient medium 7 and propagates in the ambient medium 7 as a converging beam, forming a cone 15 with an apex angle Ω. Bacteria of the enterobacteriaceae type form colonies with this morphology. Thus, depending on the type of microorganism observed and its growth stage, the morphology of the colony changes, affecting the spatial distribution of the backscattered radiation 14. One limitation of the device described in WO2016/097063 is that it has a small field of view and is stationary and is not suitable for bacterial colonies shaped like the shape illustrated in the example of FIG. 1B. The inventors have defined an observation apparatus that takes into account the variability of the spatial distribution of the backscattered radiation 14. More specifically, the device according to the invention has a field of view that can be adapted to the micro-organisms observed. In particular, the inventors have determined that for microorganisms producing backscattered radiation spatially distributed as shown in fig. 1B, a very large field of view may be required.

Fig. 2A shows a first embodiment of the device 1 according to the invention. In this case, this is the preferred embodiment. The device comprises a light source 10 capable of emitting a light beam 12, called incident light beam, said light beam 12 propagating to a sample 2 comprising an object 3 to be characterized. The light source 10 is preferably coherent in time and space. The light source 10 is preferably a laser source. According to a variant, the light source may be a light emitting diode or a white light source. Thus, the light source is preferably a spatially coherent, substantially point-like object. This may be achieved by associating the light source 10 with a spatial filter, such as an aperture or an optical fibre. The light source 10 may also be associated with a band-pass filter in order to obtain a sufficiently narrow emission band Δ λ, preferably narrower than 50nm or even 10 nm.

The incident light beam 12 emitted by the light source and propagating towards the object 3 is preferably a parallel light beam, the diameter of which can be advantageously adjusted. The incident beam 12 is preferably 100 μm to 10mm in diameter. The adjustment of the diameter makes it possible to adapt the size of the object 3 to be characterized. Thus, when the object 3 is a bacterial colony, this allows the incident light beam 12 to be resized to the morphology of the colony, which depends on the type of bacteria and growth stage. The shaping optical system 11 may be arranged between the light source 10 and the object 3. The shaping optical system 11 may allow adjusting the diameter of the incident light beam 12. It may also allow for a normalized increase in the spatial distribution of energy in the incident beam 12, making the light intensity in the beam more uniform.

The object 3 to be characterized may be a microorganism or a group of microorganisms forming a colony. The microorganism may be a bacterium, yeast, fungus or microalgae. The object to be characterized may also be a group of cells forming e.g. a cluster. The object to be characterized may be brought into contact with the culture medium 4 by being arranged in the culture medium or on the surface of the culture medium. The culture medium 4 is confined in the peripheral member 5. The culture medium 4 and/or the peripheral member 5 may be opaque or translucent. In particular, the culture medium 4 and the peripheral element 5 do not have to be transparent, which is a condition for the transport configuration-based methods described with reference to the prior art. The assembly formed by the peripheral element 5, the culture medium 4 and the object 3 forms a sample 2, which rests on a holder 6. In the example shown, the holder is a planar table that can be moved in translation along an axis Z called the incidence axis. The invention is particularly applicable to samples containing an opaque culture medium 4. When the culture medium 4 is not sufficiently opaque, the peripheral member 5 is preferably opaque and preferably absorptive in order to minimize parasitic reflections. The peripheral member 5 may comprise a cover, provided that the cover is transparent. When the peripheral member 5 is transparent, it is preferably provided on an opaque or translucent holder 6. Such a holder prevents parasitic reflections.

The device comprises a reflective element 13, for example a mirror, capable of directing an incident light beam 12 emitted by a light source along an incident axis Z substantially perpendicular to the object to be observed3 or substantially perpendicular to the XY plane, referred to as the sample plane, in which the culture medium 4 of the sample 2 extends. Substantially perpendicular means perpendicular within an angular tolerance, preferably less than ± 30 °, or preferably less than ± 20 °. Thus, the incident light beam 12 reaches the object 3 with an angle of incidence substantially equal to 90 ° within an angular tolerance. In the example shown, the incident light beam comprises a first component 12 between the light source 10 and the reflective element 13₁And a second component 12 between the reflecting element 13 and the object 3₂. The incident light beam 12 reaching the object is preferably centered with respect to the object 3 in the XY plane of the sample.

