Confocal raman microspectrometry apparatus with focusing system and method therefor

Abstract

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

Description

Technical Field

[0001] This invention relates to the technical field of Raman microspectroscopy methods and equipment.

[0002] More specifically, the present invention relates to a focusing system and method for a confocal Raman microspectroscopy device. Background Technology

[0003] As is well known, Raman spectroscopy enables non-invasive analysis of the chemical composition of samples. When combined with a conventional microscope, it forms a Raman microspectroscopy, allowing for chemical detection of objects at sub-micron dimensions with spatial and spectral resolution. A confocal aperture, positioned between the optical microscope and the Raman spectrometer entrance, allows only light from the focal plane of the microscope objective to pass through: this results in a confocal Raman microspectroscopy.

[0004] In microscopic equipment, there are many focusing (or autofocusing) devices and methods.

[0005] Active autofocus methods rely on one or more auxiliary optical devices to determine the distance between the sample and the focal plane of the microscope objective. The most common active autofocus methods are based on the use of phase masks, multicolor light sources combined with superdispersive optics, or variable focal length liquid lenses.

[0006] Unlike active autofocus methods, passive autofocus methods do not require auxiliary optics. Passive autofocus methods rely on algorithms that analyze images captured at different lens-sample distances. Depth-of-focus (DFF) and Depth-of-defocus (DFD) methods are the most commonly used. Various algorithms have been proposed to model the focus function, such as Sobel gradient, bandpass filter, Laplacian energy, and wavelet transform. Passive autofocus methods can achieve very high accuracy. However, these methods are very energy-intensive, requiring large amounts of storage and computation time due to the need to acquire a large number of images at different distances to construct the focus function, which limits their application. Furthermore, these methods are slow: the time required to determine and position the sample at the autofocus point is typically several seconds.

[0007] Document JP2010127726 describes a Raman microspectroscopy device and method, which includes a focus detection unit based on the intensity measurement of a light beam reflected from a sample.

[0008] Furthermore, these methods typically require adjustment based on the magnification of the microscope objective used. However, it is generally desirable to analyze samples at different scales by changing the magnification of the objective (e.g., changing from a 5x (5x) objective to a 10x (10x), 50x (50x), or 100x (100x) objective). However, the working distance between the sample and the objective varies depending on the objective used. When the working distance is short, the divergence of the excitation laser beam and the backscattered Raman beam is high, making adjustment more complex.

[0009] For samples exhibiting surface roughness, such as rocks or tablets containing drugs, focusing should be performed as close as possible to the Raman microspectroscopy measurement point, ideally at the measurement point itself.

[0010] One objective of this invention is to provide a focusing device that can be integrated into a confocal Raman microspectroscopy device. This focusing device is fast, has excellent accuracy in focusing the microscope onto the Raman measurement point, and does not interfere with the detection of Raman signals with very low intensity. Summary of the Invention

[0011] To remedy the shortcomings of the prior art, the present invention proposes a confocal Raman microspectroscopy device, comprising: a laser source capable of emitting an excitation laser beam; a sample holder adapted to hold a sample to be analyzed; a microscope objective having an optical axis, configured to receive the excitation laser beam and focus it onto a focal plane in the direction of the sample, the microscope objective being adapted to collect the beam reflected by the sample and transmit the reflected beam to a Raman spectrometer via a confocal aperture; a displacement system adapted to change the distance between the microscope objective and the sample stage along the optical axis; and a system for focusing the sample.

[0012] According to the present invention, the focusing system comprises: an optical beam splitter disposed on the path of the reflected beam between a microscope objective and a confocal aperture; an image detector having a pixel matrix comprising at least N × M pixels, wherein 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, wherein the optical beam splitter is adapted to extract a portion of the reflected beam, the astigmatic optical system is adapted to project a portion of the reflected beam onto a spot on the image detector, the image detector is configured to acquire an image of the spot, the image processing system is adapted to calculate a focusing error signal, and the feedback device is adapted to derive from the focusing error signal a displacement value and displacement direction to be applied to a displacement system along the optical axis to focus the excitation laser beam onto the sample.

[0013] Such a focusing system allows for the rapid and accurate determination of the focal point on the sample using the same excitation source as Raman spectroscopy measurements.

