Method for location identification and spectral imaging of microparticles, and associated system

Abstract

To provide a method for location identification and spectral imaging of microparticles, and an associated system.SOLUTION: The method for spatial location identification and spectral imaging of microparticles in a sample 200 includes the steps of: illuminating the sample by an annular lighting cone 124 coming from a light beam partially reflected by an annular mirror 13 and focused by a peripheral region 142 of a dark-field microscope objective lens 14; recording by being collected by a central region 144 of an objective lens; identifying a position of points of interest in an image; extracting coordinates of the points of interest; moving the sample to scan one of the extracted points; illuminating the point on the sample using a laser beam 160 transmitted through the annular mirror and focused by the central region of the dark-field microscope objective lens 14; and collecting an emitted Raman spectrum by the central region of the objective lens.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates generally to the field of particle analysis by spectral imaging.

[0002] More particularly, the present invention relates to methods and related systems for localization and spectral imaging of transparent particulates.

[0003] The invention finds particularly advantageous application in tracking and high speed spectral imaging of transparent particles scattered within macroscopically sized samples. [Background technology]

[0004] The detection and identification of the chemical properties of micrometer-sized solid particles, commonly called fine particles, is a topic of interest for many industries and / or applications. For example, the study of plastic fine particles, with their detection, quantification, and identification, is essential in fields such as environmental science and the food industry. Within the framework of such studies, macroscopically sized samples, ranging from several square millimeters or even several square centimeters, are taken from a medium to provide a representative overview of that medium. Such samples can be, for example, in the form of a liquid or gas, can contain solid fine particles in suspension, or can be in the form of a powder of solid fine particles.

[0005] Measurement systems have been developed to meet these analytical needs: spectral imaging systems have thus been proposed that link so-called full-field microscopic imaging modalities, used for the spatial localization of microparticles, with spectral analysis modalities, mostly by spot analysis, that use laser sources to record the spectrum of each microparticle, which makes it possible to track the chemical composition of the analyzed microparticles.

[0006] However, when using high-magnification microscope objectives to spatially resolve the particles under study, the image field of view of such spectral imaging systems remains limited to a rectangular area of ​​a few millimeters by a few millimeters or even less. Therefore, scanning the entire surface of the sample to identify the location of the particles requires creating a mosaic of the sample, which is then moved using a stage, with each tessera or tile of the mosaic being the size of the image field of the imaging system, whose dimensions depend in particular on the microscope objective and / or image sensor.

[0007] In methods generally described as static, a set of mosaic tiles representing the sample is acquired by full-field microscopy before proceeding to a second spot analysis by laser spectroscopy. Thus, the sample is first fully imaged before its spectral analysis.

[0008] Typically, macroscopic samples require the acquisition and processing of mosaics containing millions of tiles. Such image sizes require the compression of the image quality of the mosaic in order to store it and extract the coordinates of the various particles. This extraction is followed by spot analysis by acquiring spectra on the extracted coordinate points. However, this image compression comes at the expense of spatial resolution.

[0009] A dynamic approach has been proposed to correct for the degradation of spatial resolution. This involves extracting particle coordinates for each tile and acquiring a spectrum on each tile in situ before proceeding to the next tile. Because only the coordinates of interest are retained rather than the entire tile, this dynamic approach offers several advantages, including reduced image size. However, this approach doubles the acquisition time when switching from the imaging modality to the spectral analysis modality for each tile.

[0010] Known solutions for observing single particles are described for example in [1], whereas microspectrometry is described in [1]. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2018 / 138098 A1 Brochure [Non-patent literature]

[0012] [Non-Patent Document 1] Shaochuang Liu, Yilun Ying, Yitao Long.Rapid ultrasensitive monitoring the single-particle surface-enhanced Raman scattering(SERS) using a dark-field microspectroscopy assisted system[J].Chin.Chem.Lett.,2020,31(2):473-475 Summary of the Invention [Means for solving the problem]

[0013] In order to remedy the above-mentioned shortcomings of the state of the art, the present invention proposes to adapt a dynamic approach to allow both localization and spectral analysis of microparticles without switching electrically driven mechanical parts, thereby reducing acquisition times.

[0014] More particularly, a method for spatial localization in an image and spectral analysis of microparticles in a sample by a microscope system is proposed according to the invention, said method comprising the following steps: A) illuminating a region of the sample using an annular illumination cone, the annular illumination cone being obtained from a white light beam partially reflected by an annular mirror and focused by a peripheral region of a dark-field microscope objective. B) recording a first dark-field microscope image on a field of view contained within the illuminated region of the sample, the first dark-field image being collected by a central region of the dark-field microscope objective. C) Locating the particle within the first dark-field image D) extracting coordinates of points corresponding to microparticles in the recorded dark field image to form a list of points. E) Moving the specimen so that the optical axis of the darkfield microscope objective coincides with a point from the list of points. F) illuminating a measurement point on the sample using an excitation laser beam transmitted through an annular mirror and focused by a central region of a dark-field microscope objective, the measurement point corresponding to a point from the list of points. G) Collecting the Raman spectrum emitted from the measurement point, where the Raman spectrum is collected by a central region of the dark-field microscope objective.

[0015] Thus, with the present invention, no mechanical switching is required to switch from one microscopy modality to another within the context of dynamic techniques. This property is advantageous for samples with a large size relative to the microscope's field of view, within the context of the dynamic techniques defined in the introduction. This dynamic technique was introduced to avoid the need for spatial resolution compression, which is necessary to process large amounts of data, especially given the size of the sample being studied. However, the need to switch motorized mechanical parts within each tile to proceed from acquiring a wide-field image to acquiring a spot spectrum within the image leads to an incompressible idle time of the order of 5 seconds per tile. This idle time translates to several hours for millimeter- or even centimeter-sized samples. The present invention maintains the advantages of dynamic techniques, namely precise positioning at the sample scale, high spatial resolution, and measurement insensitivity to environmental fluctuations, while eliminating prohibitively long mechanical switching times. This elimination of mechanical switching times therefore removes a significant obstacle to the use of dynamic techniques on samples, especially large samples.

[0016] Furthermore, the invention improves the mechanical reliability of the system, since moving parts are in fact a potential source of misalignment and even system wear.

[0017] The proposed solution not only eliminates these idle times, which corresponds to a significant time saving and increases the reliability of the system, but also advantageously combines the properties of the two selected modalities: dark-field microscopy and Raman microspectroscopy, also called Raman microspectroscopy. On the one hand, it results in a solution without mechanical switching and without signal loss, meaning that, within the limits of optical losses, the entire Raman signal collected in epi-detection reaches the spectrometer. On the other hand, it facilitates the study of large samples containing small particles with low optical absorption.

[0018] Other non-limiting and advantageous features of the method according to the invention, taken individually or in all technically possible combinations, are: Steps A) and F) are carried out simultaneously, whereby an area on the sample is illuminated by a peripheral area of ​​the dark-field microscope objective, while a measurement point within the area is illuminated by a central area of ​​the dark-field microscope objective. The measurement point is located along the optical axis of the central region of the dark-field microscope objective. A field of view larger than the image field is reconstructed by a mosaic of dark-field microscope images, and these dark-field microscope images correspond to tiles of the mosaic. -The placement of each tile in the mosaic is predetermined upstream in the process Repeat steps E), F) and G) until all points from the list of extracted points on a given tile have been scanned, then proceed to the step of moving the specimen to focus the field of view of the darkfield microscope objective on another tile of the mosaic and start the process again from step A) to record another darkfield microscope image. -For each tile of the mosaic, the list of points contains a determined number of points - Establish the positioning of at least one tile of the mosaic as a function of the coordinates of a point from the list of points. - after the displacement in step E) to align the optical axis of the dark-field microscope objective with a point from the list of points, steps A), B), C), D), F) and G) are carried out, and a new dark-field microscope image corresponding to a new tile of the mosaic and a Raman spectrum emitted from the measurement point are recorded, these steps being followed by another displacement of the sample to align the optical axis of the dark-field microscope objective with another point from the list of points; - The additional points extracted from the new dark field microscope image recorded after the movement of step E) are added to the list of points to be scanned.