Under the effect of the illumination of the incident beam 12, the object 3 emits backscattered radiation 14 propagating along or around a central counter-propagation axis-Z, parallel to and opposite to the direction of the incident axis Z. In general, the term backscattered radiation denotes radiation propagating along a propagation axis comprising a component opposite to the incidence axis Z. The backscattered radiation 14 results from the interaction of the photons of the incident beam 12 with the object 3, the object 3 having a higher refractive index than the refractive index of the ambient medium 7, the incident beam propagating through the ambient medium 7, the ambient medium 7 typically being air. Due to the angle of incidence, a large part of the incident beam 12 propagates into the object 3, forming a refracted incident beam. The incident light beam 12 refracted in the object 3 undergoes one or more elastic scatterings in the object and may produce diffracted waves. As described with reference to fig. 1B and 1C, the backscattered radiation 14 emanates from the object and propagates through the surface 3 s. The backscattered radiation is refracted in the ambient medium 7 and then propagates around a back propagation axis-Z to the screen 20, where the backscattered radiation forms an image I representing the backscattered radiation₂₀Referred to as a diffraction pattern. The counter-propagation axis-Z is coaxial with the incident axis Z along which the incident beam reaches the object. In the ozle-saxon literature, the diffraction patterns herein are commonly referred to as "scatter patterns," or "scatter patterns," and may also be translated into the term scatter plots. Note that no focusing or imaging optics are provided between the object 3 and the screen 20.

The area of the reflective element 13 should be as small as possible toThe backscattered radiation 14 emanating from the object 3 is not disturbed. The area of the reflective element is preferably less than 5cm²More preferably less than 2cm²Or even less than 1cm². The area of the reflective element 13 is preferably adapted to the diameter of the light beam emitted by the light source 10.

The screen 20 is able to collect radiation 14 backscattered by the object 3 when the object is illuminated by the beam 12. The term screen denotes its first side 20₁An element for collecting the backscattered radiation 14, the backscattered radiation 14 being projected onto said first face 20₁The above. Thus, diffraction Pattern I₂₀Formed on the first side 20 of the screen 20₁The above. The screen 20 has at least 50cm in the XY plane of the sample²But preferably has an area of more than 100cm²Or even greater than 200cm²E.g. 400cm²I.e. a square with a side length of 20 cm.

The device comprises an image sensor 30 for acquiring a diffraction pattern I formed on a screen₂₀Image I of₃₀. The image sensor 30 may in particular be a matrix sensor comprising pixels arranged in a matrix, each pixel forming a basic photodetector. The image sensor 30 is, for example, a CCD or CMOS sensor. The image sensor 30 is connected to a processor 40, for example a microprocessor, the processor 40 comprising a memory 42, image processing instructions being stored in the memory 42, the instructions allowing analysis of the image acquired by the image sensor 30 to characterize the object 3. The processor 40 may also allow movement of the holder 6 relative to the screen 20, as described below. The monitor 44 allows the acquired image to be viewed.

In the embodiment shown in fig. 2A, to allow the projection of the diffraction pattern onto the screen 20, the screen 20 is not completely transparent: the screen 20 interacts with the scattered radiation 14 by absorption and/or scattering. Preferably, the screen transmits up to 80%, or even 90% or 95% of the backscattered radiation, the non-transmitted part being absorbed or scattered. The inventors believe that a transmission of about 75% is optimal. Transmittance refers to the ratio between the intensity of radiation transmitted by the screen and the intensity of radiation incident on the screen. The transmission of the screen is preferably less than 95%, or even less than 90% or 80 percent. Opacity is defined as the inverse of transmittance. The screen 20 comprises a first face 20 preferably parallel to the latter₁Extended second face 20₂. The screen 20 is configured such that it projects to a first side 20₁The image (in this case a diffraction pattern) on the second face 20 also appears by transmission and/or scattering₂The above. Since the screen 20 is interposed between the diffuse radiation source (in this case the object 3) and the image sensor 30, the screen 20 acts as a backlight or rear projection screen. The screen 20 may be translucent, which term means a material that is opaque (i.e. through which the elements cannot be clearly distinguished), but which allows light to pass through. The screen 20 is for example a transparency paper substrate, a substrate comprising scattering elements (e.g. beads), or even a fabric or a rough glass sheet. When the screen includes beads, the beads may be beads made of polycarbonate. Rear projection screens in the form of fabrics suitable for such applications are sold, for example, by Multivision Inc. under the reference "retrogris" or "retrocreme". When the screen 20 is a write-through paper, the screen 20 may include a rough surface having a Bendtsen roughness of 100 to 300ml/mm, as determined according to standard NF 8791-2. On the first side 20₁The diffraction pattern formed on the second surface 20₂As shown in fig. 2B.

