Method for detecting and spectrally imaging microparticles, and associated system

Abstract

The invention relates to a method for spatial tracking and spectral imaging of microparticles (202) in a sample (200).This method comprises the following steps: A) illumination of the sample by an annular illumination cone (124) from a light beam partially reflected by an annular mirror (13) and focused by a peripheral region (142) of a dark-field microscope objective (14), B) recording of a dark-field microscopy image, this being collected by a central region (144) of the objective, C) identification of the points of interest in the image, D) extraction of the coordinates of the points of interest, in order to form a list of points, E) movement of the sample, so as to scan one of the extracted points, F) illumination of the point on the sample using a laser beam transmitted through the annular mirror and focused by the central region of the dark-field microscope objective, G) collection by the central region of the objective of an emitted Raman spectrum. The invention also relates to an associated system. Figure for the abstract: Fig. 1.

Description

Title of the invention: Method for detecting and spectrally imaging microparticles, and associated system technical field

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

[0002] It relates more particularly to a method for detecting and spectrally imaging transparent microparticles, as well as an associated system.

[0003] The invention finds a particularly advantageous application in the tracking and rapid spectral imaging of transparent microparticles scattered within a sample of macroscopic dimensions. State of the art

[0004] The detection and identification of the chemical nature of micrometric solid particles, commonly called microparticles, is a subject of interest for many industries and / or applications. For example, the study of plastic microparticles, which notably involves their detection, quantification, and identification, is essential in fields such as environmental science, the food industry, etc. In such studies, a macroscopic sample, extending over a few square millimeters or even a few square centimeters, is taken from a medium to provide a representative overview of that medium. Such a sample may, for example, be in liquid or gaseous form and contain suspended solid microparticles, or it may be in the form of a powder of solid microparticles.

[0005] Measurement systems have been developed to meet such an analytical need. Spectral imaging systems have been proposed, combining a full-field microscopy imaging modality, used for the spatial localization of microparticles, with a spectral analysis modality, most often by point analysis using a laser source to record a spectrum of each microparticle, this point analysis making it possible to determine the chemical composition of the analyzed microparticles.

[0006] However, the image field of view of such spectral imaging systems remains limited to rectangular areas of a few millimeters by a few millimeters on a side, or even less, when high-magnification microscope objectives are used, in order to spatially resolve the micrometer-sized particles under study. Thus, scanning the entire surface of the sample to locate the microparticles requires mosaicking the sample, which is then moved using a stage, each tessera or tile of the mosaic being the size of the image field of view of the imaging system, the dimensions of which depend in particular on the microscope objective and / or the image sensor.

[0007] Following a process commonly described as static, all the tiles of a mosaic representative of a sample are acquired by full-field microscopy, before proceeding, in a second step, to point analysis by laser spectroscopy. Thus, the sample is imaged in its entirety first, before proceeding to its spectral analysis.

[0008] Typically, a macroscopic sample requires the acquisition and processing of a mosaic containing several million tiles. Such an image size necessitates image quality compression of the mosaic to allow its storage for the extraction of the coordinates of the different microparticles. This extraction is then followed by point analysis through spectral acquisition on the extracted coordinate points. However, this image quality compression comes at the expense of spatial resolution.

[0009] A dynamic approach has been proposed to address the deterioration of spatial resolution. This dynamic approach involves extracting the coordinates of the microparticles tile by tile and acquiring spectra on the fly for each tile before moving on to the next. This dynamic approach offers several advantages, including a reduction in image size, since only the coordinates of interest are retained, rather than an entire tile. However, this approach significantly increases acquisition times when switching from imaging to spectral analysis for each tile. Presentation of the invention

[0010] In order to remedy the aforementioned drawbacks of the prior art, the present invention proposes to adapt the dynamic approach to allow both the detection and spectral analysis of microparticles without switching a motorized mechanical part, thus reducing acquisition times.

[0011] More particularly, the invention proposes a method for spatial localization in an image and spectral analysis of microparticles in a sample by a microscopy system, the method comprising the following steps:

[0012] A) illumination of an area of ​​the sample using an annular illumination cone, the annular illumination cone being produced by a beam of white light partly reflected by an annular mirror and focused by a peripheral region of a dark-field microscope objective,

[0013] B) recording a first dark-field microscopy image on a field of view included in the illuminated area of ​​the sample, the first image in dark field being collected by a central region of the dark field microscope objective,

[0014] C) identification of microparticles in the first dark-field image,

[0015] D) extraction of the coordinates of the points corresponding to microparticles within from the recorded dark field image, in order to form a list of points,

[0016] E) displacement of the sample so as to make an optical axis of the dark-field microscope objective coincide with a point in the list of points,

[0017] F) illumination of a measurement point on the sample using an excitation laser beam transmitted through the annular mirror and focused by the central region of the dark-field microscope objective, the measurement point coinciding with the point in the point list,

[0018] G) collection of a Raman spectrum emitted from the measurement point, the Raman spectrum being collected by the central region of the dark-field microscope objective.

[0019] Thus, thanks to the invention, no mechanical switching is necessary to move from one microscopy mode to another within a dynamic approach. This property is advantageous for samples that are large relative to the field of view of a microscope, within the framework of a dynamic approach, as defined in the introduction. This dynamic approach was introduced in particular to avoid resorting to spatial resolution compression, which is necessary to process a large volume of data given the size of the samples studied. However, the need to switch a motorized mechanical part within each tile, in order to acquire a wide-field image and then to acquire point spectra within the image, resulted in unavoidable downtime of approximately 5 seconds per tile.When scaled down to millimeter or even centimeter samples, these dead times amount to hours. Thanks to this invention, the advantages of the dynamic approach—namely, precise positioning at the sample scale, high spatial resolution, and measurement minimally influenced by environmental variations—are preserved, while eliminating prohibitive mechanical switching times. This elimination of mechanical switching times thus removes a significant obstacle to the use of the dynamic approach on samples, particularly large samples.

[0020] Furthermore, the invention improves the mechanical reliability of the system. Indeed, moving parts are potential sources of misalignment, or even obsolescence of the system.

[0021] The proposed solution not only eliminates these dead times, resulting in significant time savings and increased system reliability, but also advantageously combines properties of the two chosen modalities, the mi Dark-field microscopy and Raman microspectrometry, also called Raman microspec-troscopy, are two key technologies. On the one hand, this results in a solution without mechanical switching and without signal loss, except for optical losses, as the entire Raman signal collected by epi-detection reaches the spectrometer. On the other hand, the study of large samples containing microparticles that absorb little light is facilitated.

[0022] Other advantageous and non-limiting features of the process according to the invention, taken individually or in all technically possible combinations, are as follows:

[0023] - steps A) and F) are executed simultaneously, an area is illuminated on the sample is illuminated by the peripheral region of the dark-field microscope objective, while a measurement point within the area is illuminated by the central region of the dark-field microscope objective,

[0024] - the measurement point is located along an optical axis of the central region of the dark-field microscope objective,

[0025] - a field with dimensions larger than the image field of view is reconstructed by a mosaic of dark-field microscopy images, these dark-field microscopy images corresponding to tiles of the mosaic,

[0026] - a positioning of each tile of the mosaic is predefined upstream of the process,

[0027] - steps E), F) and G) are repeated until all points in the list have been covered of points extracted on a given tile, before proceeding to a sample relocation step to center the field of view of the dark-field microscope objective on another tile of the mosaic and repeating the process from step A), in order to record another dark-field microscopy image,

[0028] - for each tile of the mosaic, the list of points includes a fixed number of points,

[0029] - a positioning of at least one tile of the mosaic is established according to the coordinates of a point in the list of points,

[0030] - after the displacement of step E) to make the optical axis of the lens coincide dark-field microscope with a point from the point list, steps A), B), C), D), F), and G) are executed, a new dark-field microscopy 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 make the optical axis of the dark-field microscope objective coincide with another point from the point list,

[0031] - additional points extracted from the new field microscopy image dark recorded after the movement of step F), are added to the list of points at sweep.