[0019] The present invention also relates to a system for spatial localization and spectral analysis of microparticles in images, comprising an optical microscope, a laser source, a white light source, an image sensor, a spectrometer and a control unit, the latter comprising an image processing unit, wherein the optical microscope comprises a sample holder mounted on a displacement stage, the sample holder adapted to receive a sample, the system comprising an optical system including an annular mirror, a dark field microscope objective, the optical system being disposed between the laser source, the white light source and the sample holder, the dark field microscope objective having a central region and a peripheral region, the annular mirror is configured to reflect a portion of the white light beam emitted by the white light source toward a peripheral region of the dark-field microscope objective and to transmit an excitation laser beam emitted by the laser source toward a central region of the dark-field microscope objective; the dark field microscope objective is adapted to transmit a portion of the white light beam towards the sample holder using its peripheral region and to transmit the excitation laser beam towards the sample holder via its central region; a dark field microscope objective adapted to focus a portion of the light beam to illuminate a first area of ​​the sample on the sample holder using an annular illumination cone, and adapted to focus the excitation laser beam onto a measurement point contained within the first area; a central region of the darkfield microscope objective adapted to collect a darkfield microscope image on an image field contained within the first illumination region, the darkfield microscope image being recorded using an image sensor; an image processing unit adapted to identify the locations of potential microparticles and to extract coordinates of points associated with these microparticles from the dark field microscope image to establish a list of points; the displacement stage is configured to move the sample holder in order to match the measurement point with a point from the list of points or to position the image field of the dark field microscope objective in another area that is at least partially different from the first area; The central region of the dark field microscope objective is also adapted to collect the Raman spectrum emanating from the measurement point, which Raman spectrum is recorded by a Raman spectrometer. It is characterized by:

[0020] Furthermore, various other features of the present invention will become apparent from the accompanying description taken in conjunction with the drawings illustrating non-limiting embodiments of the invention. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows a schematic representation of an optical microscope system adapted to implement a method for spatial localization and spectral analysis of microparticles within a sample. [Figure 2] 1 is a block diagram illustrating a first embodiment of the method of the present invention. [Figure 3] 1 shows an example of a mosaic that includes multiple tiles, each corresponding to an image recorded using the method described with respect to FIG. [Figure 4] FIG. 2 is a block diagram illustrating a second embodiment of the method of the present invention. [Figure 5] 5 illustrates the method described with respect to FIG. 4, and in particular an example of recording several consecutive tiles to form a mosaic according to the second embodiment. [Figure 6] 3 or 5 shows an image of the sample in which the positions of the particles are identified for some of the particles contained in the mosaic of FIG. 3 or 5. DETAILED DESCRIPTION OF THE INVENTION

[0022] In these figures, structural and / or functional elements that are common to different alternatives may have the same reference numerals.

[0023] Various other modifications can be made to the present invention within the scope of the appended claims.

[0024] 1 shows a system 1 for spatial localization and spectral analysis of microparticles in images, based on an optical microscope, an image sensor 15, an image processing system 60, and a Raman spectrometer 19. Such a system, referred to herein as an "optical microscope system", is based on light-matter interactions for imaging and / or analyzing a sample 200 having micrometer-sized structures. These structures are in particular in the form of microparticles 202, i.e. solid particles whose size is, for example, between a few tenths of a micrometer and a few hundredths of a micrometer.

[0025] The particles considered here are particles of plastic material 202, i.e., polymer particles 202 composed of long carbon chains and derived from fossil combustibles, nevertheless the term "particles" also refers, without limitation, to micrometer-sized mineral particles, or pharmaceutical powder particles, or solid particles suspended in the air.

[0026] These particles 202 are contained within a sample 200, which has a size ranging from a few hundred micrometers to a few tens of centimeters. The sample 200 may, for example, be macroscopic in size and contain particles 202 suspended in water or air, or may also be in the form of a powder. Such samples 200 are collected from the environment for analytical and / or research purposes in various fields, such as the food industry, the pharmaceutical industry, geology, and environmental science, to name a few. The collected sample 200 is here placed on a sample holder 20, in particular a glass microscope slide, and may be protected by the microscope slide.

[0027] 1 is configured to study such samples 200 by, among other things, imaging the microparticles 202 contained therein and thus making it possible to track morphological information about these microparticles 202. For example, images recorded using this optical microscope system 1 make it possible to see the areas, boundaries, shapes, etc. of these microparticles 202. It is also advantageous to complete this morphological study with a study of the chemical composition of each of these microparticles 202, in particular through the recording of spectra characteristic of this chemical composition, such as Raman or photoluminescence spectra. However, the recording of these characteristic spectra typically uses spot measurements.

[0028] In the study of large-sized samples 200 containing microparticles 202, it is particularly advantageous to proceed from the microscopic image to the spatial localization of these microparticles 202 and to establish characteristic spectra on the localized points by spot measurements on points, most often of micrometer dimensions. This makes it possible to spatially resolve information about the chemical composition of the sample 200. The term "large size" is used here to mean that the size of the sample is larger than the size of the microparticles 202, i.e., the sample 200 under consideration is macroscopic in size, having sides or diameters of, for example, several millimeters.

[0029] Given the small size of the points at which measurements can be made relative to the overall size of the sample 200, this initial location of the points of interest saves time, especially for samples 200 that contain low concentrations of particulates 202. Thus, recording spectra only at the points of interest, rather than taking spot measurements for each point on the sample 200, advantageously reduces acquisition time, processing time, and the storage space required to store the measured spectra.

[0030] Thus, the system 1 for spatial localization and spectral analysis of microparticles in images comprises at least two different microscopy modalities, each based on a contrast mechanism, i.e., light-matter interaction, and / or on the implementation of different light-matter interaction mechanisms. Here, in the present disclosure, a dark-field microscopy modality, associated with the Raman microspectroscopy modality, sometimes referred to as Raman microspectroscopy, is advantageously selected for imaging the sample 200.

[0031] The dark field microscopy modality is configured to illuminate the surface of a sample 200 and to acquire an image of at least a portion of this illuminated surface. Dark field microscopy imaging, or more simply dark field imaging, is based on the following contrast mechanism: the sample 200 and structures contained therein, e.g., as microparticles 202, are illuminated in such a way that only light rays deflected by the sample 200 and structures present on this sample 200 can be collected by an optical objective.

[0032] Dark-field microscopy is an imaging modality particularly suited to the study of microparticles 202, especially microparticles of plastic materials 202. In fact, dark-field microscopy is known to deteriorate contrast on transparent samples 200, i.e., samples 200 that absorb little light. Secondly, it is known that certain plastic materials, in particular microplastics and / or mineral or pharmaceutical powder particles, are transparent and have relatively little contrast when illuminated using other imaging modalities, for example with clear-field illumination.

[0033] The system 1 for spatial localization and spectral analysis of microparticles in an image includes a second microscopy modality corresponding to the Raman microspectroscopy modality. This Raman microspectroscopy modality probes the chemical composition of a sample 200 at a measurement point by recording a Raman spectrum emitted from this measurement point after focused optical excitation. The Raman spectrum is obtained by Raman scattering, which corresponds to the inelastic scattering phenomenon of light due to energy exchange with the probed medium. This change in energy represents the molecular vibrational modes of the probed medium at the measurement point. The emitted Raman spectrum therefore contains Raman lines. This Raman spectrum is characteristic of a given chemical species, and its measurement and analysis can provide information, among other things, about the chemical composition of the medium of interest.

[0034] Thus, the Raman microspectroscopy modality advantageously allows for point-by-point mapping, i.e., by scanning the measurement points of the chemical composition of the sample 200, and more particularly of the chemical composition of the microparticles 202 contained within the sample 200. For example, it is possible to see the composition of microparticles 202 of a plastic material dispersed within an aqueous sample 200.

[0035] The implementation of each of these microscopy modalities, whether in an imaging or spectral format, is based on a separate illumination means and / or a means for collecting the signal generated by the light-matter interaction.

[0036] The present invention proposes a system 1 for spatial localization and spectral analysis of microparticles in an image, in which two microscopy modalities, namely, dark-field microscopy and Raman microspectroscopy, are implemented simultaneously or sequentially without the need for any mechanical switch between two separate illumination and / or detection means to switch from one modality to the other. In fact, the mechanical switches typically proposed in optical microscope systems for switching from one microscopy modality to another cause slowness in system use. Although solutions exist to reduce the time required to switch between the two microscopy modalities, problems still exist related to the high monetary cost of these high-speed switching solutions, potential misalignment, and limited lifespan due to, among other things, the aging of the motors driving such mechanical switches.

[0037] A dynamic analysis method is also described that is particularly suited to imaging and spectral analysis of large, i.e., macroscopic, samples containing microparticles 202, benefiting from the presently disclosed system 1 for spatial localization within images and spectral analysis of microparticles. Indeed, to fully image such a sample 200, the number of mechanical switches required to switch from one microscopy modality to another to localize the microparticles 202 before proceeding to spectral analysis doubles depending on the size of the sample 200. Thanks to the disclosed method, analysis of this type of sample 200 is accelerated while maintaining high spatial resolution and minimizing the storage space required to record acquired data.

[0038] We will first describe a system 1 for spatial localization and spectral analysis of microparticles in images, as shown in FIG. 1 and intended for analyzing a sample 200 as described above. In one example, the sample 200 is a sample containing a dispersion of microparticles 202, such as mineral grains or microplastics. Nevertheless, the sample 200 can also be a sample containing a solution or liquid, e.g., aqueous, in which microparticles 202 of plastic material are dispersed. In any case, this sample 200 is placed on a sample holder 20. The optical microscope system includes, for example, a microscope stand, on which the various elements described below are arranged and mounted.

[0039] The main axis 10 of the microscope is set perpendicular to the surface of the sample 200, which is considered to be planar.

