Confocal Raman microspectrometry apparatus and method with focusing system

Abstract

The present invention relates to a confocal Raman microspectrometry apparatus (100) comprising a laser source (1), a microscope objective (3) arranged to focus the excitation laser beam (10) towards a sample (5), and a focusing system on the sample. According to the invention, the focusing system comprises an optical beam splitter (11), an image detector (13), an astigmatic optical system (12), a processor comprising an image processing system (15), and a feedback device (16). The optical beam splitter (11) is adapted to extract a fraction (21) of the reflected beam, the astigmatic optical system (12) is adapted to project the fraction (21) of the reflected beam as a spot on the image detector (13), and the image processing system (15) is adapted to calculate a focusing error signal from the image of the spot. Figure 1

Description

Title of the invention: Apparatus and method for confocal Raman micro-spectrometry with focusing system Technical field of the invention

[0001] The present invention relates to the technical field of Raman micro-spectrometry methods and apparatus.

[0002] It relates more specifically to a system and a method of development for a confocal Raman micro-spectrometry device. State of the art

[0003] In a known manner, a Raman spectrometry device makes it possible to analyze the chemical composition of samples in a non-invasive manner. Combined with a conventional microscope, a Raman micro-spectrometry device is obtained, offering the possibility of chemically examining objects of submicron size by combining spatial and spectral resolution. The use of a confocal diaphragm placed between the optical microscope and the entrance of the Raman spectrometer makes it possible to allow only the light coming from the focal plane of the microscope objective to pass: a confocal Raman micro-spectrometry device is thus obtained.

[0004] There are many focusing (or autofocus) devices and methods used in microscopy devices.

[0005] Active autofocus methods rely on one or more auxiliary optical devices to assess the distance between the sample and the focal plane of the microscope objective. The most widespread active autofocus methods are based on the use of a phase mask or a polychromatic light source combined with a hyperchromatic optical system or on the use of a liquid lens with variable focal length.

[0006] Unlike active autofocus methods, passive autofocus methods do not require an auxiliary optical device. Passive autofocus methods are based on algorithms for analyzing images taken at different lens-sample distances. The DFF (Depth From Focus) and DFD (Depth From Defocus) methods are the most commonly used. Different algorithms have been proposed to model the focus function such as the Sobel gradient, the bandpass filter, the Laplacian energy or the wavelet transform. Passive autofocus methods allow very high accuracy to be obtained. However, given the large number of images required at different distances to construct the focus function, these methods are very energy-intensive, in terms of storage capacity and in terms of computing time, which limits their application. In addition, these methods are slow: The time required to determine and position the sample at the autofocus point is typically several seconds.

[0007] Furthermore, these methods generally require adaptations depending on the magnification of the microscope objective used. However, it is generally desirable to analyze a sample at different scales by changing the magnification of the objective, for example to go from an objective having a magnification of x5, to x10, x50 or x100. However, the working distance between the sample and the objective varies depending on the objective used. When the working distance is small, the excitation laser beam and the backscattered Raman beam are strongly divergent, which makes the adjustments even more complex.

[0008] For a sample with surface roughness, such as a rock or a drug in tablet form, the focus must be carried out as close as possible to the Raman micro-spectrometry measurement point and ideally on the measurement point.

[0009] One of the aims of the invention is to propose a focusing device which can be integrated into a confocal Raman micro-spectrometry device, which is fast, has excellent precision for focusing the microscope at the Raman measurement point and which does not disturb the detection of the Raman signal whose intensity is very low. Presentation of the invention

[0010] In order to overcome the aforementioned drawbacks of the state of the art, the present invention provides a confocal Raman micro-spectrometry apparatus comprising a laser source capable of emitting an excitation laser beam, a sample holder capable of receiving a sample to be analyzed, a microscope objective having an optical axis arranged to receive and focus the excitation laser beam in a focal plane in the direction of the sample, the microscope objective being adapted to collect a beam reflected by the sample and transmit the reflected beam through a confocal aperture to a Raman spectrometer, a displacement system adapted to modify a distance between the microscope objective and the sample holder along the optical axis and a focusing system on the sample.

[0011] According to the invention, the focusing system comprises an optical beam splitter arranged on a path of the reflected beam between the microscope objective and the confocal aperture, a pixel matrix image detector comprising at least NxM pixels, where N is an integer greater than 2 and M is an integer greater than 2, an astigmatic optical system arranged between the optical beam splitter and the image detector and a processor comprising an image processing system and a feedback device, the optical beam splitter being adapted to extract a fraction of the reflected beam, the astigmatic optical system being adapted to project the fraction of the reflected beam into a spot on the image detector, the image detector being configured to acquire an image of the spot and the image processing system being adapted to calculate a focusing error signal and the feedback device being adapted to deduce from the focusing error signal a value and a direction of the displacement to be applied to the displacement system along the optical axis to focus the excitation laser beam on the sample.

