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 microspectrometry 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] As is known, a Raman spectrometer allows for the non-invasive analysis of the chemical composition of samples. Combined with a conventional microscope, this results in a Raman microspectrometer that enables the chemical examination of submicron-sized objects, combining spatial and spectral resolution. The use of a confocal diaphragm positioned between the optical microscope and the Raman spectrometer's inlet allows only light from the focal plane of the microscope objective to pass through: this results in a confocal Raman microspectrometer.

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

[0005] Active autofocus methods rely on one or more auxiliary optical devices to evaluate 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 variable focal length liquid lens.

[0006] Unlike active autofocus methods, passive autofocus methods do not require an auxiliary optical device. Passive autofocus methods rely 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. Various 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 for very high accuracy. However, given the large number of images required at different distances to construct the focus function, these methods are very energy-intensive, require significant storage capacity, and demand substantial computation time, which limits their application. Furthermore, these methods are slow: The time required to determine and position the sample at the autofocus point is generally several seconds.

[0007] Furthermore, these methods generally require adjustments depending on the magnification of the microscope objective used. It is generally desirable to analyze a sample at different scales by changing the objective's magnification, for example, from an objective with 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 short, the excitation laser beam and the backscattered Raman beam are highly divergent, making the adjustments even more complex.

[0008] For a sample exhibiting surface roughness, such as a rock or a drug in tablet form, the focus should be made 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 provide a focusing device that can be integrated onto a confocal Raman microspectrometry instrument, which is fast, offers excellent precision for focusing the microscope at the Raman measurement point, and does not interfere with the detection of the Raman signal, the intensity of which is very low. Presentation of the invention

[0010] In order to remedy the aforementioned drawbacks of the prior art, the present invention proposes a confocal Raman micro-spectrometry apparatus comprising a laser source capable of emitting an excitation laser beam, a sample holder suitable for 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 disposed along 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 disposed 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 direction of 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 determine the focus on the sample quickly and precisely, using the same excitation source as the Raman spectrometry measurement.

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

[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 an 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 / ir.tan '(V1 / V2) - 1;

[0016] - the image processing system is based on component analysis main elements of the image in the commercial;

[0017] - the image processing system is based on edge detection in the image from the spot;

[0018] - the image processing system is configured to apply filtering to the image from 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 blade with flat faces or a separator cube;

[0021] - the device includes a spatial mask disposed 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 one cylindrical lens or two lenses 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 decentered 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;

[0023] - the device 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 device 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 the excitation laser beam into a focal plane of an objective microscope pointed 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 suitable displacement system for modifying the 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 to 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 reflected beam 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 determination of a focusing error signal including a value and 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 can be combined with one another in various ways, provided they are not incompatible or mutually exclusive. Detailed description of the invention

[0034] In addition, various other features of the invention become apparent from the attached description made with reference to the drawings which illustrate one, non-limiting, embodiment of the invention and where:

[0035] [Fig-1] is a schematic view of a Raman micro-spectrometry device confocal with focusing system as described in this 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 spot of the beam reflected 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 defocusing along the optical axis;

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

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

[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 greatest contour (image F), approximation of the greatest contour by an ellipse (image G), verification by a box surrounding the spot (image H);

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

[0043] Fig. 1 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 suitable for 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 frame.

[0044] The apparatus 100 includes a displacement system 9 arranged and configured to modify the distance between the microscope objective 3 and the sample holder 4 along the optical axis 6. For example, the displacement system 9 includes a motorized stage adapted to move the sample holder 4 along the Z-axis with sub-micrometer spatial resolution. In one 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 sequential Raman measurement of the sample at several points.

[0045] We will first briefly explain the operation of the confocal Raman microspectrometer. The laser source 1 emits an excitation laser beam 10. The microscope objective 3 focuses the excitation laser beam 10 into a focal plane 24 in the direction of the sample 5. By reflection from the sample 5 at a point 18, a reflected beam is obtained propagating in the opposite direction to the excitation beam 10. The microscope objective 3 collects the beam reflected 20 by the sample. By way of non-limiting example, a filter 2 separates 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 microspectrometry apparatus further comprises a local probe near-field device having a tip. Indeed, tip-enhanced Raman spectroscopy (or TERS) amplifies a Raman signal using an evanescent electromagnetic wave confined to the tip of a metallic point (see patent document FR1653182). The near-field device is combined with the excitation laser beam focused on the point, the apparatus being configured to detect a local interaction between the sample, the point, and the excitation laser beam. As an example, an STM-TERS (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 adapted to each type of near-field microscope or tip-enhanced spectroscopy device, and possibly according to the application.