[0040] The sample holder 20 is integrated with a displacement stage 11 or positioning stage. The latter is adapted to move the sample holder 20 along at least two translation axes, the x-axis and the y-axis, which are orthogonal to each other and parallel to the main plane of the sample 200. This displacement stage 11, e.g. a piezoelectric stage, is controlled and motorized by a controller (not shown in FIG. 1 ) so that the movement of the sample holder 20 can be automated. The displacement stage 11 has a displacement amplitude of tens of millimeters to a few centimeters and a displacement accuracy in the micrometer range. A control unit 6, e.g. a computer, steers and synchronizes the displacement of the displacement stage 11.

[0041] Dark-field microscopy modality To illuminate the sample 200 according to the dark-field microscopy modality, the system 1 for spatial localization and spectral analysis of microparticles in an image comprises a light source, for example a light source that emits incoherent optical radiation, such as an incandescent lamp, a halogen incandescent lamp, or a lamp including at least one light-emitting diode (LED).

[0042] In this first embodiment, more precisely, the light source is an incandescent lamp which here emits light radiation having a light spectrum which at least partially covers the visible spectrum, such light radiation being perceived as close to white and therefore referred to as a white light source 12.

[0043] The optical radiation emitted by the white light source 12 propagates through free space, here in the form of a collimated white light beam 120. The white light beam 120 is assumed to propagate along an optical axis perpendicular to the main axis 10 of the microscope.

[0044] The optical microscope system also includes an annular mirror 13. The annular mirror 13 has a flat reflective surface, for example, a polished metal deposit on the flat surface. Here, for example, a silver deposit. The annular mirror 13 has an elliptical shape and is pierced through its center by an elliptical central opening. In other words, the annular mirror 13 has the shape of an elliptical ring. The opening is located in the center of the surface of the annular mirror 13. Therefore, the center of the annular mirror 13 does not reflect light. The annular mirror 13 is contained within, for example, a block or cube, typically known as a dark-field cube.

[0045] The position of this annular mirror 13 is fixed relative to the microscope stand.

[0046] The annular mirror 13 is arranged to be centered on the main axis 10 of the optical microscope system, on the one hand, and on the white light beam 120, on the other hand. Furthermore, the reflective flat surface of the annular mirror 13 forms an angle of 45 degrees with the main axis 10 of the microscope system. The annular mirror 13 is adapted to reflect a portion of the white light beam 122 towards the sample 200.

[0047] In fact, if white light beam 120 is initially approximated as a solid cylinder, only the portion corresponding to the hollow cylinder will be reflected towards sample 200. This portion corresponds to the portion of white light beam 122 that is incident on the reflective flat surface of annular mirror 13.

[0048] The remaining portion is transmitted through the elliptical central opening of the annular mirror 13, for example towards a beam blocker not shown in FIG.

[0049] A portion of the white light beam 122 corresponding to the hollow cylinder propagates along the main axis 10 of the optical microscope system towards the sample 200 .

[0050] The optical microscope system also includes a darkfield microscope objective 14, as is known in the art. The objective has two distinct optical regions: a peripheral region 142 and a central region 144. The optical axis of the darkfield microscope objective 14 is coincident with the major axis 10 of the microscope system.

[0051] In the embodiment described herein, the darkfield microscope objective 14 corresponds to an objective with a magnification of 50x and a numerical aperture of 0.60. The field of view of the darkfield microscope objective 14 is 0.44 millimeters in diameter. Furthermore, it is an infinity-corrected objective.

[0052] The peripheral region 142 of the darkfield microscope objective 14 corresponds to an outer region surrounding a central region 144. The central region 144 corresponds to a conventional microscope objective. Thus, the central region 144 is centered on the optical axis of the darkfield microscope objective 14 and is surrounded by the peripheral region 142.

[0053] A portion of the white light beam 122 corresponding to the hollow cylinder is transmitted through a peripheral region 142 of the darkfield microscope objective 14. The size of the hollow cylinder as well as the size of the annular mirror 13 are selected as a function of the size of the peripheral region 142. Specifically, it is ensured that all of the white light beam 122 reflected by the annular mirror 13 is transmitted through the peripheral region 142 of the darkfield microscope objective 14.

[0054] The peripheral region 142 is apertured for an annular optical element that focuses the hollow cylinder into an annular light cone 124. This optical element corresponds to, for example, an annular optical lens or a concave mirror. The sample 200 is illuminated in the dark-field microscopy modality using the annular illumination cone 124 that corresponds to this hollow cone. The sample 200 is therefore illuminated using light rays that are highly oblique with respect to the main axis 10 of the optical microscope system, i.e., light rays that have a large inclination angle with respect to the main axis 10 of the optical microscope system. The inclination angle of the light rays is selected so that these light rays cannot be captured in the central region 144 of the dark-field microscope objective 14. Here, the inclination angle is greater than or equal to 37 degrees with respect to the optical axis of the dark-field microscope objective 14.

[0055] In this way, an area of ​​the sample 200 is illuminated. This area extends parallel to the plane of the sample 200, and the dimensions of this illuminated area, specifically its diameter, are determined as a function of the properties of the dark-field microscope objective 14, such as its magnification and numerical aperture.

[0056] The illumination area considered here has a circular size ranging from a few micrometers to a few hundred micrometers in diameter. For example, the area considered here is a circular illumination area having a diameter of at least 100 micrometers, or even at least 140 micrometers, or even at least 150 micrometers. This circular illumination area has a size, here a diameter, larger than the size of the image to be recorded later.

[0057] Depending on the dark-field microscope objective 14 used in the first embodiment, the illuminated area corresponds to a circular area of ​​several tens or even several hundred micrometers, for example, a circular area with a diameter of 440 micrometers.

[0058] The sample 200 and in particular the structures contained therein, here particulates 202, more specifically particulates of plastic material 202 and / or particulates of minerals 202, scatter a portion of the oblique light rays from the annular light cone 124. Only the light rays deflected by the structures of the sample 200 are collected by the central region 144 of the darkfield microscope objective 14, in accordance with the known contrast mechanisms of darkfield microscopes. Thus, only structures that at least partially deflect the incident light provide contrast and contribute to the darkfield microscope image. The central region 144 of the darkfield microscope objective 14 functions as a conventional microscope objective and is characterized by a magnification and a numerical aperture as described herein above.

[0059] Therefore, the light rays deflected by the microparticles 202 are collected by the central region 144 of the dark-field microscope objective 14. From a spectral point of view, these deflected light rays have a spectrum identical to that of the white light beam 120, because the contrast mechanism is based on the elastic scattering phenomenon.

[0060] Since an infinity-corrected objective is used in the context of this first embodiment, the deflected light rays emerge collimated from the darkfield microscope objective 14. These deflected light rays then propagate in free space along the main axis 10 of the optical microscope system, in particular passing through an opening formed in the annular mirror 13.

[0061] The deflected light beam is then imaged by an image sensor 15. This image sensor 15 has an optical axis that coincides with the main axis 10 of the optical microscope system and thus with the optical axis of the darkfield microscope objective 14.

[0062] The image sensor 15 comprises imaging optics and a photosensitive matrix sensor 150, this unit often referred to as a camera. The optical imaging system is adapted to create an image of the sample 200 on the photosensitive matrix sensor 150. The photosensitive matrix sensor 150 comprises a matrix of light-sensitive pixels, here rectangular, capable of converting light information into accumulated electrical charges. This sensor is, for example, a CCD sensor or a CMOS sensor.

[0063] The image sensor 15 is adapted to record a dark-field microscope image corresponding to at least a portion of the dark-field illuminated area by imaging the polarized light beam onto a light-sensitive matrix sensor. The image sensor 15 has a field of view that depends, among other things, on the magnification of the optical imaging system of the image sensor 15 and the size of the rectangular matrix of pixels. This field of view of the image sensor 15, like the field of view of the central region 144 of the dark-field microscope objective 14, limits the area of ​​the sample 200 that can be imaged with the dark-field microscopy modality. This imaged area is referred to as the image field of view.

[0064] The image field of view is contained within the illuminated area and is therefore smaller in size than the size of the illuminated area.

[0065] The dark field microscope image reproduces the area of ​​the sample 200 as described above in this specification, and the contrast within this dark field microscope image represents structures, such as particles of plastic material or mineral grains, i.e., particles 202, that can deflect incident light rays.

[0066] This dark field microscope image often has a rectangular shape due to the rectangular shape of the photosensitive matrix sensor 150 .

[0067] Each dark field microscope image represents, for example, a 140×10 5 micrometer area on the sample 200 .

[0068] The image processing unit 60 shown in Fig. 1 is included in the control unit 6, which is here for example a computer, and is configured to exchange information with the image sensor 15. This image processing unit 60 is also adapted to identify and extract coordinates of points from the dark-field microscope image that may correspond to microparticles 202. More specifically, microparticles 202 having a minimum diameter of 0.5 micrometers are spatially located and morphological parameters are extracted therefrom. This minimum diameter depends in particular on the optical performance, i.e., the imaging performance, of the system. There is no upper limit to the diameter of the microparticles 202 that can be detected.

[0069] The image processing unit 60 is also configured to interact with a user via an interface integrated into the software. The user has the possibility to filter certain particles 202 of interest based on specific criteria, such as morphological parameters. For example, the user can decide to keep only those particles 202 whose diameter falls between two value limits.