[0014] Other non-limiting and advantageous features of the container according to the invention, individually or in all technically possible combinations, include the following: - The error signal is compared with a calibration curve, which is obtained by scanning displacement along the optical axis at a series of distances and calculating the focus error signal for each position in the series; - The image processing system is adapted to determine the ellipse corresponding to the light spot in an image, and the processor is adapted to derive the measured values ​​of the major axis V1 and the minor axis V2 of the ellipse, wherein the focusing error signal is equal to 4 / π.tan -1 (V1 / V2) – 1; - The image processing system is based on principal component analysis of spot images; - Image processing systems are based on edge detection in spot images; - The image processing system is configured to apply filtering to the spot image to generate a filtered image, determine the larger contour of the spot on the filtered image, and determine an ellipse from the larger contour; - The filtering applied to the spot image to generate the filtered image is Gaussian filtering or morphological gradient filtering; - Optical beam splitters include planar parallel plates, wedge-shaped planar side plates, or beam-splitting cubes; - The device includes a spatial mask disposed between the optical beam splitter and the astigmatic optical system, the mask being set and configured for internal reflection of the beam within the blocking plate; - An astigmatic optical system includes a cylindrical lens or a cylindrical lens with two intersecting optical axes, at least one concave or convex spherical mirror oriented at a non-zero incident angle, at least one toric mirror, at least one parabolic mirror, at least one simple lens pivoting about its optical axis, at least one lens eccentric relative to the optical axis, multiple cylindrical lenses, multiple spherical and cylindrical lenses, at least one Powell lens, and at least one prism or microlens array. - The device includes a localized probe near-field device with a needle tip, which is combined with an excitation laser beam focused on the needle tip. The device is configured to detect local interactions between the sample, the needle tip, and the excitation laser beam.

[0015] This invention also relates to a confocal Raman microspectroscopy method, comprising the following steps: - Emit an excitation laser beam, - Focus the excitation laser beam onto the focal plane of the microscope objective, pointing it toward the sample to be analyzed placed on the sample stage. - The light beam reflected from the sample is collected through the microscope objective and transmitted to the Raman spectrometer via a confocal aperture. - Use a suitable displacement system to change the distance between the microscope objective and the sample stage to focus the sample. The focusing process includes the following steps: - A portion of the reflected beam is extracted and guided to an astigmatic optical system positioned between the optical beam splitter and a pixel matrix image detector, wherein the image detector comprises at least N × M pixels, where N is an integer greater than 2 and M is an integer greater than 2. - A portion of the reflected light beam is projected onto a light spot on the image detector using an astigmatic optical system. - Obtain the image of the light spot on the image detector. - Process the image to calculate the focusing error signal and determine the focusing error signal, which includes the displacement value and displacement direction to be applied to the displacement system to focus the excitation laser beam onto the sample.

[0016] Of course, as long as they are not mutually exclusive or incompatible, different features, variations and embodiments of the present invention can be combined with each other in various ways. Attached Figure Description

[0017] Furthermore, various other features of the invention will become apparent from the following description with reference to the accompanying drawings, which illustrate a non-limiting embodiment of the invention, wherein: Figure 1 This is a schematic diagram of a confocal Raman microspectroscopy device with a focusing system according to the present disclosure; Figure 2 It is a perspective view formed by the cross image through an astigmatic lens; Figure 3 This is an example of a beam spot image reflected on an image detector by a combined astigmatic optical system; Figure 4 This is a theoretical curve illustrating the focus error signal (FES) as a function of defocusing along the optical axis; Figure 5 The principal component analysis method for spot images is illustrated, with an example of spot edge detection (image A) and a magnified view of the spot (image B). Figure 6 The different steps of the second method for spot image analysis are illustrated schematically; Figure 7 Explained that Figure 6 Examples of the method applied to light spot images (Image 7A), after Gaussian filtering (Image 7B), after adaptive threshold classification (Image 7C), after determining the maximum contour (Image 7D), after approximating the maximum contour with an ellipse (Image 7E), and after verification with a bounding box surrounding the light spot (Image 7F). Figure 8It is the experimental focus error signal (FES) curve as a function of the focus error along the optical axis. Detailed Implementation

[0018] Figure 1 The confocal Raman microspectroscopy apparatus 100 is schematically illustrated. The confocal Raman microspectroscopy apparatus 100 includes a laser source 1, a microscope objective 3, a sample stage 4 suitable for accommodating the sample 5 to be analyzed, a confocal aperture 7, and a Raman spectrometer 8. The microscope objective 3 has an optical axis 6, which is arranged parallel to the Z-axis of an orthogonal XYZ coordinate system.