[0032] The invention also relates to a system for spatial localization in an image and spectral analysis of microparticles, 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, the optical microscope comprising a sample holder mounted on a moving stage, the sample holder being adapted to receive a sample, the system being characterized in that it comprises an optical system comprising 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 part of a white light beam emitted by the white light source towards the peripheral region of the dark-field microscope objective, and to transmit an excitation laser beam emitted by the laser source towards the central region of the dark-field microscope objective. - The dark-field microscope objective is adapted to transmit the white light beam portion via its peripheral region towards the sample holder and to transmit the excitation laser beam via its central region towards the sample holder. - The dark-field microscope objective is adapted to focus the portion of the light beam to illuminate a first area of ​​a sample on the sample holder using an annular illumination cone, and the dark-field microscope objective is adapted to focus the excitation laser beam on a measurement point within the first area. - the central region of the dark-field microscope objective is adapted to collect a dark-field microscopy image over an image field of view included in the first illuminated area, the dark-field microscopy image being recorded using the image sensor, - The image processing unit is adapted to detect any microparticles and to extract from the dark-field microscopy image the coordinates of the points associated with these microparticles, in order to establish a list of points, - the displacement stage is configured to move the sample holder, in order to make the measurement point coincide with a point in the point list or in order to place the image field of view of the dark-field microscope objective on another area at least partially different from the first area, - the central region of the dark-field microscope objective is also suitable for collecting a Raman spectrum generated from the measurement point, the Raman spectrum being recorded by a Raman spectrometer. Detailed description of the invention

[0033] In addition, various other features of the invention become apparent from the attached description made with reference to the drawings which illustrate non-limiting embodiments of the invention and where:

[0034] [Fig. 1] schematically illustrates an optical microscopy system adapted to implement a method of spatial localization and spectral analysis of microparticles in a sample,

[0035] [Fig.2] is a flowchart representing a first embodiment of the process of the invention,

[0036] [Fig.3] represents an example of a mosaic comprising a plurality of tiles, each tile corresponding to an image recorded according to the process described in connection with [Fig.2],

[0037] [Fig.4] is a flowchart representing a second embodiment of the process of the invention,

[0038] [Fig.5] illustrates the process described in connection with [Fig.4], and notably represents an example of recording several successive tiles in order to form a mosaic according to the second embodiment,

[0039] [Fig.6] represents an image of the sample from [Fig.3] or 5, within which a position of the microparticles is identified for some of the microparticles included in mosaic 4 of figures 3 or 5.

[0040] It should be noted that in these figures the structural and / or functional elements common to the different variants may have the same references.

[0041] Various other modifications may be made to the invention within the scope of the annexed claims.

[0042] Figure 1 shows a spatial positioning system in an image and spectral analysis of microparticles 1, based on an optical microscope, an image sensor 19, an image processing system 60, and a Raman spectrometer 19. Such a system is referred to herein as an optical microscopy system and relies on light-matter interactions to image and / or analyze a sample 200 exhibiting structures of micrometer dimensions. These structures are notably in the form of microparticles 202, that is, a solid particle whose size is, for example, between a few tenths of a micrometer and a few hundred micrometers.

[0043] Here, microparticles 202 of plastic material are considered, i.e., microparticles 202 of polymer, for example, consisting of long carbon chains and derived from fossil fuels. Nevertheless, the term microparticle refers also, and without limitation, to micrometric mineral grains, or to pharmaceutical powder grains, or solid particles suspended in the air.

[0044] These microparticles 202 are contained within a sample 200, which itself has dimensions ranging from a few hundred micrometers to a few tens of centimeters. The sample 200 is, for example, macroscopic in size and comprises microparticles 202 suspended in water or air, or may be in the form of a powder. Such a sample 200 is, for example, taken from an environment for the purposes of analysis and / or investigations in various fields such as the agri-food industry, the pharmaceutical industry, geology, and environmental science, to name just a few. The sample 200 is deposited on a sample holder 20, in particular a glass microscope slide, and optionally protected by a microscope coverslip.

[0045] The spatial tracking system in an image and spectral analysis of microparticles 1, schematically represented in [Fig. 1], is therefore configured to study such samples 200, in particular by allowing imaging of the contained microparticles 202, and thus providing morphological information about these microparticles 202. For example, it is possible to study the area, perimeter, shape, etc., of these microparticles 202 via images recorded using this optical microscopy system 1. Furthermore, it is advantageous to complement this morphological study with a study of the chemical composition of each of these microparticles 202, notably via the recording of spectra, for example, Raman spectra or photoluminescence spectra, which are characteristic of the chemical composition. However, the recording of these characteristic spectra typically uses point measurement methods.

[0046] In the context of studies on large samples 200 containing microparticles 202, it is particularly advantageous to first spatially locate these microparticles 202, notably using a microscopy image, and to establish a characteristic spectrum at points located, most often, by a point measurement at a point of micrometric dimensions. It is then possible to spatially resolve information regarding the chemical composition of the sample 200. By large dimension, it is understood here that the dimensions of the sample are large compared to the dimensions of the microparticles 202; i.e., the sample 200 under consideration is macroscopic in size, extending over a few millimeters in width or diameter, for example.

[0047] This initial identification of points of interest saves time, given the small size of the point on which the measurement can be performed compared to the total dimensions of the sample 200. This is all the more true in the case of a sample 200 containing a low concentration of microparticles 202. Thus, recording a spectrum only on the points of interest rather than a point measurement on each point of the sample 200 advantageously reduces acquisition times, processing times as well as the memory space required to store the measured spectra.

[0048] The system for spatial localization in an image and spectral analysis of microparticles 1 therefore comprises at least two distinct microscopy modalities, each of the modalities relying on a different contrast mechanism, i.e., a different light-matter interaction mechanism and / or an implementation of a different light-matter interaction mechanism. Here, in the present disclosure, a dark-field microscopy modality is advantageously chosen to image sample 200, combined with a Raman microspectroscopy modality, also sometimes referred to as Raman microspectrometry.

[0049] The dark-field microscopy modality is configured to illuminate a surface of the sample 200 and acquire an image of at least a portion of this illuminated surface. Dark-field microscopy imaging, or simply dark-field imaging, is based on the following contrast mechanism: the sample 200, and the structures it comprises, such as microparticles 202, are illuminated in such a way that only light rays deflected by the sample 200 and structures present in this sample 200 can be collected by an optical objective.

[0050] Dark-field microscopy is an imaging modality particularly well-suited to the study of microparticles, especially plastic microparticles. Indeed, dark-field microscopy is known to enhance contrasts on transparent samples, i.e., those that absorb little light. However, some plastics, and in particular microplastics and / or mineral or pharmaceutical powder grains, are known to be transparent and exhibit relatively low contrast when illuminated using other imaging modalities, for example, bright-field illumination.

[0051] The spatial tracking system in an image and spectral analysis of microparticles 1 comprises a second microscopy modality corresponding to a Raman microspectroscopy modality. This Raman microspectroscopy modality probes the chemical composition of the sample 200 at a measurement point, via the recording of a Raman spectrum emitted from this measurement point following light excitation focused on this point. This Raman spectrum is obtained by Raman scattering, which corresponds to an inelastic scattering phenomenon of light, due to an exchange of energy with the probed medium. This energy variation is representative of the molecular vibration modes of the probed medium at the point of measurement. The emitted Raman spectrum therefore contains Raman lines. This Raman spectrum is characteristic of a given chemical species, and its measurement and analysis allow, among other things, information on the chemical composition of a medium of interest.

[0052] Thus, the Raman microspectroscopy modality advantageously allows for point-by-point mapping, that is to say, by scanning point by point of the measurement point, of the chemical composition of a sample 200, here, more particularly, the chemical composition of the microparticles 202 contained within the sample 200. For example, it is possible to be interested in the composition of the microparticles 202 of plastic material, dispersed within an aqueous sample 200.

[0053] The implementation of each of these microscopy modalities is based on distinct illumination means and / or means of collecting a signal generated by the light-matter interaction, whether in the form of an image or a spectrum.

[0054] The present invention proposes a system for spatial localization in an image and spectral analysis of microparticles 1 in which the two microscopy modalities, namely, dark-field microscopy and Raman microspectroscopy, are implemented simultaneously or successively, without requiring mechanical switching between the two distinct illumination and / or detection means to switch from one modality to the other. Indeed, the mechanical switching commonly used in optical microscopy systems to switch from one microscopy modality to another introduces delays during system operation.Although solutions exist to reduce the time required to switch between two microscopy modalities, problems remain related to the significant financial cost of these rapid switching solutions, their potential misalignment, and their limited lifespan, particularly due to the obsolescence of the motors driving such mechanical switches.

[0055] A dynamic analysis method is also described, benefiting from the spatial tracking system in an image and spectral analysis of microparticles 1 as herein disclosed, particularly suitable for imaging and spectral analysis of large-dimensional samples, i.e., macroscopic-sized samples, comprising microparticles 202. Indeed, in order to image such samples 200 in their entirety, the number of mechanical switches required to move from one microscopy modality to another, to locate the microparticles 202 before proceeding with their spectral analysis, is multiplied due to the size of the sample 200. Thanks to the disclosed method, the analysis of this type of sample 200 is accelerated, while maintaining high spatial resolution, and minimizing the memory space required for recording the acquired data.

[0056] The spatial tracking system in an image and spectral analysis of microparticles 1, shown in [Fig. 1], is described first. This system is intended for analyzing a sample 200 as described previously. In one example, the sample 200 is a sample comprising a dispersion of microparticles 202, such as mineral grains or microplastics. However, it could also be a sample comprising a solution or a liquid, for example aqueous, in which microparticles 202 of plastic materials are dispersed. In all cases, this sample 200 is deposited on a sample holder 20. The optical microscopy system includes, for example, a microscope stand on which the various elements described below are arranged and fixed.

[0057] A principal axis 10 of the microscope is defined perpendicular to a plane of the sample 200, which is considered to be flat.