[0070] This identification and extraction may be performed using dedicated image processing algorithms, or by automated learning or machine learning algorithms known in the art. Alternatively, the identification and extraction of the coordinates of the points of interest in each dark-field microscope image may be performed manually or partially manually by a user of the optical microscope system.

[0071] Raman microspectroscopy modality The Raman microspectroscopy modality is also illustrated in Figure 1.

[0072] Advantageously, this Raman microspectroscopy modality can operate without switching mechanical elements to switch from one modality to another, and therefore can be implemented simultaneously with dark-field microscopy modalities.

[0073] The optical microscope system includes a laser source 16. This laser source 16 emits an excitation laser beam 160, which is here continuous. In addition, the excitation laser beam 160 is here collimated at the exit of the laser source 16.

[0074] In a non-limiting manner, this laser source 16 corresponds, for example, to a diode-pumped solid-state laser.

[0075] In the Raman microspectroscopy modality, the excitation laser beam 160 is monochromatic, e.g., has a wavelength selected within the spectral window between 380 nanometers and 1064 nanometers, e.g., here, excitation laser beam 160 has a wavelength of 532 nanometers.

[0076] At the exit of the laser source 16, this excitation laser beam 160 propagates along its optical axis, which here is initially parallel to the main axis 10 of the optical microscope system. A first dichroic filter 17, also known in the art as an edge filter, reflects the excitation laser beam 160 so that the optical axis of the laser beam is perpendicular to the main axis 10 of the optical microscope system.

[0077] This first dichroic filter 17 corresponds more precisely to a high-pass filter. Such a filter is adapted to reflect a spectral range of wavelengths below a cutoff wavelength and to transmit a distinct spectral range of wavelengths that is in turn greater than the cutoff wavelength. The cutoff wavelength is selected as a function of the wavelength of the excitation laser beam 160, here to reflect the excitation laser beam 160. The first dichroic filter 17 has a cutoff wavelength established by the constructor at 532.85 nanometers, for example.

[0078] To correspond to an edge filter, also known as a laser filter, the curve of transmittance as a function of wavelength of this first dichroic filter 17 has a very narrow transition region between the transmitted and reflected wavelengths. In other words, the transmittance curve of the first dichroic filter has a steep slope at its cutoff wavelength. In this case, the transition between the transmitted and reflected wavelengths occurs over a few nanometers around the cutoff wavelength. For example, the transition occurs over a width of 10 nanometers or less around the cutoff wavelength, more ideally 5 nanometers or less around the cutoff wavelength, or even 3 nanometers or less around the cutoff wavelength.

[0079] The optical microscope system also includes a second dichroic filter 18 .

[0080] Such a second dichroic filter 18 is adapted to reflect a spectral range of wavelengths greater than the cutoff wavelength and to transmit a spectral range of wavelengths, and is therefore a low-pass dichroic filter.

[0081] Here, the cutoff wavelength is selected to be less than the wavelength of the excitation laser beam 160, i.e., precisely less than 532 nanometers. More precisely, the second dichroic filter 18 has a cutoff wavelength equal to 495 nanometers. The excitation laser beam 160 is therefore reflected by the second dichroic filter.

[0082] This second dichroic filter 18 is also selected to transmit at least a portion of the optical spectrum of the white light beam 120. Advantageously, according to one embodiment, the optical spectrum of the white light beam 120 is completely transmitted by the second dichroic filter 18.

[0083] This second dichroic filter 18 is centered on the main axis 10 of the optical microscope system between the annular mirror 13 and the image sensor 15. The second dichroic filter 18 is also centered with respect to the optical axis of the excitation laser beam 160. The surface of the second dichroic filter 18 is oriented at 45 degrees with respect to the optical axis of the laser beam in order to reflect the excitation laser beam 160 along the main axis 10 of the optical microscope system towards an annular opening formed inside the annular mirror 13.

[0084] The excitation laser beam 160 is transmitted through a hole in the annular mirror 13 along the main axis 10 of the optical microscope system towards the sample 200 .

[0085] The central region 144 of the dark field microscope objective 14, centered on the main axis 10 of the optical microscope system, transmits the excitation laser beam 160 of the Raman microspectroscopy modality and then focuses the excitation laser beam 160 to a measurement point on the sample 200.

[0086] This measurement point is centered on the optical axis of the dark field microscope objective 14, which coincides with the main axis 10 of the optical microscope system. This point coincides with the geometric center of the illuminated area, for example in dark field microscopy.

[0087] This measurement point has an extent in the xy plane that is defined as a function of the properties of the dark field microscope objective 14 , in particular its numerical aperture, as well as the wavelength of the excitation laser beam 160 .

[0088] Typically, the measurement point range varies between a few hundred nanometers and a few micrometers, where, for example, given the numerical aperture of the dark-field microscope objective 14 and the wavelength of the excitation laser beam 160, the measurement point probes a spot on the sample 200 with a diameter of approximately 564 nanometers.

[0089] Due to the Raman effect, the sample 200 emits a Raman spectrum related to inelastically scattered light. Here, we focus on the Stokes optical radiation, which corresponds to optical radiation whose wavelength is equal to or greater than the wavelength of the excitation laser beam 160.

[0090] This optical radiation corresponds to the Raman spectrum emitted from the measurement point where the excitation laser beam 160 is focused. This Raman spectrum contains Raman lines that are specific to the chemical composition of this measurement point. To access this spectral information, the scattered optical radiation is analyzed.

[0091] In this way, the Raman spectrum in the form of scattered light radiation is collected by the central region 144 of the dark-field microscope objective 14, which collimates this scattered light radiation into a light beam called the Raman beam 162. This Raman beam 162 is accompanied by a beam corresponding to the light radiation that is elastically scattered by Rayleigh scattering, which has the same wavelength as the excitation laser beam 160. Hereinafter, this beam will be referred to as the Rayleigh beam.

[0092] The Raman beam 162 and the Rayleigh beam propagate along the main axis 10 of the optical microscope system towards the hole in the annular mirror 13 .

[0093] Thus, the Raman beam 162, the Rayleigh beam, and the pump laser beam 160 are spatially overlapping and parallel to one another, yet the Raman beam 162 and the Rayleigh beam are counter-propagating relative to the pump laser beam 160, i.e., they propagate in opposite directions relative to one another.

[0094] Thus, the Raman beam 162 and the excitation laser beam 160 share the same optical path between the sample 200 and the first dichroic filter 17 .

[0095] Thus, the central opening of the annular mirror 13 allows the Raman beam 162 and the Rayleigh beam collected and collimated by the central region 144 of the dark field microscope objective 14 to pass through.

[0096] The Raman beam 162 and the Rayleigh beam continue their propagation up to the second dichroic filter 18. If the Raman beam 162 corresponds to Stokes optical radiation of a wavelength equal to or greater than the wavelength of the excitation laser beam 160, and therefore strictly above the cutoff wavelength of the second dichroic filter 18, the Raman beam 162 will be reflected at an angle of 90 degrees to the main axis 10 of the optical microscope system. This is also the case for a Rayleigh beam of the same wavelength as the excitation laser beam 160.

[0097] In this way, the Raman beam 162 and the Rayleigh beam propagate along the optical axis of the excitation laser beam 160 to the first dichroic filter 17 .

[0098] The first dichroic filter 17 transmits the Raman beam 162 and reflects the Rayleigh beam towards a Raman spectrometer 19 included in the optical microscope system. In fact, this first dichroic filter 17 is configured to reflect wavelengths below a cutoff wavelength selected equal to that of the excitation laser beam 160, while transmitting wavelengths above that cutoff wavelength.

[0099] In this way, the Raman beam 162 is spectrally analyzed by a spectrometer 19. Such a spectrometer 19 comprises, inter alia, a dispersive element, i.e. an optical element such as a prism or a diffractive network, adapted to spectrally decompose the light into monochromatic spectral components, as well as a photosensitive matrix sensor 190, such as a CCD or CMOS sensor.

[0100] Spectral analysis of the Raman beam 162 by the spectrometer 19 makes it possible to trace the Raman spectrum coming from the measurement point where the excitation laser beam 160 is focused by the central region 144 of the dark-field microscope objective 14 .

[0101] Thus, in implementations of the present disclosure of a bi-mode optical microscope system, i.e., a system that associates a dark-field microscopy modality with a Raman microspectroscopy modality for imaging, both modalities can be implemented simultaneously without mechanical switching to switch from one illumination modality to another.

[0102] According to a first alternative, in order to simultaneously implement both modalities, the optical spectrum of the white light beam 120 and the wavelength of the excitation laser beam 160 are selected to have discontinuous wavelength ranges. Specifically, in order to separate the Raman beam 162 coming from the Raman microspectroscopy modality from the polarized light coming from the dark-field microscopy modality, the optical spectrum of the white light beam 120 is selected to be strictly smaller than the wavelength of the excitation laser beam 160, for example. In particular, a low-pass filter arranged between the white light source 12 and the annular mirror 13 filters out undesired wavelengths. Alternatively, monochromatic radiation of a wavelength strictly smaller than the wavelength of the excitation laser beam 160 is selected to serve as white light. In the embodiment shown in FIG. 1 , the wavelength ranges covered by the laser source 16 and the white light source 12 are considered to be discontinuous. The wavelength range covered by the white light source 12 is considered to be strictly below the wavelength of the excitation laser beam 160.