[0019] The apparatus 100 includes a displacement system 9, which is set and configured to change the distance between the microscope objective 3 and the sample stage 4 along the optical axis 6. For example, the displacement system 9 includes a motorized stage adapted to move the sample stage 4 along the Z-axis with sub-micron spatial resolution. In one embodiment, the sample stage 4 is mounted on a multi-axis moving stage to move the sample 5 along the X, Y, and Z axes, thereby allowing sequential Raman measurements of the sample at several points.

[0020] We will first briefly describe the operation of the confocal Raman microspectrometer. Laser source 1 emits an excitation laser beam 10. Microscope objective 3 focuses the excitation laser beam 10 toward sample 5 onto focal plane 24. A reflected beam, propagating in the opposite direction to the excitation beam 10, is obtained through reflection from the sample at point 18. Microscope objective 3 collects the beam 20 reflected from the sample. As a non-limiting example, filter 2 allows separation of the excitation beam 10 from the reflected beam 20, enabling the reflected beam 20 to pass through confocal aperture 7 to the Raman spectrometer 8.

[0021] In one particular embodiment, the Raman microspectroscopy device also includes a localized probe near-field device with a tip. In fact, tip-enhanced Raman spectroscopy (TERS) allows the amplification of Raman signals using evanescent electromagnetic waves confined to a metal point tip (see patent document FR1653182). The near-field device is combined with an excitation laser beam focused on the tip, and the device is configured to detect local interactions between the sample, the tip, and the excitation laser beam. For example, STM-TERS (or scanning tunneling microscopy) combines scanning tunneling microscopy and tip-enhanced Raman spectroscopy. In another example, AFM-Raman microscopy combines atomic force microscopy (AFM) and Raman spectroscopy. Depending on each type of near-field microscope or each tip-enhanced spectroscopy device, and possibly depending on the specific application, there are correspondingly adapted different types of tips.

[0022] The device 100 also includes a system for focusing the sample. The focusing system includes: an optical beam splitter 11 located in the path of the reflected beam 20 between the microscope objective 3 and the confocal aperture 7; a pixel matrix image detector 13; an astigmatic optical system 12 located between the optical beam splitter 11 and the image detector 13; and a processor including 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 via an optomechanical support that is opaque to ambient light. Therefore, a module including the astigmatic optical system 12 and the image detector 13 within their optomechanical supports can be easily mounted onto an existing confocal Raman microscope. This module includes, for example, a wired connection for powering the image detector 13 and transmitting the images acquired by the image detector 13 to the processor for image processing.

[0023] More specifically, the optical beam splitter 11 includes, for example, a plate positioned in the path of the reflected beam 20. This plate is, for example, tilted at 45 degrees relative to the optical axis of the reflected beam 20. The optical beam splitter 11 is arranged and configured to extract a portion 21 of the reflected beam. The optical beam splitter 11 includes, for example, a plate. In one embodiment, the plate has planar and parallel sides. However, such a plate is prone to generating undesirable interference. Alternatively, the optical beam splitter 11 includes a prism plate with planar sides. For example, the angle between the two planar sides is between 0.5 degrees and 10 degrees. Optionally, the plate has an anti-reflective coating on the plate side facing the laser source and possibly on both sides of the plate. The anti-reflective treatment is adapted to make the plate have a high transmission coefficient and a low reflection coefficient to the reflected beam, in order to extract a small portion of the reflected beam.

[0024] Advantageously, the focusing system includes a spatial mask 17 located between the optical beam splitter 11 and the astigmatic optical system 12. The mask 17 is arranged and configured to block internal reflections within the lens. Specifically, when the optical beam splitter 11 has parallel sides or prisms, internal reflections of the beam within the lens can distort the spot image by generating parasitic interference. Since the beam is a monochromatic laser beam, the beam exiting the lens through direct refraction and internal reflection is coherent, thus interference occurs 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 to block portions of the beam that have already undergone internal reflection within the lens, while allowing the beam reflected only on the lens side facing the microscope objective to pass through.