[0047] The apparatus 100 also includes a focusing system for the sample. The focusing system comprises an optical beam splitter 11 arranged in a path of the reflected beam 20 between the microscope objective 3 and the confocal aperture 7, a pixel array image detector 13, 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 microscope body. Preferably, the astigmatic optical system 12 and the image detector 13 are mechanically connected to the microscope body 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 includes, for example, wired connections to power the image detector 13 and to transmit 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 disposed in the path of the reflected beam 20. The blade is inclined, for example, at 45 degrees, with respect 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 embodiment, the blade has flat, parallel faces. However, such a blade is susceptible to generating unwanted 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 has an anti-reflective coating on the face of the blade oriented towards the laser source and possibly on both faces of the blade.The anti-reflective coating 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 includes a spatial mask 17 disposed 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 within the blade. In particular, when the optical beam splitter 11 has a parallel-sided or prismatic blade, internal reflections of the beam reflected inside the blade are likely to disrupt the image of the spot by generating spurious interference. Since the light beam is a monochromatic laser beam, the beams emerging from the blade by direct refraction and by internal reflection are coherent with each other, hence the formation of interference when the two beams overlap on the image detector.Advantageously, the mask 17 is configured and positioned at the output of the beam splitter 11, so as to block part of the beam that has undergone internal reflection in the blade, while allowing the beam reflected only on the face of the blade oriented towards the microscope objective to pass through.

[0050] The mask 17 allows for the suppression of parasitic interference in the image of the spot on the image detector 13. By way of non-limiting example, the plate 11 is a glass plate with an average thickness of 5 mm, parallel faces, and no anti-reflective coating. The plate is inclined at 45 degrees with respect to the reflected beam 20. The plate advantageously has a reflection factor, for example, of 3% of the reflected beam 20. In this way, the optical beam splitter 11 takes only 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 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 along the optical path and whose optical axes are crossed, i.e., oriented perpendicular to each other and perpendicular to the optical axis of the fraction 21 of the reflected beam. Alternatively or complementaryly, 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 rotated about its optical axis, at least one lens decentered 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 microlens array.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 positioned at a fixed distance from the image detector 13. The astigmatic optical system 12 is configured to project a fraction 21 of the reflected beam as a spot onto the image detector 13. The image detector 13 is a pixel matrix and comprises at least N x M 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 x 2064 pixels. The image detector 13 comprises, for example, a Basler video camera positioned 45 mm from the astigmatic optical system. The image detector comprises, for example, a CCD-type camera that allows image acquisition at a frequency of 50 Hz, i.e., one image every 20 ms.

[0055] Figure 2 schematically illustrates the operation of the astigmatic optical system 12. For the purposes of explaining Figure 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 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 arm of the cross is sharp in a sagittal plane S, while the image of the other arm 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 arm and blurry on the vertical arm. of the cross. Conversely, when the image detector 13 is in the sagittal plane S, the image of the cross is sharp on the vertical arm and blurry on the horizontal arm. When the image detector 13 is midway between the tangential plane T and the sagittal plane S, the image of the cross is equally sharp on both arms.

[0056] As mentioned above, the image detector 13 is positioned at a fixed distance from the astigmatic optical system 12. For example, the pixel array image detector 13 is positioned midway between the sagittal plane S and the tangential plane T, which are optically conjugate with the focal 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 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 sample point 18 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. Conversely, when the sample point 18 is offset by a distance Z from the focal plane 24 of the microscope objective 3, the image 14 of the spot on the image detector 13 is generally elliptical. The distance Z is also called the focusing error. The orientation of the ellipse varies according to the sign of the focusing error with respect to the focal plane 24.

[0057] Unlike most autofocus systems, the same laser source 1 is used here for both confocal Raman measurement and focusing. This configuration offers several advantages. First, it allows focusing to be performed precisely at the point of measurement for confocal Raman microspectrometry. Using the same laser source also avoids any interference with the Raman signal from another light source, unlike other autofocus systems. Furthermore, focusing can be performed during or just before measurement, with a very short focusing time. This solution does not require pre-scanning, unlike previous autofocus systems. In addition, determining the sign of the focusing error allows the direction and value of the feedback to be applied to be determined, resulting in faster focusing compared to other previous techniques.