[0103] Conversely, according to a second alternative, the two modalities can also be implemented alternately, i.e., one after the other, by controlling the power supply of one or both of the light sources, i.e., the laser source 16 and the white light source 12. It is thus possible to selectively switch off one of the light sources in favor of the other in a fast manner compared to conventional switching times known in the art. In this case, the spectral ranges covered by each of the two light sources can be freely selected.

[0104] Dynamic Method Such optical microscope systems find particularly advantageous use in implementing methods for spatial localization and dynamic spectral analysis of microparticles 202, as described below. Note that in such dynamic techniques, rather than imaging the entire sample 200 before proceeding to spectral analysis as in static techniques, only a portion of the sample 200 is imaged before proceeding to spectral analysis within the imaged portion. These two steps are repeated until a representation of the entire sample 200 is obtained.

[0105] A first embodiment of such a method is illustrated by a first block diagram 3 shown in FIG.

[0106] In its first embodiment, the method includes the following steps, which are detailed using the following illustrative example: - illuminating an area on the sample 200 using a dark field microscopy modality (30) -Record dark field microscope images (31) - Identifying the location of the particle 202 in the image (32) - Extract the coordinates of the points corresponding to those particles 202 to form a list (33) - moving the sample 200 so that the central area 144 of the dark field microscope objective 14 corresponds to a point in the list of extracted coordinates (34); - Irradiating the sample 200 using the Raman microspectroscopy modality (35), the excitation laser beam 160 is focused to one measurement point that coincides with the point of the previous step. The central region 144 of the dark-field microscope objective 14 collects the Raman spectrum emitted from the measurement point (36).

[0107] The moving step 34, the illuminating step 35, and the collecting step 36 are then possibly repeated, for example, until all points on the list have been scanned, i.e., browsed. A new moving step 37 then moves the sample 200 and restarts the method with illumination 30 of a new dark field area that is at least partially different from the previous area.

[0108] An example application of the first embodiment of this method is shown in FIG.

[0109] In this first embodiment, multiple dark-field microscope images are used to reconstruct a mosaic 4 representing the sample 200. The mosaic 4 represents a field of view larger than the image field by assembly of several dark-field microscope images that cover at least partially discontinuous areas on the sample 200. The images considered here are dark-field microscope images that cover completely discontinuous areas. Ideally, the mosaic 4 represents the entire surface of the sample 200.

[0110] Each of the multiple dark-field microscope images is referred to herein as a tile 40, i.e., a region of the sample 200 extending in the xy plane. Each of the tiles 40 is therefore at least partially separated from the other tiles 40 included in the mosaic 4. Here, each of the tiles 40 is considered to be completely separated from the other tiles 40. The size of each of the tiles 40 is the same here and is determined by the field of view of the image sensor 15, which image field of view is contained within the illuminated area of ​​the sample 200.

[0111] Therefore, based on the characteristics of the image sensor 10 and the characteristics of the dark field microscope objective 14, each tile 40 has a rectangular shape of 140 micrometers by 105 micrometers.

[0112] In FIG. 3, the tiles 40 forming the mosaic 4 are arranged in a rectangular grid with the tiles 40 adjacent to each other.

[0113] In a first embodiment, the position of each tile 40 of the mosaic 4 is predetermined upstream of the execution of the method with respect to a coordinate system, which is represented for example by the displacement stage 11, which is integral with the sample holder 20 and thus with the sample 200. The coordinate system established by the displacement stage 11 therefore also specifies the position of points on the sample 200.

[0114] Thus, each tile 40 is located by a coordinate pair along the x and y axes in the coordinate system. i ,y i ) locates the geometric center 41 of the tile 40 relative to the sample 200. Here, the geometric center 41 of the tile 40 indicates the intersection of the diagonals of the tile 40, and the tile has a rectangular shape. For each of the tiles 40 in FIG. 3, the geometric center 41 is represented by a target, i.e., a cross and a circle.

[0115] Alternatively, each tile 40 may be located by one of its corners.

[0116] In a first embodiment, a set of coordinate pairs for each of the tiles 40 is established in advance, i.e., prior to implementation of the disclosed methods for spatially localizing and spectrally imaging microparticles 202. Each tile 40 occupies a predetermined spatial extent on the sample 200.

[0117] For illustrative purposes, only a portion of the mosaic 4 reconstructed on the sample is shown in FIG.

[0118] The portion of the mosaic 4 shown in Figure 3 includes 20 tiles 40, each of which has a size of 140 micrometers by 105 micrometers.

[0119] Typically, the sample 200 under consideration has dimensions from about 10 millimeters up to several centimeters, such as 13 millimeters, 25 millimeters, or 42 millimeters in diameter, and is represented by a mosaic 4 of up to 1000 x 1000 tiles 40, i.e., 1 million or more tiles 40.

[0120] For example, for a sample 200 having a size of 27 millimeters in diameter, if the tiles 40 are rectangular and each have a size of 100 micrometers by 100 micrometers, then π * (D / 2) 2 tiles, where D corresponds to the diameter of the sample 200. This means that approximately 58,000 tiles will be acquired to cover the entire surface of the sample 200.

[0121] Small size tiles 40, corresponding to images acquired using high magnification objectives, are preferred to increase spatial resolution, especially in the plane of sample 200.

[0122] To record each of the darkfield microscope images forming the tiles 40 of the mosaic 4, the displacement stage 11 moves the sample 200, for example, to align a coordinate pair (shown here as the geometric center 41 of each tile 40) of a set of coordinate pairs pre-established for each tile 40 with the optical axis of the darkfield microscope objective 14. More broadly, what is important is to align the field of view contained in the area of ​​the sample 200 illuminated by the darkfield microscopy modality with a given tile 40.

[0123] Within the framework of the dynamic method for spatial localization and spectral imaging of microparticles 202, coordinate points 42 associated with potential microparticles 202 are located within each imaged tile 40 by the dark-field microscopy modality, and the Raman spectra of these located coordinate points 42 are recorded thanks to the Raman microspectroscopy modality before proceeding to the acquisition of the next tile 40. The sequential order of the imaged tiles 40 is predefined before the process is implemented. For example, the surface of the sample 200 is imaged according to a line-by-line scanning pattern, also called a raster scan, or a serpentine sweep, or any other sweep pattern considered appropriate.

[0124] Therefore, according to a first implementation form in which the positions of the tiles 40 are predetermined, for each of the n tiles 40 forming the mosaic 4, the steps described below are implemented, during which there is a movement 37 of the sample 200 from one tile to another.

[0125] We now detail the application of the method to obtain, for example, the tile 40 located in the upper left corner of the mosaic 4 shown in FIG.

[0126] First, it is assumed that the geometric center 41 defined for one of the tiles 40 corresponds to the optical axis of the darkfield microscope objective 14 after positioning of the displacement stage 11 .

[0127] For each tile 40, we proceed to illuminate (30) an area of ​​the sample 200 using an annular illumination cone 124. Note that this illumination cone is generated by reflection of a portion of the white light beam 120 by an annular mirror 13. The reflected portion, which forms a hollow cylinder, is transmitted by a peripheral region 142 of the darkfield microscope objective 14 and focused onto the sample 200 so as to illuminate an area on the sample 200 using only light rays that are highly oblique to the optical axis of the darkfield microscope objective 14.

[0128] Next, the image sensor 15 is used to record (31) a first dark-field microscope image corresponding to one of the tiles 40. As described above, light rays deflected by the sample 200, and in particular by the particles 202 contained therein, are collected by the central region 144 of the dark-field microscope objective 14. These deflected light rays form a first dark-field microscope image in which contrast is present only for the structures deflecting the light, i.e., the particles 202. The first dark-field microscope image is recorded over an image field contained within the illuminated area of ​​the sample 200. This first dark-field microscope image corresponds to the first tile 40, here located in the upper left corner of the mosaic.

[0129] This dark field microscopy modality optimizes contrast on highly transparent samples 200, here on fine particles 202 of plastic material, or on mineral grains, which facilitates the next step.

[0130] Indeed, based on this first dark-field microscope image, a localization (32) of potential particles 202 in this first image is then performed. This localization (32) is carried out, for example, by means of an image processing algorithm. In particular, shape or contour detection by such algorithms can be used. Alternatively, potential particles 202 are recognized using a machine learning algorithm. This machine learning algorithm, for example a shape recognition algorithm, is pre-trained using a base of images to recognize some patterns of interest.

[0131] Once a potential particle has been identified in the first microscope image, the process continues with extracting (33) the coordinates of points in the dark field image that may correspond to the particle 202. This extraction is performed by image processing unit 60, for example using image processing or machine learning algorithms.

[0132] Thus, for this first dark field microscope image, which corresponds to one of the tiles 40 of the mosaic 4, a list of points formed by coordinate points 42 is obtained. These coordinate points 42 are defined in the coordinate system of the displacement stage 11.