[0025] Mask 17 eliminates unwanted interference in the spot image on image detector 13. As a non-limiting example, plate 11 is a glass plate with an average thickness of 5 mm, parallel sides, and no anti-reflective coating. The plate is tilted at 45 degrees relative to the reflected beam 20. The plate advantageously has, for example, a reflectance factor of 3% of the reflected beam 20. Thus, optical beam splitter 11 captures only a small portion of the reflected beam, while the majority of the reflected beam is transmitted through the plate to be guided to confocal aperture 7 and Raman spectrometer 8.

[0026] Alternatively, the optical beam splitter 11 includes a beam splitting cube.

[0027] In all example embodiments, the optical beam splitter 11 extracts a portion 21 of the reflected beam to guide it to the astigmatic optical system 12.

[0028] The astigmatic optical system 12 includes, for example, cylindrical lenses. Alternatively, the astigmatic optical system 12 includes two cylindrical lenses arranged in series along the optical path, their optical axes intersecting, i.e., oriented perpendicularly to each other and perpendicular to the optical axis of a portion 21 of the reflected beam. Alternatively or additionally, the astigmatic optical system 12 includes: at least one concave or convex spherical mirror oriented at a non-zero angle of incidence, at least one toric mirror, at least one parabolic mirror, at least one simple lens pivoting about its optical axis, at least one lens eccentric relative to the optical axis, multiple cylindrical lenses, multiple spherical and cylindrical lenses, at least one Powell lens, at least one prism or microlens array. Advantageously, the astigmatic optical system 12 is positioned at an optical distance of approximately 20 cm from the microscope objective. In one example, two plano-convex cylindrical lenses arranged in series with a focal length of 50 mm are used, positioned at a distance of 20 cm from the microscope objective.

[0029] 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 portion 21 of the reflected beam as a light spot onto the image detector 13. The image detector 13 is a pixel array comprising at least N × 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 × 2064 pixels. The image detector 13 includes, for example, a Basler camera positioned 45 mm from the astigmatic optical system. The image detector includes, for example, a CCD-type camera, allowing images to be acquired at a frequency of 50 Hz, i.e., one image every 20 ms.

[0030] Figure 2 The operation of the astigmatic optical system 12 is illustrated schematically. To explain... Figure 2Assume the sample has a cross shape at point 18, illuminated by excitation beam 10. Microscope objective 3 is not shown here. The astigmatic optical system exhibits different optical powers along the tangential and sagittal axes. The astigmatic optical system forms an image of point 18. One arm of the cross is clearly imaged in the sagittal plane S, while the other arm is clearly imaged in the tangential plane T. The sagittal and tangential planes are separated by a distance D along the longitudinal optical axis of the beam. Therefore, when image detector 13 is located in the tangential plane T, the image of the cross is clear, for example, on the horizontal arm and blurry on the vertical arm. Conversely, when image detector 13 is located in the sagittal plane S, the image of the cross is clear on the vertical arm and blurry on the horizontal arm. When image detector 13 is located at an intermediate position between the tangential plane T and the sagittal plane S, both arms of the cross image are equally clear.

[0031] As described above, the image detector 13 is located at a fixed distance from the astigmatic optical system 12. For example, the pixel array image detector 13 is located midway between the sagittal plane S and the tangential plane T, which are optically conjugate to the focal plane 24 of the microscope objective. Advantageously, the pixel rows of the image detector are parallel to the sagittal axis of the astigmatic optical system 12, and the pixel columns are parallel to the tangential axis of the astigmatic optical system 12. The sample is illuminated by the excitation beam 10 through the microscope objective 3 to form a reflected beam 20 originating from the circular point 18. The image detector 13 is configured to acquire a spot image 14 formed by a portion 21 of the reflected beam through the astigmatic optical system 12. When the sample point 18 is located within the focal plane 24 of the microscope objective 3, the spot image 14 on the image detector 13 is circular. Conversely, when the sample point 18 is deviated from the focal plane 24 of the microscope objective 3 by a distance Z, the spot image 14 on the image detector 13 is typically elliptical. This distance Z is also referred to as the focusing error. The orientation of the ellipse varies depending on the sign of the focusing error relative to the focal plane 24.