The device comprises focusing optics 25, the focusing optics 25 allowing focusing on the second side 20 of the screen 20₂Upper formed diffraction pattern I₂₀So that an image I acquired by the image sensor₃₀Corresponding to the diffraction pattern. Preferably, the image sensor 30 extends parallel to the screen 20 and the focusing optics 25 comprise an optical axis coaxial with the counter-propagation axis-Z (or with the incidence axis Z). Thus, the image formed by the image sensor and the diffraction pattern I formed on the screen₂₀Corresponding, without deformation.

According to a variant, the screen 20 comprises structured optical components defining, for example, a Fresnel (Fresnel) lens. The fresnel lens comprises concentric annular structures arranged to focus large diameter images with a short focal length. Company DNP sells screens for backscatter applications based on structured optical lenses on one or both sides of the screen. These screens are called "optical rear projection screens". Such a screen allows increasing the amount of signal collected by the image sensor.

According to a variant, the screen 20 is comprised on a first face 20₁And a second face 20₂A plurality of light guides extending therebetween to facilitate diffraction pattern from the first face 20₁To the second side 20₂. It may be included on the first side 20₁And a second face 20₂A fiber optic faceplate having an array of optical fibers extending adjacent to one another. The size of such a screen can reach several hundred cm in the XY plane²For example 32.5cm x 32.5 cm. Each fiber has a diameter of 5 to 25 μm and a numerical aperture of 0.92 to 1. Such panels are for example marketed by Schott.

Fig. 2C shows a screen formed of two layers: defining a first side 20 of the screen₁And a lower layer 21 and a second side 20 defining a screen₂The upper layer 22. For example, since the lower layer 21 consists of a rough sheet made of glass or plastic, the rough surface corresponding to the first face 20₁And therefore the lower layer 21 may be scattering. The upper layer 22 may be formed of a transparent sheet made of glass or a fresnel lens, serving as a protective layer.

Fig. 2D shows the reflective element 13, and details of the incident radiation 12 and the backscattered radiation 14. The incident radiation 12 comprises a first component 12₁Said first component propagating between the light source 10 and the reflective element 13. The incident radiation comprises a second component 12₂The second component propagates from the reflecting element 13 to the object 3. Also shown is backscattered radiation 14 emanating from the object 3 in the form of a cone 15 having a vertex angle omega. The backscattered radiation includes a first component, indicated as 14₁Referred to as the "reflected component", which substantially corresponds to the specular reflection of the incident beam 12 from the sample surface, the first component adds 0 th order diffraction. The backscattered radiation includes a surrounding first component 14₁Second component of extension 14₂The second component comprises information that can be used to characterize the object 3. The size of the reflecting element 13 depends on the light beam 12 emitted by the light source₁Is determined. This is, for example, a prism with a side length of 10 to 15mm, which is opposite to the light beam from the light source 1012₁Is inclined by 45 deg.. Preferably, the reflective element 13 is firmly fixed to the screen 20. This makes it possible to prevent the arm B for holding the reflective element from needing to extend into the cone 15 where the backscattered radiation 14 propagates, which would lead to a deterioration of the diffraction pattern formed on the screen 20. The distance δ between the reflective element 13 and the screen 20 is preferably greater than 1 mm. A too small distance delta, for example less than 1mm, may cause the laser beam 12 emitted by the light source and propagating to the reflective element 13₁Interaction with the screen 20. Preferably, the distance δ is less than 10mm or 20mm, or even 30mm, so as not to impede translation of the sample 2 in the direction of the screen 20, as described below. The reflective element 13 is preferably subjected to an antireflection treatment. The reflective element 13 may comprise an opaque rear surface 16 in order to block the propagation of non-reflected radiation. This makes it possible to avoid light leakage. The reflective element may be connected to the screen 20 by a bonding medium 17. Preferably, the bonding medium 17 extends between the reflective element and the screen, parallel to the axis Z of the incident beam 12 arriving at the sample, while advantageously being coaxial with the incident beam 12 arriving at the sample.