[0058] This sample holder 20 is attached to a displacement plate 11 or positioning plate. This plate is adapted to move the sample holder 20 along at least two axes of translation, an x-axis and a y-axis, the x and y axes being orthogonal and parallel to the principal plane of the sample 200. This displacement plate 11, for example, a piezoelectric plate, is controlled by a controller (not shown in [Fig. 1]) and is also motorized, so as to automate the movements of the sample holder 20. The displacement plate 11 has a displacement range from a few tens of millimeters to a few centimeters, as well as a displacement accuracy on the order of a micrometer. A control unit 6, for example, a computer, controls and synchronizes the movements of the displacement plate 11. Dark-field microscopy modality

[0059] In order to illuminate the sample 200 according to the dark-field microscopy modality, the spatial tracking system in an image and spectral analysis of microparticles 1 includes a light source. For example, this is a light source emitting incoherent light radiation, such as an incandescent lamp, a halogen incandescent lamp, or a lamp comprising at least one light-emitting diode, abbreviated as "LED" in French.

[0060] More specifically, in this first embodiment, it is an incandescent lamp emitting light radiation with a spectrum covering at least part of the visible spectrum. Such light radiation is then perceived as close to white, and is referred to as a white light source 12.

[0061] The light radiation emitted by the white light source 12 propagates here in free space, in the form of a white light beam 120, this beam being collimated. It is assumed that the white light beam 120 propagates along a optical axis perpendicular to the main axis 10 of the microscope.

[0062] The optical microscopy system also includes an annular mirror 13. This annular mirror 13 has a flat reflective surface, for example, a polished metallic deposit on a flat surface. In this case, it is, for example, a silver deposit. This annular mirror 13 has an elliptical shape, which is pierced in its center by a central elliptical aperture. In other words, the annular mirror 13 has the shape of an elliptical ring. The aperture is centered on the surface of the annular mirror 13. Consequently, a central portion of this annular mirror 13 does not reflect light. This annular mirror 13 is, for example, included in a block or cube, typically known by the English term "darkfield cube."

[0063] A position of this annular mirror 13 is maintained fixed relative to the microscope stand.

[0064] This annular mirror 13 is arranged so that it is centered, on the one hand, with respect to the principal axis 10 of the optical microscopy system, and on the other hand, centered with respect to the white light beam 120. Furthermore, the flat reflective surface of the annular mirror 13 forms a 45-degree angle with the principal axis 10 of the microscopy system. The annular mirror 13 is thus adapted to reflect a portion of the white light beam 122 towards the sample 200.

[0065] Indeed, if the white light beam 120 is initially approximated as a solid cylinder, only a portion corresponding to a hollow cylinder is reflected in the direction of the sample 200. This portion corresponds to a part of the white light beam 122 incident on the flat reflective surface of the annular mirror 13.

[0066] A remaining portion is transmitted through the central elliptical opening of the annular mirror 13, for example in the direction of a beam blocker, not shown in [Fig.1].

[0067] The part of the white light beam 122 corresponding to the hollow cylinder propagates along the main axis 10 of the optical microscopy system, in the direction of the sample 200.

[0068] The optical microscopy system also includes a dark-field microscope objective 14, as known in the prior art. Such an objective has two distinct optical regions, a peripheral region 142 and a central region 144. An optical axis of the dark-field microscope objective 14 is aligned with the principal axis 10 of the microscopy system.

[0069] Here, in the described embodiment, the dark-field microscope objective 14 corresponds to a 50x magnification objective with a numerical aperture of 0.60. A field of view (called field of view in English) of the dark-field microscope objective The dark 14 is 0.44 millimeters in diameter. This is more than a lens corrected to infinity.

[0070] The peripheral region 142 of the dark-field microscope objective 14 corresponds to an external region surrounding the central region 144. The central region 144 corresponds to a conventional microscope objective. Thus, the central region 144 is centered around the optical axis of the dark-field microscope objective 14, surrounded by the peripheral region 142.

[0071] The portion of the white light beam 122 corresponding to the hollow cylinder is transmitted through the peripheral region 142 of the dark-field microscope objective 14. The dimensions of the hollow cylinder, as well as the annular mirror 13, are chosen according to the dimensions of the peripheral region 142. In particular, it is ensured that the entire portion of the white light beam 122 reflected by the annular mirror 13 is transmitted through the peripheral region 142 of the dark-field microscope objective 14.

[0072] This peripheral region 142 opens onto a ring optical element, which focuses the hollow cylinder into an annular illumination cone 124. This optical element corresponds, for example, to a ring optical lens or a concave mirror. It is with this annular illumination cone 124, corresponding to a hollow cone, that the sample 200 is illuminated in the dark-field microscopy mode. The sample 200 is thus illuminated by means of light rays having a high obliquity with respect to the principal axis 10 of the optical microscopy system, that is to say, light rays having a large angle of inclination with respect to the principal axis 10 of the optical microscopy system. An angle of inclination of the light rays is chosen so that these rays cannot be collected by the central region 144 of the dark-field microscope objective 14.Here, the angle of inclination takes values ​​greater than or equal to 37 degrees with respect to the optical axis of the dark field microscope objective 14.

[0073] An area of ​​the sample 200 is therefore illuminated. This area extends parallel to the plane of the sample 200, and a dimension, in particular a diameter, of this illuminated area, is determined according to the properties of the dark field microscope objective 14, such as for example, its magnification, its numerical aperture.

[0074] Here, an illuminated area is considered to have a circular dimension with a diameter ranging from a few microns to a few hundred microns. Here, for example, a circular illuminated area is considered to have a diameter of at least 100 microns, or even at least 140 microns, or even at least 150 microns. This circular illuminated area has dimensions, here a diameter, greater than the dimensions of an image subsequently recorded.

[0075] Depending on the dark-field microscope objective 14 used in the first In this embodiment, the illuminated area corresponds to a circular zone with an area on the order of a few tens, or even a few hundred micrometers. For example, the illuminated area corresponds to a circular zone with a diameter of 440 micrometers.

[0076] The sample 200, and in particular the structures it contains, here microparticles 202, and more specifically, microparticles 202 of plastic materials and / or mineral microparticles 202, scatters a portion of the oblique light rays from the annular illumination cone 124. Only light rays deflected by the structures of the sample 200 are collected by the central region 144 of the dark-field microscope objective 14, according to the known contrast mechanism of dark-field microscopy. Thus, only the structures that deflect at least partially the incident light produce contrast and contribute to a dark-field microscopy image. The central region 144 of the dark-field microscope objective 14 behaves like a conventional microscopy objective and is characterized by a magnification and numerical aperture, as described above.

[0077] The light rays deflected by the microparticles 202 are therefore 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 a spectrum of the white light beam 120, since the contrast mechanism is based on an elastic scattering phenomenon.

[0078] The deflected light rays emerge from the dark-field microscope objective 14 in collimated form, since an infinity-corrected objective is used in this first embodiment. These deflected light rays then propagate in free space along the principal axis 10 of the optical microscopy system, and notably pass through the aperture formed within the annular mirror 13.

[0079] An image sensor 15 then images the deflected light rays. This image sensor 15 has an optical axis that is aligned with the principal axis 10 of the optical microscopy system, and therefore, by extension, with the optical axis of the dark-field microscope objective 14.

[0080] The image sensor 15 comprises an optical imaging system and a photosensitive matrix sensor 150, this assembly being frequently referred to as a camera. The optical imaging system is adapted to image the sample 200 on the photosensitive matrix sensor 150. The photosensitive matrix sensor 150 comprises an array, here rectangular, of light-sensitive pixels, capable of converting light information into an accumulated electrical charge. For example, it is a CCD sensor or a CMOS sensor.

[0081] The image sensor 15 is adapted to record a dark-field microscopy image corresponding to at least a portion of the dark-field illuminated area, by imaging the light rays deflected on the photosensitive matrix sensor. The sensor Image sensor 15 has a field of view, which depends, among other things, on the magnification of the image sensor's optical imaging system and the dimensions of the rectangular pixel array. This field of view of image sensor 15, along with the field of view of the central region 144 of the dark-field microscope objective 14, defines a region of the sample 200 that can be imaged in the dark-field microscopy modality. This imaged region is referred to as the image field of view.

[0082] Since the image field of view is included in the illuminated area, it therefore has dimensions smaller than the dimensions of the illuminated area.

[0083] The dark-field microscopy image reproduces the region of the sample 200 as previously described, and the contrast within this dark-field microscopy image represents the structures, i.e., the microparticles 202, such as, for example, microparticles of plastic material, or grains of minerals, capable of deflecting incident light rays.

[0084] This dark-field microscopy image most often has a rectangular shape, due to the rectangular shape of the photosensitive matrix sensor 150.

[0085] Each dark-field microscopy image represents, for example, a region of 140 by 105 microns on the sample 200.

[0086] An image processing unit 60, shown in [Fig. 1], and included, for example, in the control unit 6, here a computer, is configured to exchange information with the image sensor 15. This image processing unit 60 is also adapted to identify and extract from the dark-field microscopy image coordinates of points likely to correspond to microparticles 202. More specifically, microparticles 202 with a minimum diameter of 0.5 micrometers are spatially located, and morphological parameters are extracted from them. 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 detectable microparticles 202.