[0133] For each particle 202 located within the image field, a coordinate point 42 is defined, for example located at the geometric center of each particle 202. In other words, each particle 202 appearing on the dark-field image tile 40 is associated with a single coordinate point 42.

[0134] The geometric center of each of the particles 202 is located inside each of the particles 202 and is positioned as far away from the edge of the particle 202 as possible.

[0135] Alternatively, multiple coordinate points 42 can be extracted from the same microparticle 202 appearing in the first dark-field microscope image. This multiple points takes the form of a matrix of points defined on each of the microparticles 202. The spacing between the extracted coordinate points 42 on the same microparticle 202 is determined as a function of the displacement accuracy of the displacement stage 11 and the spatial resolution of the Raman microspectroscopy measurement modality. This results in a fine mesh for each microparticle 202. Using this thin mesh, a spectrum, here a Raman spectrum, can be measured for each of the points in the matrix, thereby realizing a spectral map for accessing the chemical distribution within the microparticle 202. Alternatively, all spectra acquired within a given microparticle 202 can be averaged to obtain an average spectrum representing this microparticle 202.

[0136] Optionally, a pre-filtering of the particles 202 from which the coordinate points 42 are extracted as described above can be performed. For example, filtering criteria can be established based on criteria linked to morphological parameters based on the first dark-field microscope image, such as size or shape criteria.

[0137] A variation is now shown in Figure 3 where a single coordinate point 42 is extracted for each particle 202 that appears in a different tile 40. Each coordinate point 42 located on a different tile 40 that forms the mosaic 4 is indicated using a dotted cross.

[0138] Thus, on the first dark field microscope image shown in the upper left corner of Figure 3, three coordinate points 42 are extracted: coordinate point 421, coordinate point 422 and coordinate point 423. These three points now together form the list of points for this tile.

[0139] Moving (34) the sample using the translation stage 11 allows the optical axis of the darkfield microscope objective 14 to coincide with that position. In other words, this movement (34) places the central region 144 of the darkfield microscope objective 14 at the center of a point from the list of points established in the previous step. For example, this is coordinate point 421 shown in Figure 3.

[0140] The Raman microspectroscopy modality is then used to illuminate (35) a measurement point on the sample 200. For this purpose, the excitation laser beam 160 transmitted through the annular mirror 13 is focused by the central region 144 of the dark-field microscope objective 14.

[0141] Given that the measurement point is centered with respect to the optical axis of the darkfield microscope objective 14, the measurement point coincides with a point obtained from the list of coordinate points. In other words, the excitation laser beam 160 converges on the coordinate point 421.

[0142] Afterwards, the white light source 12 is switched off, for example by its power supply, which is connected to the control unit 6 .

[0143] However, it is advantageous to simultaneously maintain the two illumination modalities, especially when the displacement stage 11 is moved. In fact, the simultaneous use of illumination by the annular illumination cone 124 and illumination of the measurement point by the focused laser beam makes it possible, among other things, to simultaneously observe the illuminated area and the measurement point on the sample 200 using the image sensor 15 of the dark-field microscopy modality. A small portion of the excitation laser beam 160 passes through the second dichroic filter 18. This very small portion corresponds, for example, to less than 0.001% of the optical power of the excitation laser beam 160. Nevertheless, this portion is sufficient to be observable on the image sensor 15. Therefore, it is possible to observe the relative position of the measurement point generated by the excitation laser beam 160 with respect to the dark-field illuminated area on the sample 200. The measurement point appears as a small spot of light on the image field.

[0144] Therefore, if the discontinuous wavelength ranges of the white light beam 120 and the excitation laser beam 160 are selected with the optical spectrum of the white light beam 120 strictly below the wavelength of the excitation laser beam 160, the two illuminations can be maintained simultaneously without risk of interference.

[0145] Next, a step 36 is performed of collecting the Raman spectrum emitted from the measurement point. This spectrum is collected by the central region 144 of the darkfield microscope objective 14 and sent along the optical path detailed above to the spectrometer 19 for analysis. With respect to the illumination of the sample 200 by the excitation laser beam 160, the collection of the Raman spectrum does not require mechanical switching, which makes it possible to avoid misalignment of optical elements along this optical path.

[0146] To collect Raman spectra, other coordinate points 422, 423... extracted from the same dark-field microscope image are also probed. To do so, the displacement stage 11 manipulates the movement (34) of the sample 200 in order to successively coincide the measurement point with each of the other coordinate points 422, 423... from the list of points of the tile being considered. Thus, within the framework of the illustrated example, the displacement stage 11 moves the sample 200 to scan the coordinate point 422 and then the coordinate point 423. The scanning pattern within the tile 40 is indicated by arrows.

[0147] A Raman spectrum is collected at each of the points from the list of points by moving (34) the displacement stage 11 and subsequently collecting (36) the spectra.

[0148] These two acquisition steps 36 and translation steps 34 are applied recursively to all coordinate points 42 from the list of points extracted from the first dark-field microscope image, i.e., extracted from the first tile 40.

[0149] After all coordinate points 42 from the list of points of the first tile 40 have been scanned, a new darkfield microscope image is recorded as part of this process by moving (37) the sample so that one of its predetermined geometric centers of coordinate pairs is aligned with the optical axis of the darkfield microscope objective.

[0150] The list of points is then reset.

[0151] This new dark-field microscope image corresponds to the next tile 40 of the mosaic 4 defined according to the selected scanning pattern, which here is, for example, the tile to the right of the first tile 40 mentioned above, i.e., the tile in row 1, column 2 in FIG.

[0152] The above described series of steps is repeated for this next tile 40 and for all tiles 40 contained in the mosaic 4 until a spectral image representing the complete sample 200 is obtained, as shown in FIG.

[0153] In this spectral image, thanks to the dark-field microscopy modality, visible structures corresponding here to the microparticles 202 are imaged, while colors, represented here by texture, are associated with each of the microparticles 202. These colors indicate the chemical nature, i.e., composition, of the microparticles 202, which in the example shown in FIG. 6 correspond, but are not limited to, to mineral materials, more specifically to chalk microparticles 202. These colors further correspond to pseudocolors arbitrarily selected as a function of the microparticle's chemical composition. The composition of each microparticle 202 is determined by analysis of spot Raman spectra collected by the Raman microspectroscopy modality, in particular based on the Raman lines present in the collected spectra.

[0154] In practice, the Raman spectrum of each particle 202 is identified and assigned to a class by a dedicated algorithm. For example and without limitation, correlation between the spectrum acquired on the particle 202 and a pre-established base of spectral data stored in memory can identify, for example, the chemical composition of the particle 202. The particle 202 is identified as corresponding to the known chemical species whose spectrum has the closest correlation.

[0155] Multivariate analysis can also be performed, for example by multivariate curve resolution (MCR), which allows the particles 202 to be distributed into different classes, which can then be identified manually or automatically.

[0156] According to another alternative, the identification of the chemical composition of the particulates 202 is performed by machine learning: a model is pre-trained based on a large amount of labeled data, allowing it to identify the spectrum of the particulates 202 to be characterized.

[0157] In this manner, for each tile 40 corresponding to a acquired dark-field microscope image, spectral information is extracted that relates to the imaged particulates 202 within the tile 40. Such spectral information allows the chemical nature of these particulates 202 to be tracked.

[0158] The dynamic acquisition method described in the first embodiment provides the following advantages in particular.

[0159] First, the morphological parameters of the microparticles 202 are determined in situ during the tile-by-tile reconstruction of the mosaic 4. It is therefore possible to fully utilize the spatial resolution of the dark-field microscopy modality during the determination of the morphological parameters, which has the effect of improving the accuracy of this determination. In fact, in the static mode mentioned above, the resolution of the dark-field microscopy image needs to be compressed during acquisition in order to limit the memory space required to store the dark-field image.

[0160] Furthermore, spectral analysis is also performed in situ during the tile-by-tile reconstruction of the mosaic 4. Therefore, the movements performed by the displacement stage 11 to position each of the microparticles are small, on the order of a few micrometers. The precision of the displacement stage 11 is then usually given in micrometers per millimeter, typically in the order of 3 micrometers per millimeter. Therefore, movements on the order of a few micrometers are more accurate than movements on a few millimeters. Given that the system described herein is applied to research objects with micrometer dimensions, a similar precision, i.e., on the order of micrometers, becomes important. Therefore, when measurements are made using the Raman microspectroscopy modality, the method offers a gain in terms of spatial resolution. Within the framework of the static method described above, the sample 200 is imaged over its entire surface, typically an area of ​​1 centimeter by 1 centimeter, before returning to scan points of interest on the microparticles 202 using the Raman microspectroscopy modality. This potentially introduces positioning errors of a few micrometers.

[0161] Furthermore, the dynamic approach described in the first embodiment also makes it possible to limit the inconveniences caused by fluctuations in environmental conditions encountered in static mode. Indeed, acquiring a full mosaic 4 using dark-field microscopy modality takes tens of minutes, or even an hour. Therefore, environmental conditions such as temperature, humidity, and vibrations can change while switching between dark-field microscopy and Raman microspectroscopy, potentially causing measurement inaccuracies. The dynamic mode described here by two implementations allows for a short connection between the two modalities, or even for the two modalities to be implemented simultaneously. This makes the system insensitive to environmental fluctuations that occur over long timescales.

