Method and system for microscopic spectroscopy measurements

Abstract

The present invention relates to an optical microspectroscopy system comprising an optical microscope (10), a spectroscopy system (50), and an optical system (14) adapted to direct an excitation light beam onto a sample through the at least one microscope objective (11, 12) and collect a Raman or PL light beam from the sample. According to the present invention, the optical microspectroscopy system comprises an imaging system (16, 41) configured to acquire a first image (71) and a second image (72) of the sample by reflection or transmission of an illumination beam from the sample surface, the first image (71) having a large field of view and the second image (72) having a small field of view, a processing system (40) configured to determine a region corresponding to the second image (72) in the coordinate system of the first image (71), and a display system (44) configured to display the region by overlaying the first image (71), the second image (72), and a third image (73) representing the region on the first image (71).

Description

Technical Field

[0001] The present invention relates to a method and system for visualizing and mapping a sample surface in two dimensions (2D) or three dimensions (3D) over a wide spatial range, for example, from millimeter scale to micron scale, with high resolution in the lateral and axial directions, and performing microscopic spectroscopic measurements in accurately selected regions of interest (ROIs).

[0002] The present invention also relates to a method and system for easily navigating over a sample surface with high precision and over a wide spatial range, for example, from millimeter scale to micron scale, and selecting a region of interest (ROI) for performing spectroscopic analysis or spectroscopic analysis imaging, such as photoluminescence (PL) or Raman imaging.

Background Art

[0003] In the last 20 years, with the leapfrog development of photon detectors and light source development, the development of PL and Raman spectrometers has advanced dramatically. Photon detection systems include, in particular, CCD, EMCCD and CMOS cameras, as well as a new generation of avalanche photodiodes and photomultiplier tubes with improved characteristics. New light sources include small solid-state lasers that cover a spectral range from UV light to near-infrared light and have an average output of several tens of mW sufficient to generate PL or Raman signals.

[0004] An important breakthrough in the development of PL and Raman technologies has resulted from the combination of spectroscopic technology and microscopy technology. Despite the fact that both technologies are very old, recent developments in electronics and photonics have enabled important innovations.

[0005] In particular, the latest microscope spectrometers are equipped with an autofocus system for detecting and tracking the sample surface. The main functions of the autofocus system have an optical or numerical origin.

[0006] Optical hardware autofocus systems measure the distance to the sample surface using a laser or maintain a constant distance using white light interference. They can also pinpoint the location of the sample surface via the maximum value of the measured signal, using the intensity of light reflected from the sample surface or the intensity of the sample's spectral response. Generally, all optical hardware autofocus systems are point-by-point measurement methods, requiring time to collect information from an N×N pixel area. As a function of the time spent per pixel and the number of pixels, the total time can vary from minutes to hours. Another drawback of optical autofocus methods is their sensitivity to the properties of the sample surface. For highly diffusive, translucent, sloped, or uneven surfaces, this method will yield inaccurate results or may not work at all. Moreover, the price of optical hardware autofocus devices increases the overall cost of the microscope spectrometer.

[0007] An alternative to using optical hardware autofocus systems is the use of digital or numerical autofocus systems. These systems do not incur any additional costs for the equipment, as they utilize existing video image hardware such as optical objective lenses, optical transmission optics, and detection devices such as CMOS or CCD cameras. The only investment is the development of mathematical algorithms integrated into the software to find and maintain focus on the studied or measured sample. Such systems have already been developed and are commonly used in digital photo cameras, smartphones, or digital microscopy. In all specific implementations, the inventors propose their specific solutions for both hardware and software implementations, and the application varies depending on the device. A key advantage of digital autofocus is the wide working area corresponding to the field of view. Digital autofocus proceeds through the simultaneous measurement of millions of pixels. Digital autofocus systems generally rely on contrast or sharpness analysis of the image. Their software includes algorithms that analyze the contrast or sharpness of the image as a function of the z-coordinate to determine the best focus position. The fact that a high-contrast or sharp image corresponds to the focus position of the sample makes this software approach possible.

[0008] For generally flat and smooth samples, autofocus allows for sharp visualization of the entire field of view. However, most samples exhibit rough surfaces with significant variation along the OZ axis, requiring optical autofocus to be activated at each point of the ROI for visualization of the sample surface and for imaging of the PL and / or Raman surface. As mentioned above, this is a major drawback of optical hardware autofocus systems because it is very time-consuming. Furthermore, because a portion of the sample surface within the field of view of the microscope objective lens is blurred, it is difficult for the user to select the region of interest (ROI) for performing PL or Raman measurements of the sample. Therefore, determining the ROI region during use of this system is difficult. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Accordingly, in order to improve upon the shortcomings of the prior art, one object of the present invention is to provide an optical microspectroscopy system comprising: an optical microscope having a sample holder for holding a sample to be analyzed and at least one microscope objective lens defining an optical axis (OZ); a spectroscopic system having a light source, a spectrometer, and a detection system adapted to generate an excitation light beam; an optical system adapted to guide the excitation light beam onto the sample through the at least one microscope objective lens and to collect a Raman or photoluminescent light beam generated by scattering of the excitation light beam on the sample, and adapted to guide the Raman or photoluminescent light beam toward the spectrometer and the detection system; and an actuation system for driving relative lateral and / or axial movement between the sample holder and the at least one microscope objective lens. [Means for solving the problem]

[0010] According to the present invention, the optical microspectroscopy system comprises: an imaging system arranged in combination with the at least one microscope objective lens and configured to acquire a first image of a sample and a second image of a sample, wherein the first and second images are formed by reflection or transmission of an illumination beam from the surface of the sample, the first image having a large field of view and the second image having a small field of view; a processing system configured to determine a region in the coordinate system of the first image corresponding to the small field of view of the second image; a display system configured to display the first and second images and to display a graphic representation of the region superimposed on the first image; and a user interface configured to select a region of interest in the first and / or second image, wherein the operating system is configured to position the region of interest to receive an excitation light beam, and the spectroscopic measurement system is configured to detect a Raman or photoluminescent light beam generated from the region of interest.

[0011] According to a particular advantageous embodiment, at least one microscope objective lens comprises a low-magnification objective lens and a high-magnification objective lens, wherein the low-magnification objective lens is configured to form a first image on the imaging system, and the high-magnification objective lens is configured to form a second image of the sample on the imaging system.

[0012] In a particular advantageous configuration, the imaging system is adapted to acquire a second image in real time, the processing system is adapted to update the region corresponding to the second image in real time, and the display system is adapted to display the second image in real time and the graphic representation of the updated region in real time.

