Apparatus and method for beam scanning microspectroscopy

Abstract

The invention relates to a device for beam scanning microspectroscopy, the device comprising a microscope objective (5) and a moving system (100) for moving an excitation beam (10). According to the invention, the moving system (100) for moving the excitation beam (10) comprises: a first focusing optical component (M1, L1) suitable for focusing the excitation beam (10) in an intermediate focal plane (20); another focusing optical component (M3, L3) suitable for forming an image of the intermediate focal plane (20) in a focal plane (51, 52) of the microscope objective (5); and a single scanning mirror (M2) arranged between the first optical component (M1, L1) and the intermediate focal plane (20), the scanning mirror (M2) being mounted on a stage and the direction of the scanning mirror (M2) being adjustable so as to move the image of the focus (61, 62) in two lateral directions in the object focal plane (51) or the image focal plane (52) of the microscope objective.

Description

Technical Field

[0001] The present invention generally relates to the field of microspectroscopy devices and methods, wherein an excitation light beam is located or moved on a sample through a microscope objective and wherein the light beam formed by scattering and / or reflection on the sample is collected for spectral analysis.

[0002] More specifically, the present invention relates to a laser beam scanning Raman microspectroscopy device and method, and also to a total internal reflection fluorescence microscope device and method. Background Art

[0003] A method and system for spectral imaging of a sample by beam scanning is known in particular from document EP198332A. More precisely, document EP1983332A describes a scanning device placed in the tube of a confocal microscope so as to be inserted between the microscope objective and the injection suppression filter of a Raman spectrometer. The scanning device comprises a double galvanometer mirror arranged in series in the optical path of the laser beam. The double galvanometer mirror has rotation axes orthogonal to each other to angularly move the laser beam in orthogonal directions on the sample surface. The double mirror optical system makes it possible to angularly move the excitation laser beam so as to position the excitation laser beam at different points on the sample surface. By the reverse return of light, this double mirror optical system can collect the Raman backscattered beam in order to transmit it to a detection system comprising a Raman spectrometer. The advantage of this double galvanometer mirror system is that the laser source and the detection system remain fixed. This device can obtain an image of a portion of the sample surface by Raman spectroscopy with a resolution of approximately 50×50 points in about ten minutes.

[0004] Other patent documents describe beam scanning microscope devices (see, for example, WO 2010 / 069987, US 2005 / 128476 or JP 2001 / 091848).

[0005] Document WO2015 / 159035A1 describes another beam scanning or beam angular motion microscopy device and method, comprising at least one first mirror and a second mirror, which are arranged in series on the optical path of the light beam between the light source and the microscope objective, the first mirror is tilted according to a first predetermined rotation angle, and the second mirror is tilted according to a second rotation angle, so that the axis of the light beam pivots around the center of the microscope objective pupil. The system can increase the beam scanning area on the sample without vignetting effect. By the reverse return of light, the system can collect a large amount of Raman flow. However, the system requires synchronization between the tilt of the first mirror and the second mirror arranged in series, and is relatively expensive. Summary of the invention

[0006] An object of the present invention is to provide a device and method for microspectroscopy with moving or scanning excitation laser beam, wherein the scanning system is simple and cheap while being able to scan a spatially and / or angularly extended area on the sample.

[0007] Another object of the invention is to increase the spatial extent of the measurement field without changing the spatial resolution of the measurement or the measurement quality.

[0008] Another object of the present invention is to improve the quality of Raman or fluorescence microspectroscopy measurements.

[0009] Another object of the invention is to reduce the acquisition duration of Raman or fluorescence microspectroscopy measurements obtained at a defined point of a sample or by scanning a sample area.

[0010] In order to remedy the above-mentioned shortcomings of the prior art, the present invention proposes a beam scanning micro-spectroscopy device, comprising: at least one light source, suitable for emitting an excitation light beam; a microscope objective, arranged along the main optical axis of the micro-spectroscopy device, the microscope objective having an object focal plane and an image focal plane; a system for moving the excitation light beam along two spatial directions X and Y transverse to the optical axis of the light beam, the microscope objective and the moving system being suitable for moving the excitation light beam on the sample; an optical system, suitable for collecting a light beam formed by reflection, scattering and / or transmission of the excitation light beam on the sample; and a spectral detection system, the spectral detection system being suitable for receiving the light beam formed by reflection, scattering and / or transmission.