[0032] Unlike most autofocusing systems, this system uses the same laser source 1 for both confocal Raman measurements and focusing. This configuration offers several advantages. First, it allows for precise focusing at the measurement point in the confocal Raman microspectroscopy measurement. Using the same laser source also prevents interference from Raman signals from other sources, unlike other autofocusing systems. Furthermore, focusing can be performed during or just before the measurement, significantly reducing focusing time. Unlike previous autofocusing systems, this solution requires no pre-scanning. Additionally, determining the sign of the focusing error allows for the calculation of the direction and value of the feedback to be applied, resulting in faster focusing compared to previous techniques.

[0033] Figure 3 This represents an example of a spot image 14 acquired by image detector 13. A portion 21 of the reflected beam projected onto image detector 13 forms a point cloud 19, which is represented as gray dots on a bright background.

[0034] Image 14 is transmitted to the image processing system 15 of the processor.

[0035] According to this disclosure, analysis of the spot image 14 allows calculation of parameters of an ellipse that approximates the shape of the spot in the detection image, and derives a focusing error signal represented as FES. V1 represents the major axis of the ellipse, V2 represents the minor axis of the ellipse, and ALPHA represents the angle between the major axis of the ellipse and the pixel X-axis of the image detector 13.

[0036] The focusing error signal depends on the ratio V1 / V2 according to the following formula: FES = 4 / π.tan -1 (V1 / V2) – 1. The focusing error signal is between -1 and +1. For a perfect astigmatic optical system and a non-diffuse sample, the image at point 18 is circular when the microscope is at its optimal focus, which corresponds to FES equal to 0.

[0037] Figure 4 A theoretical curve 23 is shown as the focusing error signal FES as a function of the distance from the sample point 18 to the focal plane 24 of the microscope objective 3. When the illuminated point 18 on the sample lies in the optical conjugate planes of the sagittal and tangential planes of the astigmatic optical system 12, the focusing error signal FES is located at point A or B on curve 23. Between points A and B, the position of the sample relative to the optimal focal point (FES=0) can be determined from the focusing error signal by reversing curve 23. Therefore, the displacement value and direction to be applied along the optical axis 6 to the displacement system 9 to focus the excitation laser beam 10 onto the sample can be calculated. Most advantageously, the focal region of length L extending between points C1 and C2 corresponds to an axial displacement linearly related to the FES variation. L is calculated for a given magnification. Curve 23 can be obtained for samples with reflective surfaces, such as polished silicon plates, polished metal plates, or glass plates.

[0038] For an astigmatic optical system 12 comprising a first cylindrical lens with a focal length of 50 mm and a second cylindrical lens with a focal length of 50 mm, positioned at an optical distance of 20 cm from a 100x microscope objective, the working area (denoted as Δz) along the Z-axis between points A and B on curve 23 is approximately 8 µm. At the optimal focusing position, optical calculation software (such as Zemax) allows for the evaluation of the laser spot diameter at the optimal focal point (FES=0).

[0039] However, the same principle can be applied to different objectives, with the displacement range adjusted according to the magnification of the microscope objective. Therefore, for a 50x microscope objective, the working area Δz along the Z-axis is approximately 39 µm. For a 10x microscope objective, the working area Δz along the Z-axis is approximately 1000 µm. Finally, for a 5x microscope objective, the working area Δz along the Z-axis is approximately 3700 µm.

[0040] Based on the elliptic parameters, the processor calculates the focusing error signal (FES). This focusing error signal is pre-calibrated to obtain curve 23, which depends on both the microscope objective and the astigmatic optics system, and on the sample, particularly its surface roughness. Using the calculated focusing error signal (FES) and calibration curve 23, the processor calculates the feedback signal to be applied to the displacement system 9 to change the distance between the microscope objective 3 and the sample stage 4 along the optical axis 6, positioning the sample at the optimal focal point (FES=0). Thus, focusing of the excitation laser beam can be achieved in a single movement. Raman measurements are then immediately performed at point 18 corresponding to the optimal focal point (FES=0) without changing the optical system or the light source (and without moving the XY stage). Therefore, focusing is very fast: it can be completed in less than a second, for example, within 200 ms or 300 ms.