Preferably, unlike the device described in patent application WO2016/097063, the backscattered radiation 14 propagating towards the screen 20 is blocked by the reflective element 13 or by the bonding medium 17. This blocks the first component 14 of the backscattered radiation₁(reflected component) transmission towards the screen. However, as previously mentioned, reflected component 14₁Substantially representing the specular reflection of the incident beam 12 from the object 3; the first component includes no or little information useful for characterizing the observed object 3. Moreover, the first component is generally intense. The absence of this first component projected onto the screen 20 allows to suppress the strong and informative contribution to the diffraction pattern. This improves the dynamic range of the diffraction pattern. Reflected component 14₁Appears in the form of a dark disk on the diffraction pattern, which is a shadow of the reflective element 13 or the bonding medium 14. This shading is indicated by the black arrows on the diffraction pattern shown in fig. 2B. Preferably, the reflective element 13 and/or the joining medium 17 absorb at least 30%, advantageously at least 50%, or even 80% or 90% of the backscattered radiation 14 emitted by the object. Adjusting the size of the reflecting element and the bonding mediumSo that they shield only the reflected component 14 of the backscattered radiation₁Without masking the component 14 containing useful information₂。

The distance d between the sample 2 and the screen 20 is advantageously variable, as shown in fig. 2E and 2F. In particular, as mentioned above, the spatial distribution of the backscattered radiation 14 may vary, and the backscattered radiation 14 may take the form of a relatively open cone 15, extending divergently or convergently from the object. Thus, the holder 6 of the sample may be mounted on a translation stage that allows translation parallel to the axis of incidence Z. Fig. 2E and 2F show the screen 20 positioned at a first distance d ═ d, respectively₁And a second distance d ═ d₂Sample 2 of (2), wherein d₁>d₂. The movement of the holder 6 may be controlled by a processor 40. The distance typically varies from 3cm to 20cm, or even 30 cm. The distance is determined from the diffraction pattern formed on the screen 20 such that the diffraction pattern extends over the largest possible area while remaining compatible with the field of view of the image sensor 30, which depends on the dimensions of the image sensor 30 and the focusing optics 25.

The distance can be adjusted manually or by implementing an algorithm based on profile recognition that defines the diffraction pattern. Such an algorithm may, for example, use a Canny filter. When the profile is detected, the distance is adjusted so that the area of the diffraction pattern on the screen 20 exceeds a predetermined threshold. The adjustment of the distance d makes it possible to take into account the variability of the backscattered radiation due to different types of objects to be characterized. According to one embodiment, when the optimal distance has been determined, an image of the diffraction pattern is acquired allowing to maximize the area of the diffraction pattern projected onto the screen. The distance is then increased in order to verify that there is no backscattered radiation outside the previously observed diffraction pattern (i.e. corresponding to the optimal distance).

Preferably, the holder 6 also moves in the XY plane of the sample. This allows the incident light beam 12 to be centered on the object 3. This allows analysis to be performed regardless of the position of the object 3 in the sample 2. This centering can be adjusted according to the symmetry criterion of the diffraction pattern. In particular, the diffraction pattern presented on the screen has rotational symmetry when the incident beam is centered on the object. Symmetry can be quantified, for example, by the shape of the diffraction pattern profile.

According to a second embodiment, as shown in fig. 3A, the screen 20 is formed by a large-sized image sensor having a sensitive area greater than 100cm²Or larger. According to this embodiment, the screen 20 also serves as the image sensor 30. The image sensor may be a sensor, for example used in a medical X-ray imaging apparatus, which is then coupled with a scintillator material to ensure conversion between X-ray radiation and visible radiation detectable by the sensor. This type of sensor is sensitive to visible radiation and has a potentially large area. The screen 20 corresponds to a photosensitive portion of the image sensor 30, in which incident light photons are converted into charge carriers.