[0087] The image processing unit 60 is also configured to exchange data with a user via an interface integrated into software. The user then has the option of filtering certain microparticles 202 of interest based on specific criteria, such as morphological parameters. For example, the user can choose to retain only the microparticles 202 with a diameter between two value limits.

[0088] This identification and extraction is performed using a dedicated image processing algorithm, or alternatively, by means of a machine learning algorithm, as known in the prior art. Alternatively, the identification and extraction of the coordinates of Points of interest within each dark-field microscopy image are made manually or partly manually by an optical microscopy system user. Raman microspectroscopy modality

[0089] The Raman microspectroscopy modality is also shown in [Fig.1].

[0090] Advantageously, this Raman microspectroscopy modality is operable without switching a mechanical element to switch from one modality to another, and can thus be implemented simultaneously with the dark-field microscopy modality.

[0091] The optical microscopy system includes a laser source 16. This laser source 16 emits an excitation laser beam 160, which is continuous. The excitation laser beam 160 is further collimated at the output of the laser source 16.

[0092] By way of non-limitation, this laser source 16 corresponds for example to a diode-pumped solid state laser, known in English as a diode-pumped solid state laser.

[0093] For the Raman microspectroscopy modality, the excitation laser beam 160 is monochromatic and has, for example, a wavelength chosen within a spectral window between 380 nanometers and 1064 nanometers. For example, this is an excitation laser beam 160 with a wavelength of 532 nanometers.

[0094] Upon exiting the laser source 16, this excitation laser beam 160 propagates along an optical axis of the beam, the optical axis of the laser beam being initially parallel to the principal axis 10 of the optical microscopy system. A first dichroic filter 17, also known in the prior art by the English term edgefilter, reflects the excitation laser beam 160 so that the optical axis of the laser beam is perpendicular to the principal axis 10 of the optical microscopy system.

[0095] 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 shorter than a cutoff wavelength and to transmit another spectral range of distinct wavelengths, which is this time longer than the cutoff wavelength. The cutoff wavelength is chosen according to the wavelength of the excitation laser beam 160, here, so as to reflect the excitation laser beam 160. The first dichroic filter 17, for example, has a cutoff wavelength set at 532.85 nanometers by the manufacturer.

[0096] Since it corresponds to an edge filter, or edge filter, also known as a laser filter, a transmission curve as a function of wavelength of this first dichroic filter 17 shows a very small transition zone between the transmitted and reflected wavelengths. In other words, the transmission curve of the first dichroic filter has a slope considered steep at its cutoff wavelength. In this case, a transition between the transmitted and reflected wavelengths occurs over a few nanometers around the cutoff wavelength. For example, the transition occurs over a width less than or equal to 10 nanometers around the cutoff wavelength, more ideally over a width less than or equal to 5 nanometers around the cutoff wavelength, or even a width less than or equal to 3 nanometers around the cutoff wavelength.

[0097] The optical microscopy system also includes a second dichroic filter 18.

[0098] This second dichroic filter 18 is adapted to reflect a spectral range with wavelengths greater than a cutoff wavelength, and to transmit another spectral range with a distinct wavelength. It is therefore a low-pass type dichroic filter.

[0099] Here, the cutoff wavelength is chosen to be less than the wavelength of the excitation laser beam 160, that is, strictly less than 532 nanometers. More precisely, the second dichroic filter 18 has a cutoff wavelength of 495 nanometers. Thus, the excitation laser beam 160 is reflected by the second dichroic filter.

[0100] This second dichroic filter 18 is also chosen so as to transmit at least part of the light spectrum of the white light beam 120. Advantageously, according to one embodiment, the light spectrum of the white light beam 120 is fully transmitted by the second dichroic filter 18.

[0101] This second dichroic filter 18 is placed centered on the main axis 10 of the optical microscopy system between the annular mirror 13 and the image sensor 15. It is also centered with respect to the optical axis of the excitation laser beam 160. A surface of the second dichroic filter 18 is oriented at 45 degrees with respect to the optical axis of the laser beam, so as to reflect the excitation laser beam 160 along the main axis 10 of the optical microscopy system, in the direction of the annular aperture formed within the annular mirror 13.

[0102] The excitation laser beam 160 is then transmitted through the hole in the annular mirror 13, along the main axis 10 of the optical microscopy system, in the direction of the sample 200.

[0103] The central region 144 of the dark field microscope objective 14, which is centered around the main axis 10 of the optical microscopy system, transmits the excitation laser beam 160 of the Raman microspectroscopy modality, before focusing this excitation laser beam 160 into a point measurement point on the sample 200.

[0104] This measurement point is centered on the optical axis of the dark-field microscope objective 14, which coincides with the principal axis 10 of the optical microscopy system. This point coincides, for example, with a geometric midpoint of the illuminated area in dark-field microscopy.

[0105] This measurement point has an extent in the xy plane defined according to the properties of the dark field microscope objective 14, such as in particular its numerical aperture, as well as the wavelength of the excitation laser beam 160.

[0106] Usually, the extent of the measurement point varies from a few hundred nanometers to a few micrometers. Here, for example, given the numerical aperture of the dark-field microscope objective 14, as well as the wavelength of the excitation laser beam 160, the measurement point probes a point approximately 564 nanometers in diameter on the sample 200.

[0107] Due to the Raman effect, the sample 200 emits a Raman spectrum, associated with inelastically scattered light. We are interested here in Stokes light, corresponding to light whose wavelength is greater than or equal to the wavelength of the excitation laser beam 160.

[0108] This light radiation corresponds to the Raman spectrum emitted from the measurement point onto which the excitation laser beam 160 is focused. This Raman spectrum notably includes Raman lines specific to the chemical composition of this measurement point. In order to access this spectral information, the scattered light radiation is analyzed.

[0109] Thus, the Raman spectrum, in the form of scattered light, is collected by the central region 144 of the dark-field microscope objective 14, which collimates this scattered light into a light beam, called the Raman beam 162. This Raman beam 162 is accompanied by a beam corresponding to the light scattered elastically by Rayleigh scattering. This beam has the same wavelength as the excitation laser beam 160. This beam is referred to in the following description as the Rayleigh beam.

[0110] The Raman beam 162 and the Rayleigh beam propagate along the main axis 10 of the optical microscopy system, in the direction of the hole in the annular mirror 13.

[0111] The Raman beam 162, the Rayleigh beam, and the exciter laser beam 160 are therefore spatially coincident, being collinear with each other. Nevertheless, the Raman beam 162 and the Rayleigh beam are contrapropagating with respect to the exciter laser beam 160, that is, they propagate in opposite directions to each other.

[0112] Thus, the Raman beam 162 and the exciter laser beam 160 share an identical optical path between the sample 200 and the first dichroic filter 17.

[0113] The opening in the center of the annular mirror 13 therefore 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.

[0114] The Raman beam 162 and the Rayleigh beam continue their propagation to the second dichroic filter 18. Since the Raman beam 162 corresponds to Stokes light radiation, with a wavelength greater than or equal to the wavelength of the excitation laser beam 160, and therefore strictly greater than the cutoff wavelength of the second dichroic filter 18, the Raman beam 162 is thus reflected, forming an angle of 90 degrees with respect to the principal axis 10 of the optical microscopy system. The same applies to the Rayleigh beam, which has the same wavelength as the excitation laser beam 160.

[0115] The Raman beam 162 and the Rayleigh beam therefore propagate along the optical axis of the exciter laser beam 160 to the first dichroic filter 17.

[0116] The first dichroic filter 17 transmits the Raman beam 162 towards a Raman spectrometer 19 included in the optical microscopy system, and reflects the Rayleigh beam. Indeed, this first dichroic filter 17 is configured to reflect wavelengths less than or equal to the cutoff wavelength, chosen to be equal to that of the excitation laser beam 160, while transmitting wavelengths greater than it.

[0117] The Raman beam 162 is therefore analyzed spectrally by the spectrometer 19. Such a spectrometer 19 includes in particular a dispersive element, that is to say an optical element adapted to spectrally decompose the light into monochromatic spectral components, such as a prism or a diffraction grating, as well as a photosensitive matrix sensor 190, such as a CCD sensor or a CMOS sensor.

[0118] Spectral analysis of the Raman beam 162 by a spectrometer 19 makes it possible to trace back to the Raman spectrum from the measurement point on which the excitation laser beam 160 was focused by the central region 144 of the dark field microscope objective 14.

[0119] Thus, in the embodiment currently disclosed for the bi-modal optical microscopy system, i.e. combining here a dark-field microscopy modality for imaging, with a Raman microspectroscopy modality, the two modalities can be implemented simultaneously, without mechanical switching to go from one illumination modality to another.