[0012] Such a focusing system makes it possible to quickly and precisely determine the focus on the sample, using the same excitation source as the Raman spectrometry measurement.

[0013] Other non-limiting and advantageous characteristics of the container according to the invention, taken individually or in all technically possible combinations, are the following:

[0014] - the error signal is compared to a calibration curve obtained by scanning the displacement at a series of distances along the optical axis and by calculating the focusing error signal for each position in the series;

[0015] - the image processing system is adapted to determine a cor ellipse corresponding to the spot in the image and the processor is adapted to deduce a measurement of the major axis VI of the ellipse, of the minor axis V2 of the ellipse and in which the focusing error signal is equal to 4 / jr.tan1(Vl / V2) - 1;

[0016] - the image processing system is based on component analysis main features of the spot image;

[0017] - the image processing system is based on contour detection in the image of the spot;

[0018] - the image processing system is configured to apply filtering to the image of the spot to generate a filtered image, determine a larger contour of the spot on the filtered image and determine the ellipse from the larger contour;

[0019] - the filtering applied to the image of the spot to generate a filtered image is a filtering Gaussian or morphological gradient filtering;

[0020] - the optical beam splitter comprises a blade with flat and parallel faces, a wedge-shaped flat-faced blade or splitter cube;

[0021] - the apparatus comprises a spatial mask arranged between the optical separator of beam and the astigmatic optical system, the mask being arranged and configured to block an internal reflection beam in the blade;

[0022] - the astigmatic optical system comprises a cylindrical lens or two lenses cylindrical having crossed optical axes, at least one concave or convex spherical mirror oriented with a non-zero angle of incidence, at least one toric mirror, at least one parabolic mirror, at least one simple lens pivoted on its optical axis, at least one lens off-center with respect to the optical axis, a plurality of cylindrical lenses lindric, a plurality of spherical and cylindrical lenses, at least one Powell lens, at least one prism, or a microlens array;

[0023] - the apparatus comprises a local probe near-field device having a tip, the near-field device being combined with the excitation laser beam focused on the tip, the apparatus being configured to detect a local interaction between the sample, the tip and the excitation laser beam.

[0024] The invention also relates to a confocal Raman micro-spectrometry method comprising the following steps:

[0025] - emission of an excitation laser beam,

[0026] - focusing of the excitation laser beam in a focal plane of a mi objective microscope towards a sample to be analyzed placed on a sample holder,

[0027] - collection, via the microscope objective, of a beam reflected by the sample and transmission of the reflected beam through a confocal aperture to a Raman spectrometer,

[0028] - focusing on the sample using a displacement system adapted for changing a distance between the microscope objective and the sample holder, the focusing step comprising the following steps:

[0029] - separation of the reflected beam by means of an optical beam splitter arranged between the microscope objective and the confocal aperture, to extract a fraction of the reflected beam and direct it towards an astigmatic optical system arranged between the optical beam splitter and a pixel matrix image detector, the image detector comprising at least NxM pixels, where N is an integer greater than 2 and M is an integer greater than 2,

[0030] - projection via the astigmatic optical system of the fraction of the beam reflected in a spot on the image detector,

[0031] - acquisition of an image of the spot on the image detector

[0032] - image processing to calculate a focusing error signal and determine mination of a focusing error signal comprising a value and a direction of a displacement to be applied to the displacement system to focus the excitation laser beam on the sample.

[0033] Of course, the various features, variants and embodiments of the invention may be combined with each other in various combinations to the extent that they are not incompatible or mutually exclusive. Detailed description of the invention

[0034] Furthermore, various other characteristics of the invention emerge from the appended description given with reference to the drawings which illustrate a non-limiting form of embodiment of the invention and where:

[0035] [Fig.l] is a schematic view of a confocal Raman micro-spectrometry apparatus with focusing system according to the present disclosure;

[0036] [Fig.2] is a perspective view of the formation of the image of a cross through an astigmatic lens;

[0037] [Fig.3] is an example of an image of the reflected beam spot on the image detector combined with the astigmatic optical system;

[0038] [Fig.4] is a theoretical curve illustrating the focusing error signal (FES) as a function of defocus along the optical axis;

[0039] [Fig.5] illustrates a method of principal component analysis of the spot image, with an example of spot contour detection (image A) and an enlarged view of the spot (image B);

[0040] [Fig.6] schematically illustrates different stages of a second method of spot image analysis;

[0041] [Fig.7]; illustrates an example of application of the method of [Fig.6] on a spot image (image C), after Gaussian filtering (image D), after sorting by adaptive threshold (image E), after determination of a largest contour (image F), approximation of the largest contour by an ellipse (image G), verification by a box surrounding the spot (image H);

[0042] [Fig.8] is an experimental curve of focusing error signal (FES) as a function of focusing error along the optical axis.