An example of manufacturing such a sensor made of silicon, the detection area of which is greater than 100cm, is given in document WO2014/006214²Or 200cm². The area of the pixel may be 50 μm to 200 μm. A transparent protective plate of small thickness, typically a few millimetres in thickness, may be placed against the screen 20. Such an embodiment may allow a significant increase in sensitivity relative to the first embodiment, but at a higher cost.

According to the second embodiment, as shown in fig. 3B, the screen 20 is not a rear projection screen but a front projection screen, and the sample 2 and the image sensor 30 are disposed facing the same face of the screen 20. In this embodiment, the backscattered radiation 14 is on the first side 20 of the screen₁Forming a diffraction pattern. Image sensor 30 is optically coupled to first face 20 using optical system 25₁. The image sensor 30 acquires an image of the diffraction pattern projected onto the first surface. However, in this embodiment, the image sensor is off-center with respect to the screen. In this embodiment, the reflection component 14 of the diffraction pattern formed on the screen 20 cannot be removed₁. In addition, this embodiment does not allow both the screen 20 and the incident light beam 12 to be centered on the object 3 to be characterized.

The spatial distribution of the backscattered radiation 14 may vary significantly depending on the object under observation. In some cases, this spatial distribution extends over a very high range of angles on both sides of the axis of incidence Z. This is particularly the case when the object, in this case a bacterial colony, has a curved morphology, such as that observed on a bacterial colony of a staphylococcus. In this case, the size of the screen 20 must be large in order to obtain a complete diffraction pattern, in particular in view of the large backscattering angle (typically greater than 65 °). The term "backscattering angle" refers to the angle between the backscattered radiation 14 emitted from the object and the axis of incidence Z. As previously mentioned, the distance between the screen 20 and the object 3 may also be adjusted. This allows, among other things, to obtain a diffraction pattern whose diameter corresponds to a predetermined template, for example a diameter of 15 to 20 cm. Fig. 3C depicts a variant that allows to maintain a reasonable size of the screen 20 while allowing to take into account the backscattered radiation 14 emitted from the object at large backscattering angles. According to this variant, a ring reflector 18 extending parallel to the axis of incidence Z is arranged around all or part of the object 3 between the object 3 and the screen 20. The ring reflector 18 allows a portion of the backscattered radiation 14 to be reflected towards the screen 20. The annular reflector 18 may be a tubular reflector coaxial with the incident axis Z. Fig. 3C shows a cylindrical ring reflector. The height and diameter of the cylindrical ring reflector may be 6cm and 17.5cm, respectively. The cylindrical ring reflector may be a cylinder, the inner wall 18i of which is reflective. For example, a thin metal layer (e.g., aluminum) may be deposited on the inner walls 18 i. The annular reflector 18 may also be conical, as shown in FIG. 3D. Such a conical reflector may have a minor diameter equal to 19cm, a major diameter equal to 20cm and a height of 3 cm. The angle of inclination of the inner wall with respect to the Z axis is, for example, 13 °. The angular dimensions of the inner walls 18i may be determined such that the backscattered radiation 14 with the largest backscattering angle undergoes only one reflection before reaching the screen 20. Preferably, at least one diameter of the annular reflector 18 is greater than 2 times the diameter of the peripheral element 5.

A space may be provided between the ring reflector 18 and the screen 20 to allow the incident light beam 12 to propagate between the light source 10 and the reflective element 13.

Fig. 3E shows a variant in which the screen 20 is not flat and has a curved shape that is curved towards the sample 2. This also helps the screen to collect radiation that is backscattered at large backscattering angles. The curvature of the screen 20 may or may not be regular. The screen 20 may, for example, describe a full or partial hemisphere. The screen 20 may have a dome shape. The screen may also describe a curve with a flat face.