[0041] We will now describe different image processing methods for determining ellipse parameters.

[0042] In the first embodiment, Principal Component Analysis (PCA) is used. PCA is a method that uses a covariance matrix to measure how each variable (here, a pixel) is related to other variables (here, other pixels in the image), the covariance matrix including the data propagation direction using eigenvectors. Eigenvalues ​​are coefficients applied to the eigenvectors, assigning length to the vectors. Here, principal component analysis is used to determine the ellipse parameters of the spot image 14 acquired by the image detector 13.

[0043] More specifically, an adaptive threshold based on the OTSU algorithm is used to eliminate insufficiently bright pixels in image 14. This produces a list of pixels with coordinates (X, Y) whose intensity is greater than or equal to the threshold, corresponding to the pixels of the light spot. Here, the parameters (X, Y) are the pixel coordinates of image detector 13.

[0044] Principal components are the eigenvectors of the data covariance matrix. The covariance matrix is ​​a square matrix that gives the covariance between X and Y after applying an adaptive threshold.

[0045] Let A be a linear transformation represented by matrix A. If there exists a vector AX = λX, then λ is called an eigenvalue of A and has a corresponding (right) eigenvector X.

[0046] We calculate the eigenvectors and sort them by eigenvalues.

[0047] The two eigenvalues ​​determined from this are: • V1, the largest eigenvalue, corresponds to the major axis of the ellipse, and • V2, the smallest eigenvalue, corresponds to the minor axis of the ellipse.

[0048] Then we calculate the angle ALPHA, the major axis of the ellipse relative to the horizontal direction (on image detector 13): ALPHA = 90 - rad2deg(arctan(V1 / V2)), Where rad2deg means converting the angle from radians to degrees.

[0049] A is the covariance matrix of the pixels (from the previously selected list X, Y).

[0050] The obtained V1 / V2 ratio is the ratio of the major axis to the minor axis of the ellipse.

[0051] If the angle ALPHA is less than 90 degrees, the values ​​of V1 and V2 remain unchanged. If the angle ALPHA is greater than 90 degrees, the values ​​of V1 and V2 are interchanged.

[0052] Figure 5 An example of spot image processing using the PCA method is shown. Image 5A shows a rectangular box containing the spot and an ellipse with major axis V1, minor axis V2, and angle ALPHA determined by the PCA algorithm. The rectangular box encloses the ellipse. Figure 5 Image 5B shows a magnified view of the spot image region acquired by the image detector, where the acquired pixel intensities correspond to the bounding boxes containing the spots. In other words, Image 5B is the cropped original image, removing all regions with pixel intensities below a threshold. All the brightest pixels, i.e., those with intensities greater than or equal to the threshold, are located within the bounding boxes. In Image 5A, the visualization of this rectangular bounding box, along with the PCA-determined ellipse superimposed on it, allows us to visually verify the calculation of the ellipse parameters.

[0053] Principal component analysis (PCA) yielded good results, especially for samples with reflective surfaces, such as polished silicon plates, polished metal plates, or glass plates.

[0054] In the second embodiment, a different image processing algorithm was used. Figure 6 Different steps of the method according to the second embodiment are illustrated schematically. The method includes a first step 30 of acquiring a spot image 14 by an image detector 13. This first step is the same in all embodiments.

[0055] Next, the spot image 14 undergoes a filtering step 40, such as Gaussian filtering, to obtain a filtered image. This filtering can smooth different parts of the image by blurring, thereby harmonizing its details.

[0056] Optionally, filtering step 40 includes an adaptive threshold classification step 45. Step 45 includes, for example, constructing a histogram of pixel intensities in the filtered image, on a scale from 0 to 255. The histogram typically has two peaks: a first peak corresponding to the darkest pixels and a second peak corresponding to the brightest pixels (i.e., flares). Then, only the second peak is retained, the intensity of the pixels in the second peak is set to the maximum value of 255, and the intensity of all other pixels is set to 0. This results in an image filtered using adaptive threshold classification.