[0120] According to a first embodiment, in order to implement the two modalities simultaneously, disjoint wavelength ranges are chosen for the light spectrum of the white light beam 120 and the wavelength of the excitation laser beam 160. In particular, in order to dissociate the Raman beam 162 from the Raman microspectroscopy modality, from the deflected light rays from the In dark-field microscopy, the light spectrum of the white light beam 120 is, for example, chosen to be strictly shorter than the wavelength of the excitation laser beam 160. Specifically, a low-pass filter placed between the white light source 12 and the ring mirror 13 eliminates unwanted wavelengths. Alternatively, monochromatic radiation with a wavelength strictly shorter than the wavelength of the excitation laser beam 160 is chosen to serve as white light. Here, 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 disjoint. Furthermore, the wavelength range covered by the white light source 12 is considered to be strictly shorter than the wavelength of the excitation laser beam 160.

[0121] Conversely, according to a second variant, the two modes can also be implemented alternately, that is, one after the other, by controlling the power supply to one or both sources, i.e., laser source 16 and white light source 12. Thus, it is possible to selectively switch off one of the sources in favor of the other rapidly, compared to the switching times usually known in the prior art. In this case, the choice of spectral ranges covered by each of the two sources is left to the discretion of the user. Dynamic process

[0122] Such an optical microscopy system finds a particularly advantageous application in the implementation of a method for spatial localization and dynamic spectral analysis of microparticles 202 as described below. As a reminder, in such a dynamic approach, rather than imaging the entire sample 200 before performing a spectral analysis, as is the case in a static approach, only a portion of the sample 200 is imaged before proceeding with the spectral analysis within the imaged portion. These two steps are repeated until a representation of the entire sample 200 is obtained.

[0123] A first embodiment of such a process is described by a first flowchart 3 shown in [Fig.2].

[0124] The process comprises the following steps in its first embodiment; these steps are detailed with an example in the following description: - illumination of an area on sample 200 using the dark-field microscopy modality, - recording 31 of a dark-field microscopy image, - identification 32 within the image of microparticles 202, - extraction 33 of the coordinates of the points corresponding to these microparticles 202, in order to form a list, - displacement 34 of sample 200, in order to match the central region 144 of the dark-field microscope objective 14, with a point from the extracted coordinate list, - illumination 35 of sample 200 using the Raman microspectroscopy modality, the excitation laser beam 160 being focused at a measurement point which coincides with the point of the previous step, - collection 36 of the Raman spectrum, emitted from the measurement point, by the central region 144 of the dark field microscope objective 14.

[0125] The displacement step 34, illumination step 35, and collection step 36 are then optionally repeated, for example, until all the points in the list have been scanned. A new displacement step 37 then moves the sample 200 to a new dark-field area, at least partially distinct from the previous area, in order to resume the process from illumination step 30.

[0126] An application of this first embodiment of the process is illustrated in [Fig.3],

[0127] In this first embodiment, a mosaic 4 representative of the sample 200 is reconstructed using a plurality of dark-field microscopy images. The mosaic 4 represents a field larger than the image field of view, thanks to an assembly of several dark-field microscopy images covering at least partially disjoint areas on the sample 200. Here, dark-field microscopy images covering completely disjoint areas are considered. Ideally, the mosaic 4 represents the entire surface of the sample 200.

[0128] Each of the images in the plurality of dark-field microscopy images is here designated as a tile 40, that is, an area of ​​the sample 200 extending in the xy plane. Thus, each of the tiles 40 is at least partially disjoint from the other tiles 40 comprising the mosaic 4. Here, each of the tiles 40 is assumed to be totally disjoint from the others. The size of each of the tiles 40 is identical here, and defined by the field of view of the image sensor 15, this image field of view being included in the illuminated area of ​​the sample 200.

[0129] Thus, according to the characteristics of the image sensor 10 and the dark field microscope objective 14, each tile 40 has a rectangular shape of 140 micrometers by 105 micrometers.

[0130] On [Fig.3], the tiles 40 forming the mosaic 4 are arranged according to a rectangular grid, where each of the tiles 40 are adjacent to each other.

[0131] In the first embodiment, a position of each tile 40 of the mosaic 4 is predefined prior to the execution of the process with respect to a coordinate system. This coordinate system is, for example, represented by the displacement plate 11, which is fixed to the sample holder 20 and therefore to the sample 200. Thus, the coordinate system established by the displacement plate 11 also locates points on the sample 200.

[0132] Thus, each of the tiles 40 is located by a pair of coordinates along the x-axis and along the y-axis in the coordinate system. This pair of coordinates, denoted (xi5 y;), positions a geometric center 41 of the tile 40 with respect to the sample 200. The geometric center 41 of the tile 40 here designates a point of intersection of the diagonals of the tile 40, the latter having a rectangular shape. This geometric center 41 is represented for each of the tiles 40 in [Fig. 3] by a target, that is to say, a cross and a circle.

[0133] Alternatively, each tile 40 can also be located by one of its corners.

[0134] In the first embodiment, a set of coordinate pairs for each of the tiles 40 is pre-established beforehand, i.e., before the implementation of the spatial tracking and spectral imaging of microparticles 202 now disclosed. Each tile 40 occupies a predefined spatial extent on the sample 200.

[0135] Here, for illustrative purposes, only a portion of the mosaic 4 reconstructed on the sample is shown in [Fig.3].

[0136] The portion of mosaic 4 shown in [Fig.3] comprises twenty tiles 40, each of the tiles 40 having a dimension of 140 micrometers by 105 micrometers.

[0137] Usually, the 200 samples considered have dimensions on the order of ten millimeters in diameter, such as 13 millimeters, 25 millimeters, or 42 millimeters, up to a few centimeters, represented by mosaics of up to 1000 by 1000 tiles, or one million tiles, or even more.

[0138] For example, for a sample 200 with a diameter of 27 millimeters, it is necessary to acquire Æ*(D / 2)2 tiles, where D corresponds to the diameter of the sample 200, if the tiles 40 are rectangular and each measure 100 microns by 100 microns. This represents approximately 58,000 tiles to be acquired in order to tile the entire surface of the sample 200.

[0139] Small tiles 40, corresponding to images acquired using lenses with high magnification are preferable, in particular to increase spatial resolution in the plane of the sample 200.

[0140] In order to record each of the dark-field microscopy images forming the tiles 40 of the mosaic 4, the displacement stage 11 moves the sample 200, for example, so as to make a pair of coordinates from the pre-established set of coordinate pairs for each of the tiles 40, indicating here the geometric center 41 of each tile 40, coincide with the optical axis of the dark-field microscope objective 14. More generally, it is a matter of making the field of view included coincide in the area of ​​sample 200 illuminated by the dark field microscopy modality with the predefined tile 40.

[0141] In a method for the spatial identification and spectral imaging of dynamic microparticles 202, coordinate points 42 associated with potential microparticles 202 are identified within each tile 40 imaged by dark-field microscopy, and Raman spectra are recorded on these coordinate points 42 identified using Raman microspectroscopy, before proceeding to the acquisition of the next tile 40. A successive order of the tiles 40 to be imaged is defined in advance, prior to the implementation of the method. For example, the surface of the sample 200 is imaged here using a line-by-line scanning pattern, known as a raster scan, or a serpentine scan, or any other scanning pattern deemed appropriate.

[0142] Thus, according to the first embodiment where the positions of the tiles 40 are predefined, for each of the n tiles 40 forming the mosaic 4, the steps described below are implemented, with between each of them, a displacement 37 of the sample 200, to go from one tile to the other.

[0143] We now detail the application of the process, for example for the acquisition of tile 40 located in the upper left corner of mosaic 4, as shown in [Fig.3].

[0144] First, it is assumed that the geometric center 41 defined for one of the tiles 40 corresponds with the optical axis of the dark field microscope objective 14, following a positioning of the displacement stage 11.

[0145] For each tile 40, an area of ​​the sample 200 is illuminated 30 using the annular illumination cone 124. This illumination cone, as a reminder, is generated by the reflection of part of a white light beam 120 by an annular mirror 13. The reflected part, forming a hollow cylinder, is transmitted by the peripheral region 142 of the dark field microscope objective 14, and focused on the sample 200, so as to illuminate an area on the sample 200 only with light rays having a high obliquity with respect to the optical axis of the dark field microscope objective 14.

[0146] A recording 31 of a first dark-field microscopy image, corresponding to one of the tiles 40, is then made using an image sensor 15. The rays deflected by the sample 200, in particular by the contained microparticles 202, are collected by the central region 144 of the dark-field microscope objective 14, as described previously. These deflected rays form the first dark-field microscopy image, where contrast is present only for the light-deflecting structures, i.e., the microparticles 202. The first dark-field microscopy image is recorded on an image field of view included in the area of ​​sample 200 illuminated. This first dark-field microscopy image corresponds to a first tile 40, located here in the upper left corner of the mosaic.

[0147] This dark-field microscopy modality optimizes contrast on samples 200 exhibiting significant transparency, such as, in this case, the plastic microparticles 202 or mineral grains. The subsequent step is thus facilitated.