By curving towards the sample is meant that the screen describes a curve whose center is located between the sample and the screen, or more generally, whose center is located in a half-space bounded by and including the screen. Thus, the screen has a concave shape so as to define a space between the screen and the sample, the space being such that, for any two points in said space, a line segment connecting said points is included in the space. Vacuum formed acrylic IRUS screens can be customized, for example, using a dome-shaped 1/4 "with a Cine25 tint and HC coating sold by Draper Inc. Preferably, the reflective element is disposed on the first side 20 of the screen at the apex of the screen 20₁Nearby.

Regardless of the embodiment, a shaping optical system 11 may be associated with the light source 10, according to principles known to those skilled in the art, so as to form a collimated incident beam 12. Fig. 2G shows an example of a shaping optical system. The forming optical system comprises a series of conventional optical components: an achromatic lens 110, a 50 μm diameter pinhole 111, a condenser lens 112 and a beam expander 113. The shaping optical system 11 may optionally include a "flat-top" type beam converter 114 and a beam thinners 115. The beam expander 113 allows the size of the laser beam to be adjusted so that the size of the laser beam approaches the size of the object to be observed. The expander 113 may be constituted by a set of two lenses with variable focal length, programmable by the processor 40. The beam converter 114 allows to adjust the intensity distribution in the beam.

The image obtained on the image sensor 20 may allow characterizing the object 3. The characterization may be an identification. To this end, features of the image are determined and compared with calibration features established on the standard object. These features may also be classified based on the calibration features. Patent application WO2014184390 describes a method for classifying bacterial colonies based on the projection of images of orthogonal Zernike (Zernike) polynomials. Other classification algorithms, such as principal component analysis algorithms, are contemplated. The purpose of this classification is to reduce the spatial information of the image to a set of coordinates on the basis of which the identification of the micro-organisms is obtained.

Because the method is not destructive, it is possible to generate multiple images of the same bacterial colony at different stages of culture in order to understand the nature of the colony growth or its ability to resist antibiotics or antimicrobials. In this case, the representation of the object represents a trend of growth of the object.

The method may also allow counting the number of objects present on the sample surface.

Experimental tests

Experimental tests carried out using the first embodiment will now be described. The main components used are as follows:

-a light source 10: an LCGFP-D-532-10C-F laser source provided by a laser component.

The shaping optical system 11: achromatic lens Thorlabs AC254-030-A-ML-A280 TM-A; pinhole Thorlabs-P50S, convergent lens Thorlabs a280 TM-a.

-a sample enclosure: pertri dishes 90mm in diameter-Biomreieux.

-a translucent screen: L80P3-12 lumineit polycarbonate diffuser or transparency paper.

-a focusing optical system: LM5JC 10M-Kowa.

-a camera: UI-1492 ME-IDS or AVGT 3300-Allied Vision.

-a light-reflecting element: a mirror tilted at 45 deg..

The assembly was placed in the dark.

In these tests, various types of bacterial colonies were observed. During each operation, the incident laser beam 12 is visually centered on the colony by the operator. The exposure time for each acquired image is 0.6ms to 1500 ms. Some images are obtained by combining different images acquired by the image sensor.

FIGS. 4A and 4B show diffraction patterns of E.coli bacterial colonies on Columbia Blood agar (COS) medium. What is needed isThe screen used is a piece of transparency paper. The reflective element 13 is supported by a transverse arm B extending parallel to the screen 20 (fig. 4A), or by a holder 17 fixed to the screen (fig. 4B), as shown in fig. 2D. In fig. 4A, it can be observed that the transverse arm B extending parallel to the screen 20 blocks the backscattered radiation 14, which produces a dark straight shadow in the diffraction pattern. It can also be observed that the diffraction pattern includes a bright central region, saturating the pixels of the image sensor, which is in contrast to the previously described reflection component 14₁And correspondingly. In fig. 4B, a central circular dark spot formed by the shading of the holder 17 can be observed, which is marked by an arrow. The shadow masks the reflected component 14₁. Thus, the dynamic range of the image is optimized and the peripheral region 14 of the diffraction pattern₂It looks more clear. These images demonstrate the arrangement of the reflective element 13 described in connection with fig. 2D.