[0043] [Fig.l] schematically represents a confocal Raman micro-spectrometry apparatus 100. The confocal Raman micro-spectrometry apparatus 100 comprises a laser source 1, a microscope objective 3, a sample holder 4 capable of receiving a sample 5 to be analyzed, a confocal aperture 7 and a Raman spectrometer 8. The microscope objective 3 has an optical axis 6 arranged parallel to the Z axis of an orthonormal XYZ reference frame.

[0044] The apparatus 100 comprises a displacement system 9 arranged and configured to modify a distance between the microscope objective 3 and the sample holder 4 along the optical axis 6. For example, the displacement system 9 comprises a motorized stage adapted to move the sample holder 4 along the Z axis with a sub-micrometer spatial resolution. In an exemplary embodiment, the sample holder 4 is mounted on a multi-axis displacement stage, to move the sample 5 along the X, Y and Z axes in order to allow a Raman measurement of the sample sequentially at several points.

[0045] We will first briefly explain the operation of the confocal Raman micro-spectrometer. The laser source 1 emits an excitation laser beam 10. The microscope objective 3 focuses the excitation laser beam 10 in a focal plane 24 towards the sample 5. By reflection on the sample 5 at a point 18, obtains a reflected beam propagating in the opposite direction to the excitation beam 10. The microscope objective 3 collects the beam 20 reflected by the sample. As a non-limiting example, a filter 2 makes it possible to separate the excitation beam 10 and the reflected beam 20 so as to transmit the reflected beam 20 through a confocal aperture 7 to the Raman spectrometer 8.

[0046] According to a particular embodiment, the Raman micro-spectrometry apparatus further comprises a near-field device with a local probe having a tip. Indeed, tip-enhanced Raman spectroscopy (or TERS) makes it possible to amplify a Raman signal using an evanescent electromagnetic wave confined to the end of a metal tip (see patent document FR1653182). The near-field device is combined with the excitation laser beam focused on the tip, the apparatus being configured to detect a local interaction between the sample, the tip and the excitation laser beam. For example, an STM-TERS microscope (or Scanning Tunneling Microscope) combines a scanning tunneling microscope and a tip-enhanced Raman spectrometer. In another example, an AFM-Raman microscope combines an atomic force microscope (AFM) and a Raman spectrometer.There are different types of tips suitable for each type of near-field microscope or tip-enhanced spectroscopy device, and possibly for the application.

[0047] The apparatus 100 also comprises a system for focusing on the sample. The focusing system here comprises an optical beam splitter 11 arranged on a path of the reflected beam 20 between the microscope objective 3 and the confocal aperture 7, an image detector 13 with a pixel matrix, an astigmatic optical system 12 arranged between the optical beam splitter 11 and the image detector 13 and a processor comprising an image processing system 15 and a feedback device 16. The optical beam splitter 11 is integrated into the body of the microscope. Preferably, the astigmatic optical system 12 and the image detector 13 are mechanically connected to the body of the microscope by an opto-mechanical mount opaque to ambient light. A module comprising the astigmatic optical system 12 and the image detector 13 in their opto-mechanical mount can thus be easily installed on an existing confocal Raman microscope.The module comprises, for example, wired connections for electrically supplying the image detector 13 and for transmitting the images acquired by the image detector 13 to the processor for image processing.

[0048] In more detail, the optical beam splitter 11 comprises for example a blade arranged on the path of the reflected beam 20. The blade is inclined, for example by 45 degrees, relative to the optical axis of the reflected beam 20. The optical beam splitter 11 is arranged and configured to extract a fraction 21 of the reflected beam. The optical beam splitter 11 comprises, for example, a blade. In one exemplary embodiment, the blade has flat and parallel faces. However, such a blade is likely to generate parasitic interference. Alternatively, the optical beam splitter 11 comprises a prismatic blade with flat faces. For example, the angle between the two flat faces is between 0.5 degrees and 10 degrees. Optionally, the blade comprises an anti-reflection treatment on the face of the blade facing the laser source and possibly on both faces of the blade. The anti-reflection treatment is adapted so that the blade has a high transmission coefficient for the reflected beam and a low reflection coefficient to extract a small fraction of the reflected beam.