[0013] Preferably, the user interface comprises an integrated computer mouse, trackball, joystick, touchpad and / or touchscreen, and the user interface is adapted to interact with a first image or a second image, respectively, using drag-and-move operations, thereby moving a large field of view in the first image or a small field of view in the second image by a predetermined amount of horizontal displacement, and the operating system is adapted to drive relative horizontal motion (dX and / or dY) proportional to the predetermined amount of horizontal displacement in the first image or the second image, respectively.

[0014] According to another particular advantageous embodiment, the operating system comprises a drive unit configured to change the axial position of at least one microscope objective lens relative to a sample holder along the optical axis, the imaging system is configured to acquire a plurality of images of a sample at a plurality of axial positions (Z) along the optical axis (OZ), the processing system is configured to calculate the mean squared difference of adjacent pixel intensities in the central region for each of the plurality of images and therefrom evaluate the image sharpness for each of the central regions of the plurality of images, the processing system is configured to estimate the focal position on the central region from the image sharpness, and the imaging system is configured to acquire the first image and / or the second image at the focal position on the central region.

[0015] According to a particular embodiment, the focus position on the central region is determined by calculating the mean squared difference (MSD) value for each of the plurality of images and the profile of the mean squared difference as a function of the plurality of axial positions (Z), and the focus position on the central region is determined by calculating the maximum value of the profile of the mean squared difference.

[0016] According to a particular embodiment, the image sharpness is evaluated by calculating the average intensity derivative and the average intensity profile derivative in the central region for each of the plurality of images as functions of the plurality of axial Z positions, and the near-focus position range is determined by the axial (OZ) displacement toward the maximum value of the average intensity profile derivative.

[0017] Advantageously, the processing system is configured to estimate a surface topography map from it.

[0018] According to another particular advantageous embodiment, the processing system is configured to determine a plurality of pixel column vectors (Px, Py) for the plurality of images of the sample as a function of the plurality of axial positions (Z) along the optical axis, the processing system (40) is configured to evaluate a sharpness profile or contrast profile for each of the plurality of pixel column vectors (Px, Py) and associated confidence level values ​​using gradient or first derivative profile or second derivative profile, mean square difference or variation, the processing system is configured to analyze the sharpness or contrast profile for each of the plurality of pixel column vectors (Px, Py) using associated confidence level values ​​and estimate the focus position therefrom for each of the plurality of pixel column vectors (Px, Py), the processing system is configured to determine the three-dimensional coordinates of the focus position and the corresponding pixel intensity for each of the plurality of pixel column vectors (Px, Py) and construct a sample topography image representing the sample surface in three dimensions.

[0019] A further objective of the present invention is, - A step to determine the current position of the sample stage in the optical microscope, - A step of generating an illumination light beam directed towards a sample placed on a sample stage, - The step of selecting the microscope objective lens on the optical microscope, - A step of acquiring a first image formed through the optical microscope by reflection or transmission of an illumination light beam from the surface of a sample, wherein the first image has a large field of view, - storing the first image in a data storage unit; - displaying the first image on a display system; - obtaining a second image formed through the optical microscope by reflection or transmission of an illumination beam from a sample surface, the second image having a small field of view smaller than the field of view of the first image; - displaying the second image on a display system; - determining a region in the coordinate system of the first image corresponding to the second image of the small field of view; - superimposing and displaying a graphic representation of the region on the first image; - selecting a region of interest in the first image and / or the second image displayed on the display system; - positioning the sample so as to direct an excitation light beam through the microscope onto the region of interest of the sample, and collecting a Raman or photoluminescence light beam generated by scattering of the excitation light beam on the region of interest of the sample; providing a microscopic spectroscopic measurement method including the above.

[0020] Preferably, the method further includes detecting the Raman or photoluminescence light beam using a spectrometer system.

[0021] In a particular embodiment, the first image and the second image are formed using the same microscope objective lens defining an optical axis, and the step of obtaining the first image includes obtaining a plurality of images at a plurality of predetermined positions in a plane transverse to the optical axis, and forming the first image by tiling the plurality of images as a function of their predetermined positions.

[0022] ​​​​​​​-A step of using a microscope objective lens to acquire multiple images of a sample at multiple relative axial (Z) positions of the sample holder with respect to an optical microscope, wherein the relative axial (Z) positions are taken along the optical axis of the microscope objective lens, - A step of evaluating the sharpness within the central region for each of the multiple images, - A step of estimating the focus position within the central region from the sharpness evaluated for the multiple axial Z positions, - The step of positioning the sample at the focal position within the central region, Includes, - The step of acquiring the first image and / or the second image is performed at the focus position.

[0024] Advantageously, the method further includes calculating the average intensity derivative within the central region for each of the plurality of images; determining the average intensity derivative profile as a function of the plurality of axial Z positions; and, before determining the focus position, displacing the sample holder in the direction of the minimum of the average intensity derivative profile, thereby determining the near-focus position range.

[0025] In certain embodiments, the near-focus range is found using the average intensity for each of the plurality of images, and the near-focus range is found by calculating the first derivative of the average intensity as a function of the plurality of axial Z positions, and further displacing the sample in the direction of the minimum value of the calculated derivative until the MSD variation is non-zero, thereby defining the near-focus Z range.

[0026] In certain embodiments, the step of evaluating sharpness is estimated from the mean squared difference (MSD) profile and from the mean squared difference in the central region for each of the plurality of images as a function of the plurality of axial Z positions, and the focus position is determined by calculating the maximum value of the mean squared difference profile.

[0027] In yet another embodiment, the step of evaluating sharpness is estimated by calculating the image gradient in the central region of each of the plurality of images and calculating the image gradient profile as a function of the plurality of axial Z positions, and the focus position is determined by calculating the maximum value of the image gradient profile.

[0028] According to another specific embodiment, the microspectroscopy method further, - A step of determining a plurality of pixel column vectors (Px, Py) for the plurality of images of the sample as a function of the plurality of axial positions (Z) along the optical axis (OZ), - A step of evaluating a sharpness profile or contrast profile for each of multiple pixel column vectors (Px, Py), using the mean squared deviation, variation, gradient, first derivative profile or second derivative profile, and associated confidence level values. -A step of analyzing the sharpness profile or contrast profile for each of the multiple pixel column vectors using the associated confidence level value, and estimating the focus position for each of the multiple pixel column vectors from there, - A step of extracting the three-dimensional coordinates of the focus position and the corresponding pixel intensity, - A step of storing the three-dimensional coordinates of the focus position and the corresponding pixel intensity in the memory unit 45, -The steps of constructing a sample topography image Z(X,Y) and / or a 3D sample surface image using the 3D coordinates of the focus position and the corresponding pixel intensity, Includes.