[0057] In step 50, the image processing steps are then applied to the filtered image or, respectively, to the image filtered by adaptive classification to determine the larger contours of the light spot. For this purpose, for example, the FindContours function from the OpenCV library is used to compute all possible contours, sort the resulting contours according to their area, and retain the contour with the largest area.

[0058] In step 60, the "fitEllipse" function from the OpenCV library is used to determine the ellipse that best approximates the contour. This algorithm is described in "A Buyer's Guide to Conic Fitting" by Andrew W. Fitzgibbon and RB Fisher, published in the Proceedings of the 5th British Machine Vision Conference, Birmingham, pp. 513–522, 1995. Subsequently, the parameters ALPHA, V1, and V2 are derived from this ellipse.

[0059] In step 70, we calculate the V1 / V2 ratio and derive the focus error signal FES.

[0060] Optionally, the calculation of ellipse parameters V1 and V2 is verified by a bounding box method that considers all pixels of the spot (described in conjunction with the first embodiment).

[0061] In step 75, the processor calculates the feedback signal to be applied to the displacement system 9.

[0062] In step 80, the displacement system changes the distance between the microscope objective 3 and the sample stage 4 along the optical axis 6 to place the sample at the optimal focus (FES=0).

[0063] In step 90, the sample is positioned at the optimal focal point (FES=0), and Raman spectral measurements are obtained at the focal point.

[0064] If necessary, in step 95, the displacement system changes the (X,Y) position of the excitation laser beam on the sample. The focusing process is repeated at the new measurement point.

[0065] Figure 7 Examples of the main steps in the spot image processing according to the second embodiment are shown. Image 7A shows the spot image 14 acquired by image detector 13. Image 7B shows the filtered image obtained after applying Gaussian filtering (step 40) to image 6A. Image 7C shows the image obtained after applying adaptive classification (step 45) to the filtered image 7B. Image 7D shows the maximum contour obtained after step 50, which calculates the maximum contour in the filtered (and possibly adaptively classified) image. Image 7E shows the ellipse determined in step 60 for approximating the maximum contour. Image 7F compares the ellipse thus obtained with a rectangular box surrounding the spot.

[0066] The image processing method according to the second embodiment yields better results on reflective samples than that of the first embodiment. However, within the operating range ( Figure 4 At the extreme points (A and B) on the middle curve 23, the outline may be difficult to determine.

[0067] According to a variation of the second embodiment, step 45 (adaptive threshold classification) is replaced by step 46 (morphological gradient). The morphological gradient is the difference between dilation and erosion.

[0068] Dilation involves convolving image A with a kernel (B), which can have any shape or size, but is typically square. Kernel B has a defined anchor point, typically the center of the kernel.

[0069] As kernel B scans across the unfiltered image, we calculate the maximum pixel value covered by kernel B and replace the image pixels at the anchor point with this maximum value. The dilation operation causes the bright areas of the image to grow.

[0070] The corrosion process is the reverse of the expansion process. The corrosion process is based on calculating local minima on a given core surface.

[0071] As kernel B scans across the dilated image, we calculate the minimum pixel value covered by B and replace the image pixels below the anchor point with this minimum value. We obtain the 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.

[0072] The subsequent steps are similar.

[0073] This variation of the second embodiment yielded very good results, especially on reflective samples, as well as on rough or diffuse reflective samples, such as tablets and rocks.

[0074] The processing described in this disclosure enables focusing with excellent precision, less than the depth of field of the lens. Figure 8 Experimental focus error signal (FES) curves are shown using a 100x lens as a function of focus error along the optical axis. For example, for a 100x lens with a depth of field of 190 nm, the focus accuracy along the Z-axis is approximately 40 nm. Furthermore, focusing is very fast. The focus error signal calculation, as well as the displacement value and direction to be applied to the moving system, are obtained in a single step. Depending on the type of moving system used, the required displacement value, and the achievable speed, focusing can be completed in less than a second, for example, within 200 ms to 500 ms. The focusing system and method described in this disclosure are both very accurate and very fast. The focusing system and method yield good results even with high-magnification microscope objectives (e.g., x50 or x100) where the reflected beam is still very divergent.