[0148] Indeed, starting from this first dark-field microscopy image, a subsequent identification 32 of any microparticles 202 within this first image is carried out. This identification 32 is performed, for example, using an image processing algorithm. In particular, it is possible to use shape detection or edge detection via such algorithms. Alternatively, the possible microparticles 202 are recognized using a machine learning algorithm. This machine learning algorithm, for example, a pattern recognition algorithm, is trained beforehand using an image database in order to recognize certain patterns of interest.

[0149] Once any microparticles have been identified in the first microscopy image, the process continues with an extraction 32 of the coordinates of the points possibly corresponding to microparticles 202 within the dark-field image. This extraction is performed, for example, by the image processing unit 60, using an image processing algorithm, or by a machine learning algorithm.

[0150] Thus, for this first dark field microscopy image corresponding to one of the tiles 40 of the mosaic 4, we obtain a list of points, formed of points with coordinates 42. These points with coordinates 42 are defined in the coordinate system of the displacement stage 11.

[0151] A point of coordinates 42 is defined for each of the microparticles 202 located in the image field of view, for example placed at the level of a geometric center of each of the microparticles 202. In other words, each microparticle 202 shown on the tile 40 pictured in dark field is associated with a single point of coordinates 42.

[0152] The geometric center of each of the microparticles 202 is located inside it, and is placed as far away as possible from the edges of the microparticle 202.

[0153] Alternatively, a multitude of points with coordinates 42 can be extracted from the same microparticle 202 appearing on the first dark-field microscopy image. This multitude of points then takes the form of a point matrix, defined on each of the microparticles 202. A spacing between the points with coordinates 42 extracted from the same microparticle 202 is defined as a function of a displacement accuracy of the displacement stage 11, as well as a resolution The spatial arrangement of the Raman microspectrometry modality yields a fine mesh of each 202 microparticle. This fine mesh then allows for the measurement of a spectrum—in this case, a Raman spectrum—at each point of the matrix, thus creating a spectral map to access the chemical distribution within the 202 microparticle. Alternatively, it is also possible to average all the spectra acquired within a given 202 microparticle to obtain a representative average spectrum for that 202 microparticle.

[0154] Optionally, it is possible to perform a preliminary filtering of the microparticles 202 from which points with coordinates 42 are extracted, as described previously. For example, a filtering criterion is established based on criteria related to the morphological parameters from the first dark-field microscopy image. These are, for example, criteria of size or shape.

[0155] Here, the variant in which a single point of coordinates 42 is extracted by microparticle 202 appearing on the different tiles 40 is illustrated in [Fig.3]. Each point with coordinates 42 located on the different tiles 40 forming the mosaic 4 is represented by means of a dotted cross.

[0156] Thus, on the first dark field microscopy image shown in the top left of [Fig.3], three points with coordinates 42 are extracted: a point with coordinates 421, a point with coordinates 422, and a point with coordinates 423. The set of these three points here forms the list of points for this tile.

[0157] A displacement 34 of the sample, using the displacement stage 11, allows the optical axis of the dark-field microscope objective 14 to coincide. In other words, this displacement 34 centers the central region 144 of the dark-field microscope objective 14 with a point from the list of points established in the previous step. For example, this is the point with coordinates 421, shown in [Fig. 3].

[0158] Next, a measurement point 33 on the sample 200 is illuminated using the Raman micro-spectrometry modality. For this, an excitation laser beam 160 transmitted through the annular mirror 13 is focused by the central region 144 of the dark-field microscope objective 14.

[0159] Since the measurement point is centered with respect to the optical axis of the dark-field microscope objective 14, the measurement point coincides with the point from the list of coordinate points. In other words, the excitation laser beam 160 is focused on the point with coordinates 421.

[0160] The white light source 12 is then switched off, for example by means of its power supply, which is connected to the control unit 6.

[0161] However, it is advantageous to maintain both illumination modes simultaneously, especially during the movement of the displacement plate 11. Indeed, the simultaneous use of illumination by the annular illumination cone 124 and of Illuminating a measurement point by focusing the laser beam allows, in particular, the simultaneous observation of the illuminated area on sample 200 and the measurement point using the image sensor 15 of the dark-field microscopy modality. A tiny portion of the excitation laser beam 160 manages to pass through the second dichroic filter 18. This tiny portion represents, for example, less than 0.001% of the optical power of this excitation laser beam 160. This portion is nevertheless sufficient to be observed on the image sensor 15. It is thus possible to observe the relative position of the measurement point generated by the excitation laser beam 160 with respect to the illuminated area on sample 200 in the dark field. The measurement point then appears as a small bright point in the image field of view.

[0162] Thus, if disjoint wavelength ranges are chosen for the white light beam 120 and the exciter laser beam 160, with the light spectrum of the white light beam 120 strictly lower than the wavelength of the exciter laser beam 160, then the two illuminations are maintained simultaneously, without risk of interference.

[0163] Next, a Raman spectrum emitted from the measurement point is collected 36. This spectrum is collected by the central region 144 of the dark-field microscope objective 14 and sent to the spectrometer 19 for analysis, following an optical path detailed previously. As with the illumination of the sample 200 by the excitation laser beam 160, the collection of the Raman spectrum does not require mechanical switching, thus preventing misalignment of the optical elements along this optical path.

[0164] The other points with coordinates 422, 423... extracted from the same dark-field microscopy image are also probed to obtain a Raman spectrum. To do this, a displacement 34 of the sample 200 is performed, via the displacement stage 11, to make the measurement point coincide successively with each of the other points with coordinates 422, 423... in the list of points of the tile under consideration. Thus, in the illustrated example, the displacement stage 11 moves the sample 200 to scan the point with coordinates 422, then the point with coordinates 423. A scanning pattern within the tile 40 is illustrated by arrows.

[0165] A Raman spectrum is collected at each of the points in the point list, by a displacement 34 of the displacement stage 11 followed by the collection 36 of a spectrum.

[0166] These two collection steps 36 and displacement steps 34 are applied recursively, for all points of coordinate 42 of the list of points extracted from the first dark-field microscopy image, i.e. extracted from the first tile 40.

[0167] After scanning all the points with coordinates 42 in the point list of the first tile 40, a new dark-field microscopy image is in recorded as part of the process. For this, a displacement of the sample 37 makes one of the geometric centers, whose coordinate pair has been predefined, coincide with the optical axis of the dark field microscope objective.

[0168] The list of points is then reset.

[0169] This new dark-field microscopy image corresponds to a subsequent tile 40 of the mosaic 4, defined according to the chosen scanning pattern. Here, for example, it is the tile to the right of the first tile 40 described previously, i.e. on the first row and second column of [Fig.3].

[0170] The sequence of steps described above is repeated for this next tile 40, and for all the tiles 40 included in the mosaic 4, until a spectral image representative of the entire sample 200 is obtained. Such a spectral image is shown in [Fig.6].

[0171] In this spectral image, the visible structures, corresponding here to 202 microparticles, are imaged using dark-field microscopy, while colors, represented here by dithering, are associated with each of the 202 microparticles. These colors indicate the chemical nature, i.e., the composition, of the 202 microparticles, which in the example illustrated in [Fig. 6] correspond, without limitation, to 202 microparticles of mineral matter, here more specifically chalk. These colors also correspond to false colors, chosen arbitrarily, according to the chemical composition of the microparticle. The composition of each 202 microparticle is determined by the analysis of the point Raman spectra collected by the Raman microspectrometry modality, in particular from the Raman lines present in the collected spectrum.

[0172] In practice, the Raman spectrum of each of the 202 microparticles is identified and assigned to a class using dedicated algorithms. For example, and without limitation, it is possible to identify the chemical composition of a 202 microparticle by correlating the spectrum acquired on the 202 microparticle with a pre-established spectral database stored in memory. The 202 microparticle is then identified as corresponding to the known chemical species whose spectrum has the closest correlation.

[0173] It is also possible to perform a multivariate analysis, for example, using multivariate curve resolution (MCR). Such a multivariate analysis allows the microparticles 202 to be divided into different classes, which are then identified manually or automatically.

[0174] According to another variant, the identification of the chemical composition of microparticles 202 is carried out by means of machine learning. A model is then trained beforehand on a large number of labeled data, in order to allow by the following the identification of the spectrum of the microparticles 202 to be characterized.

[0175] Thus, for each tile 40 obtained and corresponding to a dark-field microscopy image, spectral information associated with the microparticles 202 imaged in the tile 40 is extracted. This spectral information then makes it possible to determine the chemical nature of these microparticles 202.

[0176] This dynamic acquisition method, as described in the first embodiment, offers in particular the following advantages:

[0177] First, morphological parameters of the microparticles 202 are determined on the fly, during the tile-by-tile reconstruction of the mosaic 4. This makes it possible to fully exploit the spatial resolution of the dark-field microscopy modality when determining the morphological parameters. This improves the accuracy of this determination. Indeed, in the static mode mentioned in the preamble, the resolution of the dark-field microscopy image must be compressed during acquisition in order to limit the memory space required to store these dark-field images.