[0029] This system and method provide rapid and easy navigation on the sample surface while enabling the acquisition of Raman signals in a selected region of interest (ROI).

[0030] Preferably, the region of interest for Raman or PL measurement is selected from a rectangular region, a disc-shaped region, a user-specified region, a line, a set of randomly selected points, and a set of user-specified points.

[0031] This method and system are fast and do not require any additional hardware components.

[0032] The present invention will be better understood with reference to the attached drawings. [Brief explanation of the drawing]

[0033] [Figure 1] This diagram schematically represents an instrument combining an optical microscope, a photoluminescent or Raman spectrometer, and a visualization device. [Figure 2] Examples of the first and second images of a patterned sample displayed on a visualization device are shown, with the boundary of the region corresponding to the second image superimposed on the first image indicated. [Figure 3] In addition, we show another example of the first and second images of a different sample obtained using the autofocus process. [Figure 4A-E] The images shown are from a stack of images acquired for a rutile sample, taken at different Z-axis positions using a 10x objective lens. [Figure 4F] Each image shows a focused image reconstructed based on processing of an image stack containing the images in Figures 4A to 4E. [Figure 5] A substantially sharpened 2D image of an aspirin tablet sample, obtained using a 10x objective lens, is shown. [Figure 6] The corresponding surface topography map is shown. [Figure 7] This image shows a reconstructed 2D focused image of a gypsum sample taken using a 100x objective lens. [Figure 8] The corresponding all-focus 3D reconstructed surface topography is shown. [Figure 9] This shows a 2D surface topography image of a rutile sample at 100x magnification. [Figure 10] The corresponding Raman spectroscopy image is shown. [Modes for carrying out the invention]

[0034] This specification discloses a novel digital method for automatically finding the focus of a sample surface during sample movement or sample surface navigation, simultaneously visualizing the entire sample surface within the field of view, registering this entire focal sample surface within the field of view and combining it with a mosaic image, and easily navigating on a large sample image with zoom in on its details for the purpose of selecting ROIs in an accurate and sophisticated manner, to perform further PL and Raman measurements and imaging of the sample surface.

[0035] Device A first aspect of this disclosure relates to a novel system and method for acquiring PL or Raman signals that provides easy navigation on a sample surface.

[0036] Figure 1 schematically shows a system combining an optical microscope 10, a photoluminescence or Raman spectroscopy system 50, a processing system 40, and a display device 44.

[0037] The optical microscope 10 generally comprises a microscope stand, at least one objective lens 11, and a sample holder for holding the sample to be analyzed. The objective lens 11 generally defines a vertical optical axis OZ. Preferably, the optical microscope 10 comprises a low-magnification objective lens 11 and a high-magnification objective lens 12.

[0038] The microscope 10 generally includes an electric actuator 20 for moving the sample stage in three dimensions relative to the objective lens 11. For example, the sample stage may be mounted on an XY scanning stage to translate the sample along the X and / or Y directions, and the microscope body may include a Z stage to allow the microscope objective lens 11 to be moved along the OZ axis to adjust the focus. Alternatively, the sample stage may be mounted on an XYZ scanning stage to allow the sample to be moved in 3D. Alternatively or supplementally, the sample stage may be mounted on a rotating stage.

[0039] In this specification, we assume an XYZ orthonormal coordinate system, where the OZ axis corresponds to the optical axis of the microscope, and the OX and OY axes intersect the OZ axis. Generally, the OZ axis is perpendicular.

[0040] The optical microscope 10 includes an illumination turret comprising a beam splitter 13 used to guide illumination light into the objective lens, a switching mirror 14 used to couple the microscope to a spectrometer, and a tube lens 15 used to generate a sample image on a CMOS camera. Furthermore, the optical microscope 10 includes a white light source 18 and an illumination condenser 17. The white light source 18 is composed of, for example, an LED lamp or a halogen lamp. The white light source 18 generates a white light beam that is guided to the illumination condenser 17. The illumination condenser 17 forms an illumination light beam that is reflected on the beam splitter 13 and directed toward the sample surface.

[0041] In addition, the optical microscope 10 is equipped with a video camera 16 for acquiring video images of the sample surface. The video images of the sample surface are formed using reflected light if the optical microscope 10 is configured to operate by reflection, or using transmitted light if the optical microscope 10 is configured to operate by transmission.

[0042] The PL or Raman spectroscopy system 50 comprises a laser light source 51, a Rayleigh filter 52, a coupled optical system 54, a spectrometer 55, and a CCD camera 60. The laser light source 51 generates an excitation light beam at a predetermined wavelength. In the input path, the Rayleigh filter 52 transmits the excitation light beam toward a switching mirror 14 in the beam path of the optical microscope, thereby guiding the excitation light beam toward the sample surface. The objective lens 11 focuses the excitation light beam onto the sample. The switching mirror 14 allows the excitation light beam to be directed toward or not toward the sample surface. The objective lens 11 collects the light beam backscattered by the sample and returns the backscattered light beam toward the switching mirror 14. In the return path, the Rayleigh filter 52 separates the Rayleigh scattered light of the laser wavelength from the PL or Raman scattered light. For example, the Rayleigh filter 52 comprises a notch-type injection-rejection spectral filter. Optionally, a confocal pinhole 53 is positioned to select a Raman or PL light beam from a specific plane in the sample. A coupled optical system 54 focuses the PL or Raman scattered light onto the entrance slit of the spectrometer 55. A CCD camera 60 is positioned at the output of the spectrometer 55 to detect the PL or Raman spectrum.

[0043] Generally, a microspectroscopy system comprises a processing system 40 including an acquisition unit 41, a processing unit 42, a control unit or central processing unit 43, and a data storage unit 45. The acquisition unit 41 acquires images from a video camera 16 and / or spectra from a CCD camera 60. The processing unit 42 performs numerical processing on the acquired images and / or spectra. The central processing unit 43 synchronizes the operation of the scanning stage 20, white light source 18, switching mirror 14, laser light source 51, Rayleigh filter 52, confocal pinhole 53, switching mirror 14, and spectrometer 55.

[0044] Finally, the microspectroscopy system includes a display device 44 consisting of one or more screens. The display device 44 is connected to a central processing unit 43. For example, the display device 44 displays in real time an image of the sample acquired by the video camera 16 and / or a spectrum measured using the CCD camera 60 at a point in the sample.