[0049] Advantageously, the focusing system comprises a spatial mask 17 arranged between the optical beam splitter 11 and the astigmatic optical system 12, the mask 17 being arranged and configured to block an internal reflection beam in the plate. In particular, when the optical beam splitter 11 comprises a plate with flat and parallel faces or a prismatic plate, the internal reflections of the beam reflected inside the plate are likely to disturb the image of the spot by generating parasitic interference. The light beam being a monochromatic laser beam, the beams coming from the plate by direct refraction and by internal reflection are coherent with each other, hence the formation of interference when the two beams are superimposed on the image detector.Advantageously, the mask 17 is configured and arranged at the output of the separating plate 11, so as to block a part of the beam having undergone internal reflection in the plate, while allowing the reflected beam to pass only on the face of the plate oriented towards the microscope objective.

[0050] The mask 17 makes it possible to eliminate parasitic interference in the image of the spot on the image detector 13. As a non-limiting example, the plate 11 is a glass plate having an average thickness of 5 mm with parallel faces and without anti-reflection treatment. The plate is inclined by 45 degrees with respect to the reflected beam 20. The plate advantageously has a reflection factor of, for example, 3% of the reflected beam 20. In this way, the optical beam splitter 11 only samples a small fraction of the reflected beam, while the majority of the reflected beam is transmitted through the plate to be directed towards the confocal aperture 7 and the Raman spectrometer 8.

[0051] Alternatively, the optical beam splitter 11 comprises a splitter cube.

[0052] In all the exemplary embodiments, the optical beam splitter 11 extracts a fraction 21 of the reflected beam to direct it towards the astigmatic optical system 12.

[0053] The astigmatic optical system 12 comprises, for example, a cylindrical lens. Alternatively, the astigmatic optical system 12 comprises two cylindrical lenses. arranged in series on the optical path and whose optical axes are crossed, that is to say oriented perpendicular to each other and perpendicular to the optical axis of the fraction 21 of the reflected beam. Alternatively or complementary, the astigmatic optical system 12 comprises at least one concave or convex spherical mirror oriented with a non-zero angle of incidence, at least one toric mirror, at least one parabolic mirror, at least one simple lens pivoted on its optical axis, at least one lens off-centered with respect to the optical axis, a plurality of cylindrical lenses, a plurality of spherical and cylindrical lenses, at least one Powell lens, at least one prism or even a matrix of microlenses. Advantageously, the astigmatic optical system 12 is placed at an optical distance of approximately 20 cm from the microscope objective.In one example, two plano-convex cylindrical lenses arranged in series, having a focal length of 50 mm, are used, placed at a distance of 20 cm from the microscope objective.

[0054] The astigmatic optical system 12 is arranged at a fixed distance from the image detector 13. The astigmatic optical system 12 is configured to project the fraction 21 of the reflected beam into a spot on the image detector 13. The image detector 13 is a pixel matrix and comprises at least NxM pixels, where N is an integer greater than 2 and M is an integer greater than 2. For example, the image detector 13 is a camera comprising 3088*2064 pixels. The image detector 13 comprises, for example, a Basler video camera placed 45 mm from the astigmatic optical system. The image detector comprises, for example, a CCD type camera allowing image acquisition at a frequency of 50 Hz, i.e. one image every 20 ms.

[0055] [Fig. 2] schematically illustrates the operation of the astigmatic optical system 12. For the explanation of [Fig. 2], it is assumed that the sample has a cross at point 18 illuminated by the excitation beam 10. The microscope objective 3 is not shown here. The astigmatic optical system has a different optical power along a tangential axis and along a sagittal axis. The astigmatic optical system forms the image of point 18. The image of one branch of the cross is sharp in a sagittal plane S while the image of the other branch of the cross is sharp in a tangential plane T. The sagittal plane and the tangential plane are at a distance D from each other along the longitudinal optical axis of the beam. Therefore, when the image detector 13 is in the tangential plane T, the image of the cross is sharp, for example on the horizontal branch and blurred on the vertical branch of the cross.On the contrary, when the image detector 13 is in the sagittal plane S, the image of the cross is sharp on the vertical branch and blurred on the horizontal branch of the cross. When the image detector 13 is halfway between the tangential plane T and the sagittal plane S, the image of the cross has the same sharpness on both branches.

[0056] As indicated above, the image detector 13 is arranged at a fixed distance from the astigmatic optical system 12. For example, the pixel matrix image detector 13 is arranged midway between the sagittal plane S and the tangential plane T which are optically conjugated with the focusing plane 24 of the microscope objective. Advantageously, the pixel rows of the image detector are parallel to the sagittal axis and the pixel columns are parallel to the tangential axis of the astigmatic optical system 12. The sample is illuminated with the excitation beam 10 via the microscope objective 3, so as to form a reflected beam 20 coming from a circular point 18. The image detector 13 is configured to acquire an image 14 of the spot formed by the fraction 21 of the beam reflected through the astigmatic optical system 12.When the point 18 of the sample is located in the focal plane 24 of the microscope objective 3, the image 14 of the spot on the image detector 13 is circular. On the contrary, when the point 18 of the sample is offset by a distance Z relative to the focal plane 24 of the microscope objective 3, the image 14 of the spot on the image detector 13 is generally elliptical in shape. The distance Z is also called the focusing error. The orientation of the ellipse varies according to the sign of the focusing error relative to the focal plane 24.