[0178] Furthermore, spectral analysis is also performed on the fly, during the reconstruction of the mosaic tile by tile. The displacement made by the displacement stage 11 to position itself on each of the microparticles is therefore small, on the order of a few micrometers. However, the precision of a displacement stage 11 is usually given in micrometers per millimeter, typically 3 micrometers per millimeter. Thus, a displacement on the order of a few micrometers is more precise than a displacement of several millimeters. Given that the system described here is applied to objects of study with micrometer dimensions, a precision of the same order of magnitude, that is to say, on the order of micrometers, becomes critical. Thus, the method allows for a gain in terms of spatial resolution when measuring using the Raman microspectrometry modality.In a static process, as described in the preamble, sample 200 is imaged over its entire surface, typically a region of one centimeter by one centimeter, before scanning the points of interest on the microparticles 202 again using the Raman microspectroscopy modality. This results in a positioning error potentially of several micrometers.

[0179] Furthermore, the dynamic approach as described in the first embodiment also makes it possible to limit the inconveniences caused by variations in environmental conditions encountered in static mode. Indeed, acquiring a complete mosaic 4 using the dark-field microscopy modality requires several tens of minutes, or even more like an hour. Thus, it is possible that environmental conditions, such as temperature, humidity, and vibrations, may vary and cause measurement inaccuracies during the transition between the two modality modes. Dark-field microscopy and Raman microspectroscopy are used in this system. The dynamic mode, described here through two implementations, allows the two modalities to be chained together in a short timeframe, or even to be implemented simultaneously. Thus, the system is rendered insensitive to environmental variations occurring over long timescales.

[0180] In addition to these advantages over the static approach, the present invention eliminates the downtime associated with mechanical switching times when moving from one modality to another within a tile 40. Indeed, the transition from the dark-field microscopy modality to the Raman microspectroscopy modality occurs without switching any mechanical elements, thanks to the system detailed above. Thus, no mechanical switching time is required, representing a considerable time saving.

[0181] Indeed, for microscopy applications, high-precision mechanical switching systems are used. Usually, such mechanical switching systems require a few seconds, for example 6 seconds, to switch from dark-field microscopy to Raman microspectrometry, and a few seconds, for example 6 seconds, to switch back; this amount of time is lost per imaged tile. Here, 12 seconds are lost per imaged tile. On the scale of a mosaic representing a large sample, i.e., on the order of a few centimeters by a few centimeters, comprising several hundred, or even millions, of tiles, this represents a considerable amount of measurement time lost.

[0182] Even for more efficient, and therefore more expensive, mechanical switching systems, a mechanical switching time of a few hundred microseconds remains. In fact, even with such mechanical switching systems, a mechanical switching time on the order of a few milliseconds remains. On the scale of a large sample size, these switching times are not negligible.

[0183] The proposed method implemented by the spatial tracking system in an image and spectral analysis of microparticles 1 described therefore allows a saving of time on the analysis of large dimension samples, i.e. of a few tens of millimeters on a side, or even a few centimeters and comprising low contrast microparticles 202. The “Little Thumb” method

[0184] A second embodiment of the disclosed process is illustrated by a flowchart 5 in [Fig.4].

[0185] This flowchart 5 includes the following steps, as initialization: - 50 illumination of an area on sample 200 using the dark-field microscopy modality, - recording 51 of a dark-field microscopy image, - identification 52 within the image of microparticles 202, - extraction 53 of the coordinates of the points corresponding to these microparticles 202, in order to form a list, - displacement 54 of sample 200, in order to match the central region 144 of the dark-field microscope objective 14, with a point from the extracted coordinate list, - illumination 55 of sample 200 using the Raman microspectroscopy modality, the excitation laser beam 160 being focused at a measurement point which coincides with the point of the previous step, - collection 56 of the Raman spectrum, emitted from the measurement point, by the central region 144 of the dark field microscope objective 14.

[0186] Once initialized, the process according to the second embodiment is continued by moving the sample 54 to align the optical axis of the dark-field microscope objective 14 with another point in the extracted coordinate list. Then, the steps of illumination 50, recording 51, and localization 52 using the dark-field microscopy modality are performed, either concurrently with, before, or following the steps of illumination 55 and collection 56 using the Raman microspectroscopy modality.

[0187] The process is thus looped back to the displacement step 54, for example, as long as the list of points contains points of coordinates 42 which have not yet been scanned and measured by Raman microspectroscopy.

[0188] In this second embodiment, a mosaic 4 representing the sample 200 is reconstructed using a plurality of dark-field microscopy images, similar to what was described for the first embodiment. Thus, this mosaic 4 represents a field larger than the field of view of a single dark-field microscopy image. This mosaic 4 consists of tiles 40, that is, dark-field microscopy images arranged to reproduce the analyzed sample 200. In the second embodiment, some of these tiles 40 overlap at least partially. In other words, some of the tiles 40 of the mosaic 4 cover partially spatially identical areas.

[0189] Just as in the first embodiment, according to the characteristics of the image sensor 15 and the dark field microscope objective 14 used to collect a dark field microscopy image, each tile 40 has a rectangular shape of 140 micrometers by 105 micrometers.

[0190] A portion of mosaic 4 obtained according to the second process is reproduced on the [Fig.5]. A rectangle indicates the boundaries of mosaic portion 4 of [Fig.3], this rectangle also being reproduced on [Fig.6].

[0191] In this embodiment, the geometric centers 41 of the tiles 40 are not predefined. Similarly, the arrangement of the tiles 40 relative to each other is not predefined prior to the process. Indeed, in this second embodiment, pairs of coordinates defining the position of each tile 40 are defined during the process, based on information collected during the implementation of the process.

[0192] More specifically, geometric centers 41 are chosen from points of coordinates 42 extracted during the process, which leads to the acquisition of new dark-field microscopy images, and the extraction of new points of coordinates 42. These new points of coordinates 42 potentially become in turn geometric centers 41 for tiles 40, and so on.

[0193] Thus, unlike the first embodiment, the passage from one tile 40 to another tile 40 is not necessarily done via a dedicated movement, and a tiling of the mosaic 4 is done as the movements 54 progress. These movements 54 aim to sweep the different points of coordinates 42 of the list of points, which then constitute geometric centers 41 for tiles 40.

[0194] The steps previously described for the second embodiment of the process are now detailed with the example of [Fig.5].

[0195] Finally, to start the process, the displacement plate 11 places the sample 200 at an arbitrary position. For example, a geometric center 41 of a first tile 40, shown in [Fig. 5], is chosen here at the midpoint of a displacement amplitude of the displacement plate 11 along the x-axis and along the y-axis. This position corresponds to a midpoint of the displacement plate 11.

[0196] The next step of the process consists of illuminating 50 an area of ​​the sample 200 according to the dark-field microscopy modality. As described previously, the illumination 50 of the area of ​​the sample 200 centered around the geometric center 41 of the first tile 40, is done by an annular illumination cone 124, focused on the sample 200 by the peripheral region 142 of the dark-field microscope objective 14.

[0197] In [Fig. 5], the geometric center 41 of each tile 40 is indicated by a target drawn with a solid line, that is to say, a cross surrounded by a circle.

[0198] Illuminating 50 of this area by the dark-field microscopy modality leads to a recording 51 of a first dark-field microscopy image. Once again, the field of view of this image is included in the illuminated area of ​​the sample 200. This first dark-field microscopy image is obtained by collection through the central region 144 of the field microscope objective dark 14 of the light rays deflected.

[0199] From this first dark-field microscopy image, a 52-point identification of the potential 202 microparticles within the first image is carried out. This identification 52 is assumed to be identical to that described for the first embodiment.

[0200] After identifying any microparticles 202 contained within the image field of view, for example, using a dedicated image processing algorithm, an extraction 53 of the coordinates of the points corresponding to any microparticles 202 is performed. This extraction 53 is carried out as described above, by the processing unit, using appropriate algorithms, and / or by a user. As in the first embodiment, a single point with coordinates 42 is associated with each of the microparticles 202 visualized in the dark field, although variations are possible.

[0201] These extracted coordinate points 42 are added to a list of points.

[0202] On the first tile 40, two points with coordinates 42 are extracted, the coordinate point 421 and coordinate point 422. These coordinate points 42 are indicated on [Fig.5], using targets in dotted lines.

[0203] A displacement 54 of the sample 200 by the displacement stage 11 aligns the optical axis of the dark-field microscope objective 14 with one of the coordinate points in the point list. Specifically, a coordinate point is chosen that has not yet been scanned by the Raman microspectroscopy modality. For example, this is the point with coordinates 421, which corresponds to the coordinate point 421 associated with the first microparticle 202 identified and located within the tile 40.

[0204] The next step involves illuminating a measurement point on the sample 200 using Raman microspectroscopy. The laser beam is focused onto the sample 200 at a measurement point by the central region 144 of the dark-field microscope objective 14. This measurement point is located along the optical axis of the dark-field microscope 14.

[0205] Thus, the displacement 54 makes the illuminated measurement point coincide with the point of coordinates 421 from the list of points.

[0206] Following illumination 55 and displacement 54, a Raman spectrum emitted from the measurement point is collected 56, this Raman spectrum being collected by the central region 144 of the dark field microscope objective 14.