Claims

1. A confocal Raman microspectroscopy device (100), comprising: Laser source (1), said laser source (1) is adapted to emit an excitation laser beam (10); A sample stage (4) adapted to hold a sample (5) to be analyzed; a microscope objective (3) having an optical axis (6), the microscope objective (3) being configured to receive an excitation laser beam (10) and focus the excitation laser beam (10) in the direction of the sample (5) onto a focal plane (24), the microscope objective (3) being adapted to collect a light beam (20) reflected by the sample and transmit the reflected light beam to a Raman spectrometer (8) through a confocal aperture (7); and a displacement system (9) adapted to change the distance between the microscope objective (3) and the sample stage (4) along the optical axis (6). And a system for focusing the sample; Its features are: The focusing system includes: an optical beam splitter (11) disposed in the path of the reflected beam (20) between the microscope objective (3) and the confocal aperture (7); and an image detector (13) having a pixel matrix comprising at least N × M 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 including an image processing system (15) and a feedback device (16), the optical beam splitter (11) being adapted to extract a portion (21) of the reflected beam, the astigmatic optical system (12) being adapted to project a portion (21) of the reflected beam onto a spot on the image detector (13), the image detector (13) being configured to acquire an image (14) of the spot, the image processing system (15) being adapted to calculate a focus error signal, and the feedback device (16) being adapted to derive from the focus error signal the displacement value and displacement direction to be applied to the displacement system (9) along the optical axis (6) to focus the excitation laser beam (10) onto the sample (5).

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

3. The device according to claim 1 or 2, wherein, The image processing system (15) is adapted to determine an ellipse in the image (14) corresponding to the light spot, and the processor is adapted to derive from it a measured value of the major axis V1 and a measured value of the minor axis V2 of the ellipse, wherein the focus error signal is equal to 4 / π.tan -1 (V1 / V2) – 1.

4. The device according to claim 3, wherein, The image processing system (15) performs principal component analysis on the image (14) based on the light spot.

5. The device according to claim 3, wherein, The image processing system (15) performs edge detection in the image (14) based on the light spot.

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

7. The device according to claim 6, wherein, The filter (40) applied to the image of the light spot to generate the filtered image is a Gaussian filter or a morphological gradient filter.

8. The device according to any one of claims 1 to 7, wherein, The optical beam splitter (11) includes a planar parallel plate, a wedge-shaped planar side plate, or a beam splitting cube.

9. The device 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 disposed and configured to block internally reflected beams within the plate.

10. The device according to any one of claims 1 to 9, wherein, The astigmatic optical system (12) includes: a cylindrical lens or a cylindrical lens with two intersecting optical axes, at least one concave or convex spherical mirror oriented at a non-zero incident angle, at least one toric mirror, at least one parabolic mirror, at least one simple lens pivoting about its optical axis, at least one lens eccentric relative to the optical axis, multiple cylindrical lenses, multiple spherical and cylindrical lenses, at least one Powell lens, and at least one prism or microlens array.

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

12. A confocal Raman microspectroscopy method (100), comprising the following steps: - Emit an excitation laser beam (10). - The excitation laser beam (10) is focused onto the focal plane (24) of the microscope objective (3) toward the sample (5) to be analyzed placed on the sample stage (4). - The light beam (20) reflected by the sample is collected through the microscope objective (3) and transmitted to the Raman spectrometer (8) via the confocal aperture (7). - The sample is focused using a displacement system (9) adapted to change the distance between the microscope objective (3) and the sample stage (4), the focusing step comprising the following steps: - The reflected beam is separated by an optical beam splitter (11) disposed between the microscope objective (3) and the confocal aperture to extract a portion (21) of the reflected beam and guide the portion (21) of the reflected beam to an astigmatic optical system (12) disposed between the optical beam splitter (11) and the pixel matrix image detector (13), the image detector (13) comprising at least N × M pixels, where N is an integer greater than 2 and M is an integer greater than 2. - A portion (21) of the reflected beam is projected onto a light spot on the image detector (13) by the astigmatic optical system (12). - Obtain an image (14) of the light spot on the image detector (13). - Process the image (14) to calculate the focus error signal and determine the focus error signal, which includes the displacement value and displacement direction to be applied to the displacement system (9) to focus the excitation laser beam (10) onto the sample (5).

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