[0057] Unlike most autofocus systems, the same laser source 1 is used here for the confocal Raman measurement and for focusing. This configuration has several advantages. First, this configuration allows focusing to be done exactly at the confocal Raman microspectrometry measurement point. The use of the same laser source also avoids any disturbance of the Raman signal by another light source, unlike other autofocus systems. In addition, focusing can be done during the measurement or just before the measurement, with a very short time for focusing. This solution does not require a pre-scan unlike previous autofocus systems. In addition, determining the sign of the focusing error makes it possible to determine the direction and value of the feedback to be applied, which allows faster focusing, compared to other previous techniques.

[0058] [Fig.3] represents an example of image 14 of the spot acquired by the detector of image 13. The fraction 21 of the reflected beam which is projected onto the image detector 13 here forms a cloud 19 of light points represented here by gray points on a light background.

[0059] The image 14 is transmitted to the image processing system 15 of a processor.

[0060] According to the present disclosure, the analysis of the image 14 of the spot makes it possible to calculate the parameters of an ellipse approaching the shape of the spot in the detected image and to deduce therefrom a focusing error signal, denoted FES. We denote VI the major axis of the ellipse, V2 the minor axis of the ellipse and ALPHA the angle between the major axis of the ellipse and an X axis of the pixels of the image detector 13.

[0061] The focusing error signal depends on the ratio V1 / V2 according to the following formula: FES = 4 / ir.tan '(V1 / V2) - 1. The focusing error signal is between the value -1 and the value +1. For a perfect astigmatic optical system and a non-diffusing reflective sample, the image of point 18 is circular when the microscope is at the point of best focusing, which corresponds to a value of FES equal to 0.

[0062] [Fig. 4] illustrates a theoretical curve 23 of the focusing error signal FES as a function of the distance of the point 18 of the sample from the focal plane 24 of the microscope objective 3. The focusing error signal FES is located at point A, respectively at point B, of the curve 23 when the illuminated point 18 on the sample is in a plane optically conjugated with the sagittal plane, respectively the tangential plane, of the astigmatic optical system 12. Between points A and B, it is possible to determine the position of the sample relative to the point of best focusing (FES = 0) from the focusing error signal, by inverting the curve 23. It is thus possible to calculate the value and the direction of the displacement to be applied to the displacement system 9 along the optical axis 6 to focus the excitation laser beam 10 on the sample.Particularly advantageously, the focusing zone of length L which extends between points C1 and C2 corresponds to the axial displacement for which the FES variations are linear. L is calculated for a given magnification. Curve 23 can be obtained for a sample which has a reflective surface, such as for example a polished silicon plate, a polished metal plate, a glass plate.

[0063] For the astigmatic optical system 12 consisting of a first cylindrical lens with a focal length of 50 mm and a second cylindrical lens with a focal length of 50 mm, placed at the optical distance of 20 cm from the microscope objective xl00, the working area, denoted Az, along the Z axis between point A and point B on the curve 23 is approximately 8 pm. At the best focus position, optical calculation software, such as Zemax, makes it possible to evaluate the diameter of the laser spot at the best focus point (FES=0).

[0064] However, the same principle can be used with different objectives, the movement range being adapted according to the magnification of the microscope objective. Thus, with a x50 microscope objective, the working area Az along the Z axis is approximately 39 pm. With an x10 microscope objective, the working area Az along the Z axis is approximately 1000 pm. Finally, with a x5 microscope objective, the working area Az along the Z axis is approximately 3700 pm.

[0065] From the parameters of the ellipse, the processor makes it possible to calculate the focusing error signal FES. The focusing error signal is previously calibrated to obtain a curve 23 which depends, on the one hand, on the microscope objective and the astigmatic optical system, and, on the other hand, on the sample considered, in particular its surface roughness. From the calculation of the focusing error signal FES and the calibration curve 23, the processor calculates a feedback signal to be applied to the displacement system 9 so as to modify the distance between the microscope objective 3 and the sample holder 4 along the optical axis 6, to place the sample at the point of best focusing (FES = 0). The focusing of the excitation laser beam can thus be carried out in a single movement. The Raman measurement is then carried out immediately at point 18 which corresponds to the best focusing (FES = 0), without changing the optical system and without changing the light source (and without moving the XY table).Focusing is therefore very fast: it can be done in less than a second, for example in 200 ms or 300 ms.