[0207] Advantageously, since no mechanical switching is required to switch from one modality to another, it is possible to make a new recording 50 of a new tile 40, whose geometric center 41 coincides with one of the points with coordinates 42 from the list of points L.

[0208] Here, in the example shown in [Fig. 5], the geometric center 41 of the new Tile 40 coincides with the point of coordinates 421. This point is indicated by a target in solid line, as long as the geometric center 41 of the first tile 40 is indicated by a cross in dotted lines.

[0209] To acquire a new tile 40, in a first variant of this second embodiment, if the spectral ranges of the laser source 16 and the white light source 12 are disjoint, and if the components of the white light spectrum have a wavelength shorter than the wavelength of the excitation laser beam 160, then the two illuminations are used simultaneously. It is assumed here that this is the case presented.

[0210] We then proceed simultaneously with an illumination 55 of a measurement point by the excitation laser beam 160, followed by the collection 36 of the emitted Raman spectrum, and with a recording 51 of the new dark-field microscopy image, the field of view of which is included in the illuminated area of ​​the sample 200, as described previously.

[0211] This recording 51 is followed by a tracking 52 of the microparticles 202, an extraction 53 of the coordinates of the points corresponding to microparticles 202. Here, no point corresponding to a microparticle 202 is detected.

[0212] According to a second variant, following the displacement 54, illumination 50, recording 51, tracking 52 and extraction 53 are carried out first according to the dark field imaging modality, then illumination 55 and collection 56 are carried out according to the Raman micro spectrometry modality.

[0213] Regardless of the order of use of the two modalities, the process is continued by moving the sample 54, in order to make the measurement point coincide with another of the points in the list of points L.

[0214] This is the point with coordinates 422, as shown in [Fig. 5]. This point with coordinates 422 then becomes the geometric center 41 of a new tile 40. Here, this is the third tile 40 shown.

[0215] The steps of the process are then repeated, via optional illumination 50, recording 51 of a new dark-field microscopy image which constitutes the third tile 40, identification 52, and extraction 53 of the coordinates of the points corresponding to any microparticles 202, these steps being carried out in parallel with the steps of illumination 55 of the measurement point by the excitation laser beam and collection 56 of the Raman spectrum. Here, in particular, the point with coordinates 423 and the point with coordinates 424 are detected and extracted from the third tile 40. These points with coordinates 423 and 424 are added to the list of points to be scanned.

[0216] A further movement 54 of the stage then places the measurement point on one of the points in the list of points, and the steps of the process described are thus repeated.

[0217] Mosaic 4 representative of both the structures and the chemical nature of Sample 200 is thus reconstructed on the fly. In other words, the list of points L is continuously updated during the implementation of the process, and not reset between each tile 40.

[0218] A fourth tile 40, corresponding to a fourth dark-field microscopy image, is acquired with the point at coordinates 423 as its geometric center 41. The points at coordinates 425 and 426 are extracted from this fourth image, and the displacement stage 11 moves the sample 200 so as to probe the point at coordinates 424 using the Raman microspectrometry modality. This point at coordinates 424 becomes the geometric center 41 of a fifth tile 40, for which the steps of the process are repeated. The point at coordinates 425 of the point list L is then scanned to collect both a Raman spectrum and a sixth dark-field microscopy image corresponding to a sixth tile 40. The point at coordinates 427 is then extracted from this sixth image.

[0219] The process results in obtaining a spectral image corresponding to a mosaic 4 representing the entire surface of the sample 200. This spectral image is shown in [Fig.6], that is to say, it corresponds to the same spectral image as obtained with the first embodiment described above.

[0220] The structures of the microparticles 202 are obtained using the dark-field microscopy modality, which highlights contrasts even on these highly transparent samples 200, while false colors are associated with a composition determined by Raman spectrum analysis.

[0221] In this second embodiment, the simultaneity of the two illuminations is exploited in order to use the necessary displacements for a probing of the points of interest by the Raman microspectroscopy modality, to also image the sample 200 by dark field microscopy.

[0222] A scanning order for the points with coordinates 42 in the list of points L is chosen, for example, so as to minimize the distances traveled by the stage during a movement. For example, it is advantageous to minimize the Euclidean distance between two points with coordinates 42 that are scanned successively. It is also possible to choose to minimize the total travel distance required to traverse all the points with coordinates 42.

[0223] This scanning order is thus required to be reorganized during the process, in particular if new points with coordinates 42 are extracted.

[0224] A suitable protocol can also be implemented in the event that there are no points with coordinates 42 in the list of points L that have not yet been scanned. For example, it is then decided to move the sample 200 by a sufficient amount in order to probe a new area of ​​the sample 200.

[0225] This second embodiment has the same advantages as the first embodiment of implementation described, in particular by eliminating prohibitive mechanical switching times when switching from dark field microscopy mode to Raman microspectroscopy mode. Variants

[0226] The present invention is in no way limited to the embodiments described and represented, but a person skilled in the art will be able to make any variation in accordance with the invention.

Claims

Demands

1. A method for spatial localization in an image and spectral analysis of microparticles (202) in a sample (200) by an optical microscopy system, the method comprising the following steps: A) illumination (30, 50) of an area of ​​the sample (200) using an annular illumination cone (124), the annular illumination cone (124) being derived from a beam of white light (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) of a first dark-field microscopy image on an image field of view included in the illuminated area of ​​the sample (200), the first dark-field image being collected by a central region (144) of the dark-field microscope objective (14), C) localization (32, 52) in the first dark-field image of microparticles (202), D) extraction (33,53) coordinates of points corresponding to microparticles (202) within the recorded dark-field image, so as to form a point list, E) displacement (34, 54) of the sample (200) so as to make an optical axis of the dark-field microscope objective (14) coincide with a point in the point list, F) illumination (35, 55) of a measurement point on the sample (200) using an excitation laser beam (16) transmitted through the annular mirror (13) and focused by the central region (144) of the dark-field microscope objective (14), the measurement point coinciding with the point in the point list, G) collection (36, 56) of 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).

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

3. A method according to any one of claims 1 to 2, wherein a field of dimensions greater than the field of view image is reconstructed by a mosaic (4) of dark field microscopy images, these dark field microscopy images corresponding to tiles (40) of the mosaic (4).

4. A method according to claim 3, wherein a positioning of each tile (40) of the mosaic (4) is predefined upstream of the method.

5. A method according to 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), before proceeding to a step of moving the sample (200) in order to center the field of view of the dark field microscope objective (14) on another tile (40) of the mosaic (4) and resuming the method from step A), in order to record another dark field microscopy image.

6. A method according to claim 5, wherein for each tile (40) of the mosaic (4), the list of points comprises a fixed number of points.

7. A method according to claim 3, wherein a positioning of at least one tile (40) of the mosaic (4) is established as a function of the coordinates of a point in the list of points.

8. A method according to claim 7, wherein after the displacement (54) in step E) to make the optical axis of the dark field microscope objective (14) coincide with a point in the point list, steps A), B), C), D), F), and G) are performed, a new dark field microscopy image, corresponding to a new tile (40) of the mosaic (4), and a Raman spectrum emitted from the measurement point, are recorded, these steps being followed by another displacement (54) of the sample (200) to make the optical axis of the dark field microscope objective (14) coincide with another point in the point list.

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

10. A system for spatial tracking in an image and spectral analysis of microparticles (1) 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 comprising an image processing unit (60), the optical microscope comprising a sample holder (20) mounted on a moving stage (11), the sample holder (20) being adapted to receive a sample (200), - the system being characterized in that it comprises: - an optical system comprising an annular mirror (13) and a dark-field microscope objective (14), the optical system being disposed between the laser source (16), the white light source (12) and the sample holder (20), the dark-field microscope objective (14) having a central region (144) and a peripheral region (142), - the annular mirror (13) is configured to reflect part of a 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 white light beam portion using its peripheral region (142) towards the sample holder (20) and to transmit the excitation laser beam (16) via its central region (144) towards the sample holder (20), - the dark-field microscope objective (14) is adapted to focus the portion of the light beam to illuminate a first area of ​​a sample (200) on the sample holder (20) using an annular illumination cone (124) and the dark-field microscope objective (14) is adapted to focus the excitation laser beam (160) on a measurement point within the first area, - the central region (144) of the dark-field microscope objective (14) is adapted to collect a dark-field microscopy image over an image field of view included in the first illuminated area, the dark-field microscopy image being recorded using the image sensor (15), - the image processing unit (60) is adapted to detect any microparticles (202) and to extract from the dark-field microscopy image the coordinates of the points associated with these microparticles (202), in order to establish a list of points, - the displacement stage (11) is configured to move the sample holder (200), in order to make the measurement point coincide with a point in the point list or in order to place the image field of view of the dark field microscope objective (14) on another area at least partially different from the first area; - the central region (144) of the dark-field microscope objective (14) is also adapted to collect a Raman spectrum generated from the measurement point, the Raman spectrum being recorded by a Raman spectrometer (19).