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

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

[0060] According to this disclosure, analysis of the image 14 of the spot makes it possible to calculate the parameters of an ellipse approximating the shape of the spot in the detected image and to deduce a focusing error signal, denoted FES. VI denotes 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 focus, which corresponds to a value of FES equal to 0.

[0062] Figure 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 conjugate 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 focus (FES = 0) from the focusing error signal, by inverting the curve 23. It is thus possible to calculate the value and 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.Most advantageously, the focal zone of length L extending 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 with a reflective surface, such as a polished silicon plate, a polished metal plate, or 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 an optical distance of 20 cm from the microscope objective xlOO, the working area, denoted Az, along the Z-axis between point A and point B on curve 23 is approximately 8 pm. At the best focus position, optical calculation software, such as Zemax, allows the diameter of the laser spot at the best focus point (FES=0) to be evaluated.

[0064] However, the same principle can be used with different objectives, the range of movement being adapted according to the magnification of the microscope objective. Thus, with a 50x microscope objective, the working area Az along the Z-axis is approximately 39 pm. With an xl0 microscope objective, the working area Az along The Z-axis is approximately 1000 pm. Finally, with a 5x microscope objective, the working area Az along the Z-axis is approximately 3700 pm.

[0065] Based on the ellipse parameters, the processor calculates the focusing error signal FES. The focusing error signal is pre-calibrated to obtain a curve 23 that 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 movement system 9 so as to modify the distance between the microscope objective 3 and the sample holder 4 along the optical axis 6, in order to place the sample at the point of best focus (FES = 0). The focusing of the excitation laser beam can thus be achieved in a single movement.The Raman measurement is then performed immediately at point 18, which corresponds to the best focus (FES = 0), without changing the optical system or the light source (and without moving the XY table). Focusing is thus very fast: it can be achieved 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, principal component analysis (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 data propagation by means of eigenvectors. The eigenvalues ​​are coefficients applied to the eigenvectors that give the vectors their length. Principal component analysis is adapted here to determine the parameters of the ellipse of the image 14 of the spot acquired by the image detector 13.

[0068] More specifically, an adaptive threshold based on the OTSU algorithm is used to eliminate pixels that are not bright enough in image 14. This yields 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. 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 (right) eigenvector X.

[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 with respect to the horizontal (on the image detector 13):

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

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

[0078] We denote 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 angle ALPHA is less than 90 degrees, the values ​​of VI and V2 are unchanged. If angle ALPHA is greater than 90 degrees, then the values ​​of VI and V2 are interchanged.

[0081] Figure 5 illustrates an example of image processing of a spot using the PCA method. Figure 5A shows a rectangular box containing the spot and an ellipse with major axis VI, minor axis V2, and angle ALPHA determined by the PCA algorithm. The rectangular box surrounds the ellipse. Figure 5B shows a zoomed-in view of the area of ​​the spot image acquired by the image detector, with the intensity of the acquired pixels corresponding to the box containing the spot. In other words, Figure 5B is the raw image cropped to remove areas where all pixels have an intensity below the threshold. All the brightest pixels, i.e., those 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 allows for visual validation of the calculation of the ellipse's parameters.

[0082] Principal component analysis (PCA) gives good results, particularly for samples with a reflective surface, such as a polished silicon plate, a polished metal plate, or a glass plate.

[0083] In a second embodiment, a different image processing algorithm is used. Figure 6 schematically represents different steps of the method according to the second embodiment. The method includes 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, for example Gaussian filtering, is then applied to the image 14 of the spot in order to obtain a filtered image. This filtering makes it possible to uniformize the parts of an image by blurring them and thus harmonizing its details.

[0085] Optionally, the filtering step 40 includes an adaptive threshold sorting step 45. Step 45 includes, for example, the construction of a histogram of the intensity of the pixels in the filtered image, for example on a scale ranging 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, only the second peak is retained, with the intensity of the pixels in the second peak set to a maximum of 255 and the intensity of all other pixels to 0. This results in 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 area, and the largest contour is retained.

[0087] In step 60, an ellipse approximating the largest contour is determined using the fitEllipse function from 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.

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

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

[0091] At 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 focus (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 changes the (X, Y) position of the excitation laser beam on the sample. The focusing procedure is repeated at the new measurement point.