[0066] We will now describe different image processing methods for determining the parameters of the ellipse.

[0067] In a first embodiment, a principal component analysis (or PCA) is used. PCA is a method that measures how each variable (here a pixel) is associated with the other variables (here, the other pixels of the image) using a covariance matrix, which includes the directions of propagation of the data using eigenvectors. The eigenvalues ​​are coefficients applied to the eigenvectors which give the vectors their length. The principal component analysis is here adapted to determine the parameters of the ellipse of the image 14 of the spot acquired by the image detector 13.

[0068] More precisely, an adaptive threshold based on the OTSU algorithm is used to eliminate pixels that are not bright enough in the image 14. A list of pixels with coordinates (X,Y) that have an intensity greater than or equal to the threshold, corresponding to the pixels of the light spot, is obtained. The parameters (X,Y) are here the coordinates in pixels of the image detector 13.

[0069] The principal components are the eigenvectors of the data covariance matrix. The covariance matrix is ​​a square matrix giving the covariance between X and Y after application of the adaptive threshold.

[0070] Let A be a linear transformation represented by a matrix A. If there exists a vector such that AX = 2 X then 2 is called the eigenvalue of A with the corresponding eigenvector X (right).

[0071] We calculate the eigenvectors and sort them by eigenvalues.

[0072] The two eigenvalues ​​thus determined are:

[0073] • VI the largest eigenvalue, corresponding to the major axis of the ellipse, and

[0074] • V2 the smallest eigenvalue, corresponding to the minor axis of the ellipse.

[0075] We then calculate the angle ALPHA of the major axis of the ellipse relative to the horizontal (on the image detector 13):

[0076] ALPHA = 90 - rad2deg(arctan(V1 / V2))

[0077] Where rad2deg represents the conversion from angle in radians to degrees.

[0078] We note A the covariance matrix of the pixels (list X,Y selected previously).

[0079] The V1 / V2 ratio obtained is the ratio of the major axis to the minor axis of the ellipse.

[0080] If the angle ALPHA is less than 90 degrees, the values ​​of VI and V2 are unchanged. If the angle ALPHA is greater than 90 degrees, then the values ​​of VI and V2 are interchanged.

[0081] [Fig.5] illustrates an example of processing image 14 of a spot by the PCA method. Image 5A) shows a rectangular box inside which the spot is inscribed and an ellipse with major axis VI, minor axis V2 and angle ALPHA determined by the PCA algorithm. The rectangular box surrounds the ellipse. In image 5B) of [Fig.5], the area of ​​the image of the spot acquired by the image detector is zoomed in, with the intensity of the acquired pixels corresponding to the box in which the spot is inscribed. In other words, image 5B corresponds to the raw image cropped to remove the areas in which all the pixels have an intensity lower than the threshold. All the brightest pixels, i.e. with an intensity greater than or equal to the threshold, are located inside the box.In image 5A), the visualization of this rectangular box and the ellipse determined by PCA superimposed on this box allow us to visually validate the calculation of the parameters of the ellipse.

[0082] Principal component analysis (PCA) makes it possible to obtain good results, particularly for samples which have a reflective surface, such as, for example, a polished silicon plate, a polished metal plate, a glass plate.

[0083] In a second embodiment, another image processing algorithm is used. [Fig. 6] schematically represents different steps of the method according to the second embodiment. The method comprises a first step 30 of acquiring an image 14 of the spot by the image detector 13. This first step is identical in all embodiments.

[0084] A filtering step 40 is then applied to the image 14 of the spot, for example Gaussian filtering so as to obtain a filtered image. This filtering makes it possible to standardize the parts of an image by buttressing them and therefore harmonizing the details of the latter.

[0085] Optionally, the filtering step 40 includes a step 45 of sorting by adaptive threshold. Step 45 includes, for example, the construction of a histogram of the intensity of the pixels of the filtered image, for example on a scale extending from 0 to 255. The histogram generally has two peaks: a first peak corresponds to the darkest pixels and a second peak corresponds to the brightest pixels. (i.e. the spot). Then, we keep only the 2nd peak by setting the intensity of the pixels of the 2nd peak to a maximum of 255 and the intensity of all the other pixels to 0. We thus obtain an image filtered by adaptive sorting.

[0086] In step 50, an image processing step is then applied to the filtered image or, respectively, to the image filtered by adaptive sorting, to determine a larger contour of the spot. For this purpose, all possible contours are calculated using, for example, the FindContours function of the OpenCV library, the contours thus obtained are sorted according to their surface area and the largest contour is kept according to the surface area.