[0045] The configuration and operation of a microspectroscopy system according to a first aspect of this disclosure are described here, which provides a sample-scale navigation map for selecting an ROI and performing Raman or PL spectroscopy measurements within that ROI.

[0046] For example, the microscope uses a first objective lens 11 having a low magnification of 4x, 5x, or 10x.

[0047] An electrically operated or piezoelectrically operated XY scanning stage 20 is used to drive displacement in the X and / or Y directions during the examination or visualization of the sample surface. Preferably, the microscope includes an electrically operated Z stage or a piezoelectrically operated Z stage for relative displacement along the OZ axis between the sample stage and the objective lens. A control unit 43 receives the current position of the XYZ stage in real time.

[0048] Using the first objective lens 11 of the microscope, the microscope camera 16 detects a first image 71 of the sample surface, obtained by the reflection of a white light beam off the sample surface. The microscope camera and the first objective lens are calibrated to determine a coordinate transfer function between the XY stage and the coordinates of the pixel array of the camera 16 combined with the first objective lens. Thus, each pixel of the first image 71 has a fixed position in the XY coordinate system of the sample stage. The first image 71 is stored in a virtual image space allocated within the data storage unit 45. This first image 71 is used to initialize the contents of the navigation map, as shown in Figure 2. The first image 71 is also displayed as a still image on the display device 44.

[0049] Next, the second objective lens 12 replaces the first objective lens 11. The second objective lens 12 is, for example, a high-magnification objective lens of 50x or 100x. Preferably, the first objective lens 11 and the second objective lens 12 have the same optical axis (OZ). Alternatively, calibration is used to determine the offset between the optical axis of the first objective lens 11 and the optical axis of the second objective lens 12. The processing unit 40 stores the magnification and, optionally, the offset corresponding to each objective lens. Similar to the first objective lens, the microscope camera and the second objective lens are calibrated to determine the coordinate transfer function between the XY stage and the coordinates of the pixel array of the camera 16 combined with the second objective lens. Optionally, the focus is adjusted using an electric Z-stage or a piezoelectric Z-stage.

[0050] The second objective lens 12 allows zooming in on a specific area on the sample and obtaining a second image 72 of the same sample surface by reflection of a white light beam on the sample surface. Each pixel of the second image 72 has a fixed position in the XY coordinate system of the sample stage. The CMOS video camera 16 records the zoomed area as live video or as an image. This second image 72 (static or live) is displayed on the screen 44 as shown in Figure 2 (right side). Due to the higher magnification, the second image 72 preferably exhibits a narrow field of view that is at least partially included in the field of view of the first image 71. The field of view on the camera 16 has a substantially rectangular shape.

[0051] Alternatively, the microscope system uses a single microscope objective lens to form a first image 71, which is a panoramic image, and a second image 72, which has a smaller field of view. In this case, the first image 71 is obtained by acquiring multiple images taken at multiple X,Y positions and panning these multiple images as a function of their respective positions, thereby obtaining a panoramic first image 71 that provides a large field of view of the sample.

[0052] In all cases, the first image 71 is acquired in a wide field of view, and the second image 72 is acquired in a narrow field of view of the sample. The first and second images may be displayed on separate screens. Alternatively, the first and second images may be displayed in separate windows on the same screen, for example, using a mosaic window.

[0053] Furthermore, the processing unit 40 calculates the position of a rectangular box corresponding to the field of view of the second image 72 in the coordinate system of the first image 71. The image 73 of the boundary of this rectangular box is superimposed on the first image 71 on the display device 44. Thus, a navigation map is obtained that includes the first image 71 and the superimposed rectangular box 73. The navigation map may also include the second image 72, which is simultaneously displayed next to the first image 71. The second image 72 generally provides zoom over a narrow field of view with higher spatial resolution and lower depth of field than the first image 71. The second image 72 is updated in real time as a function of the current position of the XY stage. At the same time, the position of the boundary of the rectangular box 73 is also updated in real time as a function of the current position of the XY stage.

[0054] The processing unit 40 is also used to drive actuators for moving the sample stage relative to the microscope objective lens. For example, the processing unit moves the sample stage in the X and / or Y directions. The camera 16 records the updated second image 72 in real time. The processing unit also calculates and updates the position of the rectangular box 73 on the display device 44 in real time.

[0055] The user visualizes images 71, 72, and 73 simultaneously and uses these images for easy navigation on the sample surface using any input device such as an integrated computer mouse, trackball, joystick, touchpad, or touchscreen. The user can easily use the "drag-and-move" function on the second image 72 (microscope image). The XY stage moves in real time to display the real-time second image at the desired XY position. The user can also click or touch to select an area on the first image 71 and move the sample toward that area.

[0056] Therefore, the user can easily control the relative movement on the sample surface by simultaneously viewing the first image 71, the second image 72, and the rectangular box 73.

[0057] The user can easily select a region of interest on the sample and determine the direction of movement.

[0058] As an alternative and / or supplement, the rectangular box 73 can be moved to a target position using a suitable user interface. The user interface may include, for example, a computer mouse, or a touchpad, or a touchscreen, or any other interactive device. The control unit 43 detects the movement of the rectangular box 73 on the display device 44 and calculates the corresponding displacement of the XY stage. The control unit 43 drives the scanning stage 20 to impart the corresponding displacement to the XY scanning stage. Thus, the automatic displacement of the XY stage to the target position can be controlled visually by the user interface on the display device 44.

[0059] Once the XY stage moves to the desired ROI, the user can initiate PL or Raman spectrum acquisition within the selected ROI.

[0060] Therefore, the system provides visually interactive navigation through a map of the sample. This system and method offer a distinctive experience in terms of sample exploration and analysis. It makes it extremely easy to locate areas of interest for Raman or PL measurements.

[0061] This system allows for navigating the surface of a zoomed-in sample and visualizing details on the sample surface. The sample shown in Figure 2 has a nearly flat surface and exhibits high-contrast features.

[0062] Therefore, the first image 71 of the sample obtained with a low-magnification objective lens and a large field of view provides a navigation map with a coordinate system. Simultaneously, a second image 72 of the zoomed-in sample, obtained with a high-magnification objective lens and including details of the sample surface, is positioned within the coordinate system of the first image. This technique allows the user to simultaneously view a global image of the sample and a detailed image of the sample surface within the region of interest.