Fig. 5A to 5C illustrate a method of centering the object 3 with respect to the incident light beam 12 and with respect to the screen. FIG. 5A is an image of a diffractogram of a bacterial colony of the Escherichia genus on COS medium, and the screen used is a piece of transparency paper. By applying the Canny filter to detect edges, fig. 5B is obtained. The diameter of the diffractogram was estimated to be 8.2 cm. The center of the diffraction pattern is determined, and the sample is moved so that the center of the diffraction pattern is located at the center of the image acquired by the image sensor (fig. 5C). This allows the colonies to be aligned with the incident beam 12 and with the optical axis of the image sensor.

Fig. 6 is an image called high dynamic range obtained by acquiring 11 images of the same diffractogram, the exposure time varying between 8ms and 495 ms. A High Dynamic Range (HDR) algorithm is implemented to combine the acquired images and form the image of fig. 6. The image shows bacterial colonies of staphylococcus epidermidis on COS medium, the screen being a piece of transparency paper.

Fig. 7A to 7F are examples of diffraction pattern images obtained by observing bacterial colonies growing on the previously described COS agar. The size of each diffraction pattern is 20cm²x 20cm². The screen used to obtain these diffraction patterns was a piece of perspective paper. The parameters for each figure are now listed:

-figure 7A: staphylococcus wavorei-diameter of laser beam: 900 μm;

FIG. 7B: diameter of saprophytic staphylococcus-laser beam: 900 μm;

-figure 7C: staphylococcus epidermidis-diameter of laser beam: 900 μm;

fig. 7D: coli-diameter of laser beam: 1800 mu m;

FIG. 7E: pseudomonas putida — diameter of laser beam: 2800 μm;

-figure 7F: enterobacter cloacae-diameter of laser beam: 2800 μm.

Fig. 8A to 8D show observations of colonies of various sizes. FIG. 8A shows microscopic observations of microcolonies of Staphylococcus epidermidis with a diameter of 760 μm. Fig. 8B shows a diffraction pattern of the microcolonies. FIG. 8C shows microscopic observation of microcolonies of E.coli 1160 μm in diameter. Fig. 8D shows a diffraction pattern of the microcolonies.

Fig. 9A to 9C show the diffraction patterns obtained on various agars:

FIG. 9A shows the diffractogram of Staphylococcus saprophyticus on Polyvitex chocolate agar (PVX);

FIG. 9B shows the diffractogram of Pseudomonas putida on Mueller Hinton agar;

FIG. 9C shows the diffractogram of E.coli on Tryptic Soy Agar (TSA).

These figures show the compatibility of the present invention with various media 4, whether they are opaque (COS, PVX) or Transparent (TSA).

Fig. 9D shows a diffraction pattern of colonies of staphylococcus saprophyticus grown on a surface formed by a pseudomonas putida layer. This result indicates that the present invention allows for non-destructive observation of colonies in situ without the need for sampling.

The observation method of the present invention is non-destructive and can be applied directly to colonies in a culture medium. This allows observation of the course of colony growth. FIG. 10 shows the diffractogram of the same Staphylococcus epidermidis colony on COS medium as a function of time. Each image in this figure is a diffractogram of a colony with a time interval of 1h between two successive images. The first image (top left) corresponds to a 16 hour incubation time and the last image (bottom right) corresponds to a 24 hour incubation time. Incubation times are marked in the upper right corner of each image.

Fig. 11A shows an experimental setup used during the experiment to compare the image obtained with a flat screen with the image obtained with a dome-shaped screen, as described in connection with fig. 3E. The screen 20 includes a curved portion 20a and a flat portion 20 b. The curved portion 20a is a portion of a polished glass hemisphere. The flat portion 20b is formed of a sheet of write-through paper. The experiments were performed using pseudomonas putida colonies on TSA agar. The diffraction pattern obtained is shown in fig. 11B. The diffraction pattern was observed to extend beyond the flat write-through paper, but was contained in the dome.

The present invention may be implemented to support various types of examinations for identification purposes or enumeration purposes, such as sterility tests, antibiotic susceptibility tests, antibacterial or phage susceptibility tests, and screening for antibacterial substances. The invention may also be applied to the observation and characterization of other types of microorganisms, such as yeast, fungi or microalgae.