[0162] In addition to these advantages over static approaches, the present invention avoids idle times corresponding to mechanical switching times for switching from one modality to another within the tile 40. In fact, switching from the dark field microscopy and Raman microspectroscopy modalities is done without switching mechanical elements thanks to the system described above. Therefore, mechanical switching times are eliminated, which represents a significant time saving.

[0163] In fact, high-precision mechanical switching systems are used in microscopy applications. Typically, such mechanical switching systems take several seconds, e.g., 6 seconds, to switch from dark-field microscopy to Raman microspectroscopy and several seconds, e.g., 6 seconds, to switch in the opposite direction, which is time wasted for each tile 40 that is imaged. Here, 12 seconds are lost for each tile 40 that is imaged. At the scale of a mosaic 4 representing a large sample 200, i.e., on the order of several centimeters by several centimeters, including hundreds or even millions of tiles 40, this represents a significant amount of measurement time that is lost in this manner.

[0164] Even more powerful, and therefore more expensive, mechanical switching systems still have mechanical switching times of hundreds of microseconds. In reality, such mechanical switching systems still have mechanical switching times on the order of milliseconds. On the scale of large sample sizes 200, these switching times are not negligible.

[0165] Therefore, the proposed method implemented by the described system 1 for spatial localization and spectral analysis of microparticles within an image provides time savings in the analysis of samples containing large, low-contrast microparticles 202, i.e., tens of millimeters on a side, or even several centimeters.

[0166] "Tom Thumb" method A second embodiment of the disclosed method is illustrated by block diagram 5 in FIG.

[0167] This block diagram 5 includes the following steps as an initial setting: - Illuminating an area on the sample 200 using a dark field microscopy modality (50) -Record dark field microscope images (51) - Identifying the location of the particle 202 in the image (52) - Extract the coordinates of the points corresponding to those particles 202 to form a list (53) - moving the sample 200 so that the central area 144 of the dark field microscope objective 14 corresponds to a point in the list of extracted coordinates (54); - Irradiating the sample 200 using the Raman microspectroscopy modality (55), the excitation laser beam 160 is focused to one measurement point that coincides with the point of the previous step. The central region 144 of the dark-field microscope objective 14 collects the Raman spectrum emitted from the measurement point (56).

[0168] Once initialized, the method according to the second embodiment continues by moving 54 the sample to align the optical axis of the darkfield microscope objective 14 with another point in the list of extracted coordinates. An illumination step 50, a recording step 51, and a localization step 52 using the darkfield microscopy modality are then performed in parallel with, before, or after an illumination step 55 and a collection step 56 using the Raman microspectroscopy modality.

[0169] Thus, as long as the list of points contains coordinate points 42 that have not yet been scanned and measured, for example by Raman microspectroscopy, the method loops to transfer step 54 .

[0170] In this second embodiment, as described for the first embodiment, a mosaic 4 representing the sample 200 is reconstructed using multiple dark-field microscope images. This mosaic 4 therefore represents a field of view larger than that of the dark-field microscope images. This mosaic 4 is made up of tiles 40, i.e., dark-field microscope images arranged to represent the sample 200 being analyzed. In the second embodiment, some of these tiles 40 at least partially overlap. In other words, some of the tiles 40 of the mosaic 4 partially cover the same spatial area.

[0171] As in the first embodiment, each tile 40 has a rectangular shape of 140 micrometers by 105 micrometers, according to the characteristics of the image sensor 15 and the dark field microscope objective lens 14 used to collect the dark field microscope image.

[0172] A portion of the mosaic 4 obtained according to the second method is reproduced in Figure 5. The rectangle indicates the boundary of the portion of the mosaic 4 in Figure 3, and this rectangle is also reproduced in Figure 6.

[0173] In this embodiment, the geometric centers 41 of the tiles 40 are not predetermined. Similarly, the placement of the tiles 40 relative to one another is not determined upstream in the process. In fact, in this second embodiment, coordinate pairs for defining the location of each tile 40 are determined during the process based on information gathered during implementation of the process.

[0174] More specifically, a geometric center 41 is selected based on coordinate points 42 extracted during this process, which triggers the acquisition of a new dark-field microscope image and the extraction of new coordinate points 42. Those new coordinate points 42 may themselves potentially become the geometric center 41 of the tile 40, and so on.

[0175] Thus, unlike the first embodiment, moving from one tile 40 to another tile 40 does not necessarily involve a dedicated move, but rather the tiling of the mosaic 4 occurs as moves (54) are made. These moves (54) aim to scan different coordinate points 42 from a list of points, which form the geometric center 41 of the tile 40.

[0176] The steps described above for the second embodiment of the method will now be explained in detail by way of example in FIG.

[0177] To start the process, the displacement stage 11 positions the sample 200 at an arbitrary position. For example, the geometric center 41 of the first tile 40 shown in Figure 5 is chosen here as the midpoint of the displacement amplitude of the displacement plate 11 along the x and y axes. This position corresponds to the middle of the displacement stage 11.

[0178] The next step of the method consists of illuminating 50 an area of ​​the sample 200 according to the dark-field microscopy modality. As described above, illuminating 50 an area of ​​the sample 200 centered on the geometric center 41 of the first tile 40 is performed by an annular illumination cone 124 that is focused onto the sample 200 by a peripheral region 142 of the dark-field microscope objective 14.

[0179] In FIG. 5, the geometric center 41 of each tile 40 is indicated by a solid target, ie, a cross within a circle.

[0180] Illumination (50) of this area with a dark-field microscopy modality leads to the recording (51) of a first dark-field microscopy image, whose field of view is again contained within the illuminated area of ​​sample 200. This first dark-field microscopy image is obtained by the central region 144 of dark-field microscope objective 14 collecting the polarized light rays.

[0181] Based on this first dark field microscope image, a localization (52) of potential particles 202 in the first image is performed, which localization (52) shall be identical to that described for the first embodiment.

[0182] After locating potential particles 202 contained within the image field, for example by dedicated image processing algorithms, extraction (53) of the coordinates of points corresponding to the potential particles 202 is performed. This extraction (53) is performed by the processing unit and / or by the user using suitable algorithms, as described herein above. As with the first embodiment, a single coordinate point 42 is associated with each particle 202 seen in the dark field, although alternatives are possible.

[0183] These extracted coordinate points 42 are added to a list of points.

[0184] In the first tile 40, two coordinate points 42 are extracted: coordinate point 421 and coordinate point 422. In Figure 5, these coordinate points 42 are indicated using dotted targets.

[0185] The movement (54) of the sample 200 by the displacement stage 11 causes the optical axis of the darkfield microscope objective 14 to coincide with a coordinate point from the list of points. In particular, a point that has not yet been scanned by the Raman microspectroscopy modality is selected, such as here coordinate point 421 corresponding to coordinate point 421 associated with the first microparticle 202 identified and located within tile 40.

[0186] The next step involves using the Raman microspectroscopy modality to illuminate (55) a measurement point on the sample 200. The laser beam is focused onto the sample 200 at the measurement point by the central region 144 of the darkfield microscope objective 14. The measurement point is here in the extension of the optical axis of the darkfield microscope 14.

[0187] The movement (54) therefore makes the illuminated measurement point coincident with the coordinate point 421 taken from the list of points.

[0188] After illumination (55) and movement (54), collection (56) of the Raman spectrum emitted from the measurement point is performed, which Raman spectrum is collected by the central region 144 of the dark-field microscope objective 14.

[0189] Advantageously, given that a non-mechanical switch is required to switch from one modality to another, it is possible to proceed to a new record (50) of a new tile 40, whose geometric center 41 overlaps one of the coordinate points 42 coming from the list of points L.

[0190] 5, the geometric center 41 of the new tile 40 overlaps with the coordinate point 421. This point is indicated by a solid target, while the geometric center 41 of the first tile 40 is indicated by a dotted cross.

[0191] In a first alternative to this second embodiment, the two illuminations are used simultaneously to obtain a new tile 40 if the spectral ranges of the laser source 16 and the white light source 12 are discontinuous and if components of the white light spectrum have wavelengths less than the wavelength of the excitation laser beam 160. Such a case is assumed here.

[0192] Illumination (55) of the measurement point with the excitation laser beam 160, followed by collection (36) of the emitted Raman spectrum and recording (51) of a new dark-field microscope image whose field of view is included within the illuminated area of ​​the sample 200, are both performed as described above.

[0193] Following this recording (51), the position of the particle 202 is identified (52) and the coordinates of the point corresponding to the particle 202 are extracted (53). In this case, no point corresponding to the particle 202 is detected.

[0194] According to a second alternative, the transfer (54), illumination (50), recording (51), localization (52), and extraction (53) are first performed according to the dark-field imaging modality, followed by illumination (55) and collection (56) according to the Raman spectroscopy modality.

[0195] Regardless of the order in which the two modalities are used, the method continues with a movement of the sample (54) in order to match the measurement point with another point from the list of points L.