[0063] However, some samples may exhibit rough or uneven surfaces and / or surface topography with features larger than the depth of focus of the microscope objective lens. Such samples generally produce at least partially blurred video images. Furthermore, using a confocal system, the first image 71 and / or the second image 72 may be at least partially blurred due to being out of focus. A blurred image of the sample hinders simple navigation on the sample surface because fine features are not visible. Determining ROIs on the sample surface and obtaining accurate Raman or PL microspectroscopy data also becomes more difficult.

[0064] A second aspect of this disclosure relates to Z-stage displacement combined with high-speed image acquisition using a CMOS camera 16 for keeping the field of view of the objective lens constantly focused on the sample surface during navigation (see Figure 3).

[0065] In this specification, this technique is referred to as NavSharp and means sharp navigation on rough, uneven or surface-relieving sample surfaces. This technique is combined with the navigation mapping technique disclosed in the first aspect to provide sharp navigation on rough, uneven or surface-relieving samples.

[0066] This system uses a computer-controlled motorized or piezoelectric XY stage. The Z stage of the motorized or piezoelectric microscope is used for displacement along the OZ axis. The control unit 43 moves the XY stage to a position selected by the user. The video camera 16 acquires a sample image at the current axial Z position. The control unit 43 moves the Z stage by a predetermined distance from the objective lens, and the camera 16 acquires another image. For each acquired image, the processing unit 42 performs image analysis. The processing algorithm is applied to the central region of the image. In the following description of the second aspect (NavSharp description), the term image analysis means that the analysis is applied to the central region of the image, consisting of several tens of pixels.

[0067] Here, the image processing unit 42 evaluates the sharpness of the image based on a combination of the mean squared difference (MSD) of the image pixel intensity in the X and Y directions in the central region of each acquired image, and its value at the corresponding axial Z position.

[0068] Within the scope of this disclosure, MSD is defined as follows: MSD=(Σ(I x+d,y -I x-d,y ) 2 +Σ(I x,y+d -I x,y-d ) 2 ) / (2*M*N)

[0069] Here, I x,y represents the image intensity of a pixel with {x,y} coordinates, Σ represents the sum of all x and y coordinates inside the central region of M×N pixels, and d represents the lateral distance parameter.

[0070] In detail, to evaluate image sharpness at different axial Z positions, the MSD for short distances (a few pixels) and long distances (tens of pixels) are calculated, respectively. By comparing the MSD over short distances for images at different axial Z positions, it becomes possible to find the maximum MSD value that defines the best focal position for the image. By comparing the MSD over long distances for out-of-focus images at different Z positions, it becomes possible to evaluate which of them is closer to the focal point. The treatment unit 42 makes a decision regarding the next Z movement based on both MSDs. If the current axial Z position is too far from the focal point, it is not possible to find the direction to the focal plane from the MSD value over long distances. In this case, the inventors use the intensity of reflected light acquired by the camera 16. The treatment unit 42 calculates the average image intensity at each axial Z position and its derivative with respect to Z. The control unit 43 moves the sample stage in the direction of the minimum value of the derivative, and in parallel, the treatment unit 42 calculates the MSD for the acquired image. Once the ratio of the MSD for a newly acquired image to the MSD for a previously acquired image exceeds a predetermined threshold level, the treatment unit 42 switches to an MSD method for focus search. In this disclosure, the inventors assert that the position where the derivative is minimized lies within the range of axial Z positions close to the focus, and that this position corresponds to a position where MSD variation is significantly observed. The inventors have empirically confirmed this statement in experiments using different types of samples.

[0071] To make a decision regarding the next movement, two or more images are required at different axial Z positions and nearly identical XY positions (shifted by less than a quarter of the analysis area). The treatment unit 42 analyzes the Z profile consisting of the MSD and mean intensity of the acquired images.

[0072] If the MSD Z profile clearly indicates that the maximum value is located within the profile zone, the control unit 43 moves the Z stage to the axial Z position corresponding to the found MSD maximum value and stores this Z focus position and the corresponding XY position. In other cases, if the MSD profile has a maximum value at the lowest or highest axial Z position within the measurement range, the control unit 43 moves the Z stage to another axial Z position below or above the measurement profile range, respectively. Image acquisition as a function of Z is resumed and the MSD profile is updated. If the maximum value is located within the measured profile range, the new MSD profile maximum value is searched using the reduced Z displacement until a Z focus position is found. The control unit 43 moves the Z stage to a new axial Z position corresponding to the found MSD maximum value and stores this Z focus position and the corresponding XY position.

[0073] If the MSD profile is nearly flat, the direction of Z movement cannot be determined. In this case, the treatment unit 42 analyzes the Z profile of average intensity. This analysis requires measurements at at least three axial Z positions with a large distance between them in order to evaluate the Z profile of average intensity. The control unit 43 moves the Z stage to these axial Z positions, and the camera 16 acquires the corresponding images. The treatment unit 42 determines the direction of movement along the OZ axis according to the minimum value of the Z profile of the derivative of average intensity. The control unit 43 moves the sample stage in this direction, and in parallel, the treatment unit 42 calculates the MSD for the acquired images and finds their maximum value, which determines the focus position. The Z stage is moved to the found focus position.

[0074] For every image acquired, the processing unit 42 calculates the MSD and average image intensity and stores them in the data storage unit 45 along with the current 3D (X,Y,Z) position. Each time the user moves the stage to a certain X,Y position, the control unit 43 checks the stored values ​​and selects those values ​​if the shift of those X,Y values ​​from the actual position is less than a quarter of the size of the central region. If such selected values ​​for different measurements are sufficient to define the focus position, the control unit 43 moves the stage directly to the predetermined position. Otherwise, the processing system 40 acquires the missing data to proceed with finding the focus position. The proposed algorithm provides a clever and effective procedure for focus discovery. When navigating over a previously scanned region of interest, the best plane of focus is selected directly.

[0075] Therefore, the processing system 40 acquires several images at various Z-axis positions by rapidly moving the Z-stage and using one or a combination of methods to detect each of the Z-values ​​of the next potential target candidates, select the sharpest plane, and then move the stage, so that the user can automatically view the sample in the best focus.

[0076] Thus, the sharp navigation method enables automatic focusing on the sample surface within the central region of the image by adjusting the controllable Z-axis position in real time. This automatic focusing is applied during XY stage movement and real-time updating of acquired images. Furthermore, the user can select an ROI that is not in the center of the image, use the drag-and-move function to center the ROI in the camera's field of view, and thereby perform automatic focusing on the selected ROI.

[0077] Furthermore, to speed up the selection of the focal plane and facilitate its discovery, the Z-direction for determining the focal position is determined using the Z-profile of the derivative of the average intensity of the image in reflected light.