[0094] Figure 7 shows an example of the main steps in processing a spot image according to the second embodiment. Figure 7A shows a spot image 14 acquired by the image detector 13. Figure 7B shows the filtered image obtained after applying Gaussian filtering (step 40) to Figure 6A. Figure 7C shows the image obtained after adaptive sorting (step 45) applied to the filtered image 7B. Figure 7D shows the largest contour obtained after step 50, which calculates the largest contour in the filtered (and possibly sorted by adaptive sorting) image. Figure 7E shows an ellipse determined in step 60 to approximate the largest contour. Figure 7F 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 can be difficult to determine at the extremes of the operating range (points A and B on curve 23 of [Fig. 4]).

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

[0097] The dilation consists of convolving an image A with a kernel (B), which can have any shape or size, generally a square. The kernel B has a defined anchor point, generally 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. We obtain a so-called morphological gradient image 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 this disclosure makes it possible to obtain focusing with excellent precision, less than the depth of field of the lens. Figure 8 shows an experimental focusing error signal (FES) curve. depending on the focusing error along the optical axis using an xlOO lens. For example, with the xlOO lens, having a depth of field of 190 nm, a focusing accuracy along the Z-axis of approximately 40 nm is achieved. Furthermore, focusing is very fast. The result of the focusing error signal calculation, along with the value and direction of the displacement to be applied to the movement system, is obtained in a single step. Depending on the type of movement system used, the required displacement value, and the achievable movement speed, focusing can be accomplished in less than one second, for example, in 200 ms to 500 ms. The focusing system and method described in this disclosure are both very precise and very fast.The focusing system and method gives good results even for high magnifications of microscope objective (e.g. x50 or xl00) in which the reflected beam is nevertheless strongly divergent.

Claims

1. Demands Confocal Raman microspectrometry apparatus (100) comprising a laser source (1) adapted to emit an excitation laser beam (10), a sample holder (4) adapted to receive 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) into a focal plane (24) in the direction of the sample (5), the microscope objective (3) being adapted to collect a beam reflected (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 includes an optical beam splitter (11) disposed 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) disposed 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 as 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 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), 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 a measurement of the major axis, VI of the ellipse, of the small axis V2 of the ellipse and in which the focusing error signal is equal to 4 / ir.tan '(V1 / V2) - 1.

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 by calculating the focusing error signal for each position in the series.

3. Device according to claim 1, wherein the image processing system (15) is based on a principal component analysis of the image (14) of the spot.

4. Device according to claim 1, wherein the image processing system (15) is based on edge detection in the image (14) of the spot.

5. Apparatus according to claim 1, wherein the image processing system (15) is configured to apply a 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.

6. Apparatus according to claim 5, wherein the filtering (40) applied to the image of the spot to generate a filtered image is a Gaussian filter or a morphological gradient filter.

7. Apparatus according to any one of claims 1 to 6, wherein the optical beam splitter (11) comprises a parallel-sided flat-faced blade, a wedge-shaped flat-faced blade, or a splitter cube.

8. Apparatus according to claim 7 comprising a spatial mask (17) disposed between the optical beam splitter (11) and the astigmatic optical system (12), the mask being disposed and configured to block an internal reflection beam in the blade.

9. Apparatus according to any one of claims 1 to 8, wherein the astigmatic optical system (12) comprises one 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 about its optical axis, at least one lens decentered with respect to the optical axis, a plurality of cylindrical lenses, a plurality of lenses spherical and cylindrical, at least one Powell lens, at least one prism, or a microlens array.

10. Apparatus according to any one of claims 1 to 9, comprising 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.

11. A method for confocal Raman micro-spectrometry (100) comprising the following steps: - emission of an excitation laser beam (10), - focusing of the excitation laser beam (10) into a focal plane (24) of a microscope objective (3) towards a sample (5) to be analyzed arranged 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 change the 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) disposed between the microscope objective (3) and the confocal aperture,to extract a fraction (21) of the reflected beam and direct it to an astigmatic optical system (12) disposed between the optical beam splitter (11) and a pixel-matrix image detector (13), 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), - acquisition of an image (14) of the spot on the image detector (13) - image processing (14) to calculate a focusing error signal and determination of a focusing error signal comprising a value and direction of a displacement to be applied to the displacement system (9) to focus the excitation laser beam (10) on the sample (5), the image processing (14) being adapted to determine an ellipse corresponding to the, spot in image (14) and to deduce a measurement of the major axis VI of the ellipse, of the minor axis V2 of the ellipse and the focusing error signal being equal to 4 / ir.tan '(V1 / V2) - 1.

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