[0196] 5, this point becomes coordinate point 422. This coordinate point 422 becomes the geometric center 41 of the new tile 40. The tile being imaged here is the third tile 40.

[0197] The steps of the method are then repeated by potential illumination (50), recording (51) a new dark-field microscope image forming the third tile 40, locating (52) and extracting (53) the coordinates of the point corresponding to the potential particle 202, these steps being carried out in parallel here with a step 55 of illuminating the measurement point with an excitation laser beam and a step 56 of collecting a Raman spectrum, where in particular coordinate points 423 and 424 are detected and extracted from the third tile 40. These coordinate points 423 and 424 are added to a list of points to be scanned.

[0198] A new movement (54) of the stage then places the measurement point at one of the points from the list of points, and the described steps of the method are then repeated.

[0199] Thus, the mosaic 4, which represents both the structure and chemistry of the sample 200, is reconstructed on the fly. In other words, the list of points L is continuously updated during the implementation of the method and is not reset between each tile 40.

[0200] A fourth tile 40, corresponding to a fourth dark-field microscope image, is acquired with coordinate point 423 as its geometric center 41. Coordinate points 425 and 426 are extracted from this fourth image, and the displacement stage 11 moves the sample 200 to probe coordinate point 424 using the Raman microspectroscopy modality. Coordinate point 424 becomes the geometric center 41 of a fifth tile 40, and the steps of the method are repeated for this tile 40. Next, coordinate point 425 from the list of points L is scanned to collect not only a Raman spectrum, but also a sixth dark-field microscope image corresponding to the sixth tile 40. Coordinate point 427 is then extracted from this sixth image.

[0201] This method results in a spectral image corresponding to a mosaic 4 representing the entire surface of the sample 200. This spectral image is shown in Figure 6, i.e. it corresponds to the same spectral image as that obtained in the first embodiment described above.

[0202] The structure of the microparticles 202 is obtained using dark-field microscopy modalities that enhance contrast even on these highly transparent samples 200, while false colors are related to composition as determined by analysis of the Raman spectra.

[0203] Within the framework of this second embodiment, the simultaneity of the two illuminations is exploited to use the movements necessary to probe points of interest by the Raman microspectroscopy modality and also to image the sample 200 by dark-field microscopy.

[0204] For example, the order in which coordinate points 42 from the list of points L are scanned is selected to minimize the distance the stage travels during translation. For example, it may be advantageous to minimize the Euclidean distance between two consecutively scanned coordinate points 42. It may also be decided to minimize the total translation distance required to scan all coordinate points 42.

[0205] Therefore, the scan order is rearranged during the process, especially when new coordinate points 42 are extracted.

[0206] If coordinate point 42 is not in the list L of points that have not yet been scanned, an appropriate protocol may be implemented, for example, it may be decided to move sample 200 an amount sufficient to probe a new area of ​​sample 200.

[0207] This second embodiment has the same advantages as the first embodiment described, in particular by eliminating prohibitively long mechanical switching times when switching from the dark-field microscopy modality to the Raman microspectroscopy modality.

[0208] Alternative Embodiments The invention is in no way limited to the embodiments described and shown, but a person skilled in the art will know how to apply any of the modifications according to the invention. [Explanation of symbols]

[0209] 1 System 3 Block diagram 4. Mosaic 5 Block diagram 6. Control Unit 10 spindle 11 Displacement stage 12 White light source 13 Annular Mirror 14 Darkfield microscope objectives 15 Image Sensor 16 Laser Source 17 First dichroic filter 18 Second Dichroic Filter 19 Spectrometer 20 Sample holder 40 tiles 41 Geometric Center 42 coordinate points 60 Image Processing Unit 120 white light beams 122 White Light Beam 124 Circular Lighting Cone 142 Surrounding Area 144 Central area 150 Photosensitive matrix sensor 160 Excitation laser beam 162 Raman beam 190 Photosensitive matrix sensor 200 samples 202 Fine particles 421 coordinate points 422 coordinate points 423 coordinate points 424 coordinate points 425 coordinate points 426 coordinate points 427 coordinate points

Claims

1. 1. A method for spatial localization within an image and spectral analysis of a particulate (202) within a sample (200) by an optical microscope system, comprising: A) illuminating (30, 50) an area of ​​the sample (200) using an annular illumination cone (124), the annular illumination cone (124) being obtained from a white light beam (120) partially reflected by an annular mirror (13) and focused by a peripheral region (142) of a dark-field microscope objective (14); B) recording (31, 51) a first dark-field microscope image of the sample (200) on a field of view included in the illuminated area, the first dark-field image being collected by a central area (144) of the dark-field microscope objective (14); C) locating (32, 52) the particle (202) within the first dark-field image; D) extracting (33, 53) the coordinates of the points corresponding to the particles (202) in the recorded dark field image to form a list of points; E) moving (34, 54) the sample (200) so that the optical axis of the dark field microscope objective (14) coincides with a point on the list of points; F) illuminating (35, 55) a measurement point on the sample (200) using an excitation laser beam (160) transmitted through the annular mirror (13) and focused by the central region (144) of the dark-field microscope objective (14), the measurement point corresponding to the point from the list of points; G) collecting (36, 56) a Raman spectrum emitted from the measurement point, the Raman spectrum being collected by the central region (144) of the dark-field microscope objective (14); A method comprising:

2. 2. The method of claim 1, wherein steps A) and F) are performed simultaneously, and wherein the peripheral region (142) of the dark-field microscope objective (14) illuminates an area on the sample (200), while the central region (144) of the dark-field microscope objective (14) illuminates a measurement point within the area.

3. 3. The method according to claim 1 or 2, wherein a field of view larger than the image field is reconstructed by a mosaic (4) of dark-field microscope images, these dark-field microscope images corresponding to tiles (40) of the mosaic (4).

4. 4. The method of claim 3, wherein the placement of each tile (40) of the mosaic (4) is predetermined upstream in the process.

5. 5. The method of claim 4, wherein steps E), F) and G) are repeated until all points of the extracted point list have been scanned on a given tile (40), and then proceeding to a step (36) of moving the sample (200) to focus the field of view of the dark-field microscope objective (14) on another tile (40) of the mosaic (4) and to resume the process from step A) to record another dark-field microscope image.

6. 6. The method of claim 5, wherein for each tile (40) of the mosaic (4), the list of points comprises a determined number of points.

7. 4. The method of claim 3, further comprising establishing a positioning of at least one tile (40) of said mosaic (4) as a function of said coordinates of a point from said list of points.

8. 8. The method of claim 7, wherein after the movement (54) of step E) to align the optical axis of the dark-field microscope objective (14) with a point on the list of points, steps A), B), C), D), F) and G) are performed and a new dark-field microscope image corresponding to a new tile (40) of the mosaic (4) and a Raman spectrum emitted from the measurement point are recorded, which steps are followed by another movement (54) of the sample (200) to align the optical axis of the dark-field microscope objective (14) with another point from the list of points.

9. 9. The method of claim 8, wherein additional points extracted from the new dark-field microscope image recorded after the movement (54) of step E) are added to the list of points to be scanned.

10. 1. A system (1) for spatial localization and spectral analysis of microparticles in images, comprising an optical microscope, a laser source (16), a white light source (12), an image sensor (15), a spectrometer (19) and a control unit (6), the latter including an image processing unit (60), wherein the optical microscope comprises a sample holder (20) mounted on a displacement stage (11), the sample holder (20) being adapted to receive a sample (200); said system being: - an optical system including an annular mirror (13) and a dark-field microscope objective (14), said optical system being arranged between said laser source (16), said white light source (12) and said sample holder (20), said dark-field microscope objective (14) having a central region (144) and a peripheral region (142); the annular mirror (13) is configured to reflect a portion of the white light beam (120) emitted by the white light source towards the peripheral region (142) of the dark-field microscope objective (14) and to transmit an excitation laser beam (160) emitted by the laser source (16) towards the central region (144) of the dark-field microscope objective (14), the dark field microscope objective (14) is adapted to transmit the part of the white light beam towards the sample holder (20) using its peripheral region (142) and to transmit the excitation laser beam (160) towards the sample holder (20) via its central region (144); the dark field microscope objective (14) is adapted to focus the portion of the light beam to illuminate a first area of ​​the sample (200) on the sample holder (20) using an annular illumination cone (124), and is adapted to focus the excitation laser beam (160) at a measurement point contained within the first area; the central area (144) of the dark field microscope objective (14) is adapted to collect a dark field microscope image on an image field included in the first illumination area, the dark field microscope image being recorded using the image sensor (15); - said image processing unit (60) is adapted to identify the locations of potential particles (202) and to extract the coordinates of the points associated with these particles (202) from said dark field microscope image in order to establish a list of points; the displacement stage (11) is configured to move the sample holder (20) in order to match the measurement point with a point from the list of points or to position the image field of the dark field microscope objective (14) in another area that is at least partially different from the first area, - the central area (144) of the dark field microscope objective (14) is also adapted to collect the Raman spectrum emanating from the measurement point, said Raman spectrum being recorded by a Raman spectrometer (19); System (1).

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