[0078] Detecting the Z-axis position of the optimal focus allows for easy navigation on the sample surface, especially when using a confocal hole and / or when the sample has a rough, uneven, or undulating surface.

[0079] This method allows users to easily locate regions of interest (ROIs) on a sample. It also enables visualization of surface details of the sample using an objective lens and camera with a limited field of view, and a motorized or piezoelectric XY stage.

[0080] As an example, Figure 3 shows a first image 71 of a gypsum mineral sample in the right corner of the display device. The gypsum sample has an uneven surface and complex surface topography. Figure 3 also shows a second image 72 of a zoomed-in area on the same gypsum sample, and a rectangular box 73 superimposed on the first image, showing the area corresponding to the field of view of the second image 72. Figure 3 also shows the position of pixel 56 at the center of the second image. The second image 72 shown here is in focus on the central area. However, the second image 72 appears blurred at other X,Y positions outside its central area. Figure 3 demonstrates the effectiveness of sharp navigation techniques for navigating on complex sample surfaces.

[0081] On the second image 72, the user can graphically define regions 74 corresponding to ROIs for Raman or PL measurements.

[0082] The example in Figure 3 shows that for samples with a Z variation greater than the depth of field of objective lenses 11, 12 and a rough, irregular, and uneven surface topography, the sample image is in focus only in the central region and blurred in other parts, thereby hindering navigation and accurate determination of ROI, as well as limiting access to the sample surface for PL and / or Raman measurements.

[0083] Accordingly, a third aspect of the present disclosure has been developed and a method is provided for visualizing the sample surface across the entire field of view.

[0084] This third aspect is called view sharp, meaning a sharper view. This is used in combination with sharp navigation and navigation maps, as detailed above.

[0085] This system uses a computer-controlled, motorized or piezoelectrically operated microscope Z-stage to displace along the Z-axis and position the sample to be explored at the desired height. The system also uses a camera 16 for rapid, high-resolution image acquisition. One of the objective lenses 11 or 12 is selected to proceed with image acquisition in white light in a wide-field microscope configuration, from a user-defined Z-range. Depending on the Z-variation of the sample surface, the user selects the z-scanning range from the options of "short," "standard," "wide," and "maximum." The minimum value, zmin, and maximum value, zmax, of these ranges, as well as the scan step, ΔZ, are predefined depending on the selected objective lens and surface roughness.

[0086] The control unit 43 scans the sample surface within the selected Z range using a motorized or piezoelectrically operated microscope Z-stage, using a selected ΔZ step. For each Z position, the video camera 16 acquires images 131, 132, 133, 134, and 135 of the same XY region of the sample. In other words, the relative XY position of the sample remains unchanged during the Z scan. Figure 4A shows, for example, image 131 of the sample region captured in a plane at Z=140 μm. Figure 4B shows another image 132 of the same sample region captured in a plane at Z=70 μm. Figure 4C shows another image 133 of the same sample region captured in a reference plane at Z=0 μm. Figure 4D shows another image 134 of the same sample region captured in a plane at Z=-70 μm. Also, Figure 4E shows another image 135 of the same sample region captured in a plane at Z=-140 μm.

[0087] Image stacks acquired at multiple axial Z positions above and below the reference plane or work plane corresponding to the central focus region are stored in the data storage unit 45. Each acquired image includes a focused region and an out-of-focus or blurred region. These image stacks contain information about the entire focal sample surface inside the field of view, and this information can be extracted and presented as a 2D image if information about the pixel's Z position is not taken into consideration, or as a 3D image if the image topography is reproduced using the pixel's Z position information. Pixel values ​​stored in the form of image stacks can be represented in column vector form for any X,Y position. The column vector corresponding to an X,Y position, i.e., the X,Y column vector, consists of pixel intensity values ​​at all Z positions used for image acquisition for the same X,Y position. For a field of view characterized by N×M pixels, all pixels in the acquired image stack will be organized into N×M column vectors.

[0088] The image processing unit 42 analyzes all acquired images. Therefore, after acquiring a stack of sample images, real-time processing of them follows. Each acquired image is processed to selectively remove noise and artifacts, and then sharpened by applying a Laplacian operator in the Z direction. Each pixel value l(z) is replaced by l(z) + dl(z), where dl(z) is the local derivative calculated on the pixel binning region. More precisely, dl(z) is calculated as dI(z) = (I(z) × 2 - I(z-1) - I(z+1)) × coefficient, where the coefficient corresponds to different levels of sharpness. This operation amplifies the observed sharpness to facilitate the discovery of maximum sharpness. Depending on the mathematical representation of the Laplacian operator, it is possible to sharpen the acquired image, represented by the intensity value matrix I(X,Y,Z), to different levels. The sharpened pixel values ​​can then be used for the final ViewSharp image reconstruction.

[0089] The image processing unit 42 applies pixel binning to all images to improve sharpness detection. The pixel binning area, for example, 3x3 pixels or 5x5 pixels, is used to calculate the MSD value. The obtained MSD value is assigned to the corresponding X,Y column vector at the corresponding Z position. The column vector thus calculated represents the X,Y MSD Z profile. The MSD Z profile is analyzed for its first and second maximum values, and based on a confidence level criterion, the found first maximum value is selected or not selected. The processing unit 42 calculates a confidence value for each of the found first maximum values. Here, the confidence value, called CV (confidence value), is defined as the ratio of the first MSD maximum value to the sum of the second MSD maximum value and the average MSD value.

number

[0090] Based on the data obtained from the experiment, confidence levels (CL) equivalent to 2 (low), 3 (medium), and 4 (high) are introduced. The maximum selection criterion is defined as follows: If the calculated CV is higher than the CL, the treatment unit (42) selects the maximum value. Selecting a low confidence level may result in noise and false positive signals in the maximum MSD value. In contrast, selecting a very high confidence level may result in failure to detect any maximum MSD value. For surfaces that are uneven, bright, and uniformly illuminated, a low or medium confidence level may be sufficient to find the maximum sharpness and reconstruct a sharp image of the entire field of view. For smooth or poorly illuminated surfaces, the treatment unit 42 uses a high confidence level, as the CV may not be high enough to overcome the CL. In this case, interpolation is used to determine the MSD value using the maximum adjacent X,Y MSD values ​​found for the same high confidence level.

[0091] Defining the criteria for selecting maximum sharpness is important because it affects the accuracy of sample surface determination. The criteria also define the time required to run the ViewSharp procedure. This criterion effectively defines the computer's memory and processor resources used for efficient procedure execution.