[0087] In step 60, an ellipse approximating the largest contour is determined using the fitEllipse function of the OpenCV library. This algorithm is described in the publication Andrew W. Fitzgibbon, RBFisher. A Buyer's, Guide to Conic Fitting, Proc.5th British Machine Vision Conference, Birmingham, pp. 513-522, 1995. The parameters ALPHA, VI and V2 are deduced from this ellipse.

[0088] In step 70, the ratio V1 / V2 is calculated and the focusing error signal FES is deduced from it.

[0089] Optionally, the calculation of the parameters VI and V2 of the ellipse is validated by the method of the box which surrounds the spot so as to take into account all the pixels of the spot (described in connection with the first embodiment).

[0090] In step 75, the processor calculates a feedback signal to be applied to the movement system 9.

[0091] In step 80, the displacement system modifies the distance between the microscope objective 3 and the sample holder 4 along the optical axis 6, to place the sample at the point of best focusing (FES = 0).

[0092] At step 90, the sample is at the point of best focus (FES = 0) and a Raman spectrometry measurement is acquired at the point of focus.

[0093] If necessary, in step 95, the displacement system modifies the position in (X, Y) of the excitation laser beam on the sample. The focusing process is repeated at the new measuring point.

[0094] [Fig.7] shows an example of the main steps of processing a spot image according to the second embodiment. Image 7 A) shows a spot image 14 acquired by the image detector 13. Image 7 B) shows the filtered image obtained after applying Gaussian filtering (step 40) to image 6 A). Image 7 C) shows the image obtained after adaptive sorting (step 45) applied to the filtered image 7 B). Image 7 D) shows the largest contour obtained after step 50 of calculating the largest contour to the filtered image (and possibly sorted by adaptive sorting). Image 7 E) shows an ellipse determined in step 60 to approximate the largest contour. Image 7 F) compares the ellipse thus obtained with the rectangular box surrounding the spot.

[0095] The image processing method according to the second embodiment gives better results than the first embodiment on reflective samples. However, the contours may be difficult to determine at the ends of the operating range (points A and B on curve 23 of [Fig.4]).

[0096] According to a variant of the second embodiment, the adaptive threshold sorting step 45 is replaced by a morphological gradient step 46. A morphological gradient is the difference between a dilation and an erosion.

[0097] Dilation consists of convolving an image A with a kernel (B), which can have any shape or size, usually a square. Kernel B has a defined anchor point, usually the center of the kernel.

[0098] When kernel B is scanned over the unfiltered image, we calculate the maximum pixel value covered by kernel B and replace the image pixel at the anchor point position with this maximum value. The dilation operation has the effect of causing the bright regions of an image to grow.

[0099] The erosion operation is the inverse of dilation. The erosion operation is based on the calculation of a local minimum on the surface of a given core.

[0100] When kernel B is scanned over the dilated image, we calculate the minimum pixel value covered by B and replace the image pixel under the anchor point with this minimum value. A so-called morphological gradient image is obtained by calculating the difference between the pixel values ​​of the dilated image and the pixel values ​​of the eroded image.

[0101] The following steps are analogous.

[0102] This variant of the second embodiment gives very good results, in particular on reflective samples but also on rough or diffusing samples, such as pharmaceutical tablets, rocks.

[0103] The processing according to the present disclosure makes it possible to obtain focusing with excellent precision, less than the depth of field of the objective. [Fig. 8] represents an experimental curve of focusing error signal (FES) as a function of the focusing error along the optical axis using an xl00 objective. For example, with the xl00 objective, having a depth of field of 190 nm, a focusing precision along the Z axis of the order of 40 nm is obtained. In addition, the focusing is very fast. The result of the focusing error signal calculation is obtained with a value and a direction of the displacement to be applied to the displacement system in a single step. Depending on the type of displacement system used, the value of the displacement to be carried out and the accessible displacement speed, it is possible to carry out the focusing in less than one second, for example in 200 ms to 500 ms.The system and method for developing the present disclosure are at . both very precise and very fast. The focusing system and method give good results even for high microscope objective magnifications (e.g. x50 or x100) in which the reflected beam is nevertheless strongly divergent.