[0092] The processing unit 42 performs post-processing on the measured MSD maximum value, which represents the maximum sharpness. Optionally, smoothing is applied to remove artifact MSD values. For any MSD maximum value found, the processing unit 42 captures the corresponding X, Y, and Z coordinates and intensity values. The obtained coordinates can be used to present the sample surface topography Z(X,Y). Because the control system 40 uses a ΔZ step to scan the sample surface, the captured Z position values ​​are spaced along the Z axis, thereby forming terraces on the sample topography image. To approach the actual surface, an interpolation step along the OZ axis is applied based on the analysis of the nearest MSD value in the OZ direction. Once interpolation along the Z axis is performed, a continuous sample topography is obtained. The obtained sample surface topography image is reduced in the X and Y directions by pixel binning during the sharpness calculation procedure. To restore the obtained sample topography image to its original size with high spatial resolution, the treatment unit 42 applies another interpolation procedure along the X and Y directions to restore all Z values ​​corresponding to the original pixel positions. The treatment unit 42 provides a full-focus surface topography image, where the new image has the same number of pixels as the original image. Using the X, Y, and Z values ​​thus obtained, full-focus intensity 2D and 3D sample surface images are constructed, where the new pixel intensity values ​​correspond to the intensity values ​​of the maximum sharpness found or the interpolated intermediate intensity values, respectively.

[0093] The obtained X, Y, Z and corresponding intensity values ​​are stored in the memory unit. The surface topography or Z-map of the sample surface is stored in memory by unit 45 and visualized by the display unit 44. Full-focus 2D and 3D sample surface intensity images are stored in memory by unit 45 and visualized by the display unit 44. Figure 4F shows an example of such a 2D reconstructed image 141. The reconstructed image 141 appears sharp across the entire field of view. This reconstructed image 141 can at least partially replace the first image 71 and / or the second image 72 displayed on the display device 44. The reconstructed image 141 allows for easy navigation on a sample surface having a rough surface texture and / or non-planar surface topography. The reconstructed image 141 can also be used to update at least a portion of the first image in the navigation map. Thus, the reconstructed image makes it possible to visualize details of the sample surface and more accurately determine the ROI for Raman or PL measurements.

[0094] Figure 5 shows an example of a 2D all-focus surface intensity image of another sample. The image shown corresponds to the field of view of camera 16. All image surfaces can be seen in sharp detail. All blurred, out-of-focus image areas have been replaced with sharp, in-focus image areas. Figure 6 shows a reconstructed surface topography image of the same sample. Sample surface topography images can provide an overall image of the sample, thus allowing for the visualization of surface details and overall evaluation of the method's performance.

[0095] Figure 7 shows another example of a 2D all-focus surface intensity image. A very sharp image of the sample surface can be seen where the Z variation is many times the field of view of the objective lens. Figure 8 shows the corresponding 3D image surface intensity profile based on the captured surface focus Z position. This image represents the detail of the surface relief and allows for precise selection of ROIs in 3D space. The selected ROIs are used to acquire Raman or PL spectra at any selected pixel. Thus, Raman or PL spectroscopic measurements with high horizontal and axial resolution are obtained. The Raman or PL spectra are acquired using a spectrometer 55 and a CCD camera 60.

[0096] Raman or PL mapping is performed by scanning each pixel of the ROI using the found Z value, so that spectra from every pixel are acquired confocally. Alternatively, the maximum or integral value of the spectrum can be used as the pixel value for image sharpening. In fact, if the excitation laser light does not penetrate into the sample, all-focus image reconstruction can be performed using the Raman response with contrast or intensity values ​​instead of white light reflection.

[0097] Figure 9 shows another example of a sharpened image processed according to the above disclosure. White light reflectance is used to generate a 3D surface profile using the ViewSharp method disclosed herein. Figure 10 shows the corresponding Raman mapping measured point by point at the focused Z-axis position determined from the topography map. The Raman spectral map is observed to have excellent spatial resolution. A comparison of Figure 9 and Figure 10 shows that considering Raman or PL measurements that provide additional information about chemical composition makes it possible to analyze morphological features clearly revealed by topography.

Claims

1. An optical microscope (10) comprising a sample holder for holding the sample to be analyzed, and at least one microscope objective lens (11, 12) for defining the optical axis (OZ), A spectroscopic system (50) comprising a light source (51) adapted to generate an excitation light beam, a spectrometer (55), and a detection system (60), An optical system (14) adapted to guide the excitation light beam onto the sample through at least one microscope objective lens (11, 12) and to collect a Raman or photoluminescent light beam generated by scattering of the excitation light beam on the sample, wherein the optical system (14) is adapted to guide the Raman or photoluminescent light beam toward the spectrometer (55) and the detection system (60), An optical microspectroscopy system comprising: an actuation system (20) for driving relative lateral (dX, dY) and axial (dZ) movement between the sample holder and the at least one microscope objective lens (11, 12); The aforementioned optical microspectroscopy system is Another light source (18) for generating an illumination light beam, An imaging system (16, 41) is arranged in combination with the at least one microscope objective lens (11, 12) and is configured to acquire a plurality of images formed by the reflection or transmission of the illumination light beam from the sample surface at a plurality of axial positions (Z) along the optical axis (OZ) using a selected ΔZ step, wherein the plurality of images form an image stack. The system comprises an image processing unit (42) and a processing system (40) including a data storage unit (45), The data storage unit (45) stores the image stack in the form of column vectors of all X,Y positions, i.e., X,Y column vectors, where each X,Y column vector consists of pixel intensity values ​​at all Z positions used to acquire the image stack for the same X,Y position. The aforementioned image processing unit is configured to perform the following: - Input the data of the image stack stored in the data storage unit (45); - Apply pixel binning to all images in the image stack to form pixel binning regions; - Using the pixel binning region of one image in the aforementioned image stack, the MSD value is calculated using the following formula: Here, I ｘ，ｙ represents an image of pixel intensity values ​​at {x,y} coordinates, and Σ represents M × N pixels. This represents the sum of all x and y coordinates inside the central region, where d is horizontal. Represents the direction distance parameter; - Assign the calculated MSD value to the corresponding X,Y column vector of the corresponding Z position. Form the X, Y MSD Z profile; - By analyzing the X,Y MSD Z profile, the first and second maximum values ​​of MSD(Z) are found; The confidence value CV, defined by the ratio of the first maximum value of MSD(Z) to the sum of the second maximum value of MSD(Z) and the mean MSD value, is calculated according to the following formula: - After or before calculating the CV, select one confidence level CL from the pre-established confidence levels CL1 (2 (low)), CL2 (3 (medium)), and CL3 (4 (high)); If the calculated CV is higher than CL, select the first maximum value of the MSD(Z); If the calculated CV is lower than CL, apply interpolation to the maximum MSD value found in adjacent X,Y and determine MSD(Z)max using the same CL; - For the first maximum value of the MSD(Z) found, the corresponding X, Y, Z coordinates and The pixel intensity values ​​are captured, and the resulting X, Y, Z coordinates represent the sample surface topography Z(X,Y); - Apply interpolation steps along the optical axis (OZ) to obtain a continuous sample surface topography; and - Applying a different interpolation procedure along the X and Y directions, the resulting sample topography image is restored to its original size with high spatial resolution. The image processing unit (42) is configured to construct a full-focus sample surface topography image representing the sample surface in three and / or two dimensions by restoring the X, Y, Z coordinates of the obtained sample surface topography image to their original size with high spatial resolution, where the pixel intensity value of each new pixel corresponds to the pixel intensity value of the maximum sharpness discovered or the interpolated intermediate pixel intensity value. The data storage unit (45) stores the maximum sharpness and the corresponding pixel intensity value. The aforementioned optical microspectroscopy system further A display system (44) configured to display a sample topography image, The system includes a user interface configured to select a region of interest within the sample topography image, The operating system (20) is configured to position the region of interest to receive the excitation light beam, and The spectroscopic system (50) is configured to acquire Raman or photoluminescence signals generated from the region of interest for each axial Z-focal position determined from the sample topography image. Optical microspectroscopy system.