Claims

Claims

1. Confocal Raman micro-spectrometry apparatus (100) comprising a laser source (1) capable of emitting an excitation laser beam (10), a sample holder (4) capable of receiving a sample (5) to be analyzed, a microscope objective (3) having an optical axis (6) arranged to receive and focus the excitation laser beam (10) in a focal plane (24) towards the sample (5), the microscope objective (3) being adapted to collect a reflected beam (20) by the sample and transmit the reflected beam through a confocal aperture (7) to a Raman spectrometer (8), a displacement system (9) adapted to modify a distance between the microscope objective (3) and the sample holder (4) along the optical axis (6) and a focusing system on the sample,characterized in that: the focusing system comprises an optical beam splitter (11) arranged on a path of the reflected beam (20) between the microscope objective (3) and the confocal aperture (7), an image detector (13) with a pixel matrix comprising at least NxM pixels, where N is an integer greater than 2 and M is an integer greater than 2, an astigmatic optical system (12) arranged between the optical beam splitter (11) and the image detector (13) and a processor comprising an image processing system (15) and a feedback device (16), the optical beam splitter (11) being adapted to extract a fraction (21) of the reflected beam, the astigmatic optical system (12) being adapted to project the fraction (21) of the reflected beam into a spot on the image detector (13),the image detector (13) being configured to acquire an image (14) of the spot and the image processing system (15) being adapted to calculate a focusing error signal and the feedback device (16) being adapted to deduce from the focusing error signal a value and a direction of the displacement to be applied to the displacement system (9) along the optical axis (6) to focus the excitation laser beam (10) on the sample (5).,

2. Apparatus according to claim 1, wherein the error signal is compared to a calibration curve (23) obtained by scanning the displacement at a series of distances along the optical axis and calculating the focusing error signal for each position in the series.

3. Apparatus according to claim 1 or 2, wherein the image processing system (15) is adapted to determine an ellipse corresponding to the spot in the image (14) and the processor is adapted to deduce therefrom a measurement of the major axis VI of the ellipse, of the minor axis V2 of the ellipse and wherein the focusing error signal is equal to 4 / ir.tan '(V1 / V2) - 1.

4. Apparatus according to claim 3, wherein the image processing system (15) is based on a principal component analysis of the image (14) of the spot.

5. Apparatus according to claim 3, wherein the image processing system (15) is based on edge detection in the image (14) of the spot.

6. Apparatus according to claim 3, wherein the image processing system (15) is configured to apply filtering (40) to the image (14) of the spot to generate a filtered image, determine a larger contour (50) of the spot on the filtered image and determine the ellipse (60) from the larger contour.

7. Apparatus according to claim 6, wherein the filtering (40) applied to the spot image to generate a filtered image is Gaussian filtering or morphological gradient filtering.

8. Apparatus according to one of claims 1 to 7, wherein the optical beam splitter (11) comprises a plate with parallel planar faces, a wedge-shaped plate with planar faces or a splitter cube.

9. Apparatus according to claim 8 comprising a spatial mask (17) disposed between the optical beam splitter (11) and the astigmatic optical system (12), the mask being arranged and configured to block an internal reflection beam in the plate.

10. Apparatus according to one of claims 1 to 9, wherein the astigmatic optical system (12) comprises a cylindrical lens or two cylindrical lenses having crossed optical axes, at least one concave or convex spherical mirror oriented with a non-zero angle of incidence, at least one toric mirror, at least one parabolic mirror, at least one simple lens pivoted on its optical axis, at least one lens off-center with respect to the optical axis, a plurality of cylindrical lenses, a plurality of spherical and cylindrical lenses, at least one Powell lens, at least one prism, or a microlens array.

11. Apparatus according to one of claims 1 to 10, comprising a device near-field device with local probe having a tip, the near-field device being combined with the excitation laser beam focused on the tip, the apparatus being configured to detect a local interaction between the sample, the tip and the excitation laser beam.

12. A method of confocal Raman micro-spectrometry (100) comprising the following steps: - emission of an excitation laser beam (10), - focusing the excitation laser beam (10) in a focal plane (24) of a microscope objective (3) towards a sample (5) to be analyzed placed on a sample holder (4), - collection, via the microscope objective (3), of a beam reflected (20) by the sample and transmission of the reflected beam through a confocal aperture (7) to a Raman spectrometer (8), - focusing on the sample using a displacement system (9) adapted to modify a distance between the microscope objective (3) and the sample holder (4), the focusing step comprising the following steps: - separation of the reflected beam by means of an optical beam splitter (11) arranged between the microscope objective (3) and the confocal aperture, to extract a fraction (21) of the reflected beam and direct it towards an astigmatic optical system (12) arranged between the optical beam splitter (11) and an image detector (13) with a pixel matrix, the image detector (13) comprising at least NxM pixels, where N is an integer greater than 2 and M is an integer greater than 2, - projection via the astigmatic optical system (12) of the fraction (21) of the reflected beam into a spot on the image detector (13), - acquiring an image (14) of the spot on the image detector (13) - processing the image (14) to calculate a focusing error signal and determining a focusing error signal comprising a value and a direction of a displacement to be applied to the displacement system (9) to focus the excitation laser beam (10) on the sample (5).

Images