2. The optical microspectroscopy system according to claim 1, wherein the imaging system (16) is configured to acquire a first image (71) of the sample and a second image (72) of the sample, the first image (71) and the second image (72) are formed by the reflection or transmission of the illumination light beam from the surface of the sample, the first image (71) has a large field of view and the second image (72) has a small field of view, and the display system (44) is configured to display the first image (71) and the second image (72) and to display a graphic representation (73) of the region overlapping the first image.

3. The optical microspectroscopy system according to claim 2, wherein the imaging system (16, 41) is adapted to acquire the second image (72) in real time, the processing system (40) is adapted to update the region corresponding to the second image (72) in real time, and the display system (44) is adapted to display the second image (72) in real time and the graphic representation (73) of the updated region in real time.

4. The optical microspectroscopy system according to claim 3, wherein the user interface comprises an integrated computer mouse, trackball, joystick, touchpad and / or touchscreen, and the user interface is adapted to interact with the sample topography image, the first image (71), or the second image (72), respectively, using drag-and-move operations, thereby moving the large field in the first image (71) or the small field in the second image (72), respectively, by a predetermined amount of horizontal displacement, and the actuation system (20) is adapted to drive relative horizontal motion (dX, dY) in the sample topography image, in the first image (71), or in the second image (72), respectively, proportional to the predetermined amount of horizontal displacement.

5. The steps include determining the current position of the sample stage in the optical microscope, The steps include generating an illumination light beam directed toward a sample placed on the sample stage, The steps include selecting the microscope objective lenses (11, 12) on the optical microscope (10), The steps include: using an actuator system (20) for driving relative lateral (dX, dY) and axial (dZ) movement between a sample holder and at least one microscope objective lens (11, 12), acquiring a plurality of images formed by the reflection or transmission of the illumination light beam from the sample surface at a plurality of axial positions (Z) along the optical axis (OZ), wherein the plurality of images form an image stack; The data storage unit (45) stores the image stack in the form of column vectors of all X,Y positions, i.e., X,Y column vectors, where each X,Y column vector consists of pixel intensity values ​​at all Z positions used to acquire the image stack. The steps involve using an image processing unit (42) configured to perform the following: - Input the data of the image stack stored in the data storage unit (45); - Apply pixel binning to all images in the image stack to form pixel binning regions; - Using the pixel binning region of one image in the aforementioned image stack, the MSD value is calculated using the following formula: Here, I ｘ，ｙ represents an image of pixel intensity values ​​at {x,y} coordinates, and Σ represents M × N pixels. This represents the sum of all x and y coordinates inside the central region, where d represents the lateral distance parameter; - Assign the calculated MSD values ​​to the corresponding X,Y column vectors at the corresponding Z positions to form an X,Y MSD Z profile; Find the first and second local maximums of MSD(Z); The confidence value CV, defined by the ratio of the first maximum value of MSD(Z) to the sum of the second maximum value of MSD(Z) and the mean MSD value, is calculated according to the following formula: - After or before calculating the CV, select one confidence level CL from the pre-established confidence levels CL1 (2 (low)), CL2 (3 (medium)), and CL3 (4 (high)); If the calculated CV is higher than CL, select the first maximum value of the MSD(Z); If the calculated CV is lower than CL, apply interpolation to the maximum MSD value found in adjacent X,Y and determine MSD(Z)max using the same CL; - For the first maximum value of the MSD(Z) found, the corresponding X, Y, Z coordinates and The pixel intensity values ​​are captured, and the resulting X, Y, Z coordinates represent the sample surface topography Z(X,Y); - Apply interpolation steps along the optical axis (OZ) to obtain a continuous sample surface topography; and - Applying a different interpolation procedure along the X and Y directions, the resulting sample topography image is restored to its original size with high spatial resolution. The steps include displaying the sample topography image on a display system (44), The steps include selecting a region of interest within the sample topography image displayed on the display system (44) using a user interface, The steps include: positioning the sample using the operating system (20) so as to guide the excitation light beam through the microscope onto the region of interest of the sample, and collecting the Raman or photoluminescent light beam generated by the scattering of the excitation light beam on the region of interest of the sample; The steps include using a spectrometer system (55, 60) to acquire a Raman or photoluminescence signal generated from the region of interest for each axial Z-focus position determined from the sample topography image, A microscopic spectroscopy measurement method that includes this.

6. A step of obtaining a first image (71) and a second image (72) of the sample formed by the reflection or transmission of an illumination light beam from the surface of the sample, wherein the first image (71) has a large field of view and the second image (72) has a small field of view. The steps include storing the first image (71) in a data storage device (45), The steps include displaying the first image (71) on a display system (44), The steps include displaying the second image (72) on the display system (44), A step of determining the region corresponding to the small field of view of the second image (72) in the coordinate system of the first image (71), The steps include displaying a graphic representation (73) of the region that overlaps the first image, It further includes, The step of acquiring the first image (71) and / or the second image (72) is performed at the in-focus position. The microspectroscopy measurement method according to claim 5.

Images