[0011] More specifically, according to the present invention, an excitation beam moving system is proposed, which includes: a first focusing optical component, suitable for receiving an excitation beam and focusing the excitation beam to a focus in an intermediate focal plane; another focusing optical component, arranged in the optical path of the beam between the intermediate focal plane and the microscope objective, the other focusing optical component being suitable for forming an image of the intermediate focal plane in the object focal plane or the image focal plane of the microscope objective, respectively; and a single scanning mirror, arranged in the optical path of the excitation beam between the first focusing optical component and the intermediate focal plane, the scanning mirror being a plane and mounted on a stage, rotatable around two lateral rotation axes, the two rotation axes being within the plane of the scanning mirror, the scanning mirror being suitable for moving the focus in two lateral directions in the intermediate focal plane, so as to move the image of the focus in two lateral directions in the object focal plane or the image focal plane of the microscope objective, respectively.

[0012] Further non-limiting and advantageous features of the beam scanning microspectroscopy device according to the invention are as follows, alone or in all technically possible combinations:

[0013] - the first focusing optic is a toric mirror;

[0014] - a first toric mirror focusing optic adapted to reduce geometrical optical aberrations in an intermediate focal plane;

[0015] - the first focusing optics comprises at least one lens;

[0016] - the first focusing optics is mounted on a rail adapted to adjust the distance between the first focusing optics and the microscope objective;

[0017] - the other focusing optical component is a spherical mirror, a toric mirror or a lens;

[0018] - another focusing optical component is mounted on another guide rail, the other guide rail being suitable for adjusting the distance between the other focusing optical component and the microscope objective;

[0019] - the scanning mirror is arranged to receive the excitation light beam with an incident angle less than or equal to 15 degrees;

[0020] - the device further comprises a first plane deflection mirror and / or a further plane deflection mirror, the first plane deflection mirror being arranged upstream of the first focusing optics and / or the further plane deflection mirror being arranged between the further focusing optics and the microscope objective, respectively;

[0021] - The rotatable stage includes a two-axis rotary actuator of the piezoelectric or voice coil type;

[0022] - said at least one light source comprises one or more sources of laser source type and / or light emitting diode type;

[0023] - the microspectroscopy device comprises a confocal aperture arranged upstream of the first focusing optics to collimate the excitation beam incident on the first focusing optics;

[0024] - The spectral detection system includes a Raman spectrometer, a coherent anti-Stokes Raman spectrometer, a fluorescence spectrometer, a photoluminescence spectrometer, a cathodoluminescence spectrometer, a hyperspectral camera, a spectral filter or a tunable bandpass filter;

[0025] - a spectral detection system adapted to measure and analyze the light beam formed by reflection, scattering and / or transmission according to the movement of the excitation light beam on the sample;

[0026] - the microscope objective is a total internal reflection objective and wherein the further focusing optical component is adapted to form an image of the intermediate focal plane in the object focal plane of the total internal reflection objective in order to angularly shift the collimated excitation beam in the image focal plane and collect the total internal reflection beam on the sample.

[0027] The present invention also proposes a beam scanning micro-spectroscopy, comprising the following steps: emitting an excitation beam through a light source; guiding the excitation beam toward a first focusing optical component and reflecting the excitation beam onto a single plane scanning mirror so that after the excitation beam is reflected on the scanning mirror, the excitation beam is focused to a focus in an intermediate focal plane, guiding the excitation beam toward another focusing optical component, and then guiding the excitation beam toward a microscope objective so as to form an image of the intermediate focal plane in an object focal plane or an image focal plane of the microscope objective, respectively, and tilting the scanning mirror around two lateral rotation axes to move the focus in two lateral directions in the intermediate focal plane so as to move the image of the focus in two lateral directions in the object focal plane or the image focal plane of the microscope objective, respectively; collecting a light beam formed by reflection, scattering and / or transmission of the excitation beam, and receiving the light beam formed by reflection, scattering and / or transmission on a spectral detection system.

[0028] The excitation beam movement system and method according to the present invention can locate and / or scan the position or tilt of the excitation beam in the front focal plane of the microscope objective. The movement system is based on the same scanning mirror. The system is simpler, faster and cheaper than the prior art systems based on at least two scanning mirrors arranged in series. In addition, this movement system provides an alternative solution to the problem of beam vignetting during beam scanning.

[0029] The systems and methods of the present invention are applicable to spectral measurements by reflection, transmission, backscattering or forward scattering.

[0030] Of course, the different features, alternatives and embodiments of the invention can be associated with each other according to various combinations, as long as they are not compatible or do not exclude each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following description given by way of non-limiting example in relation to the accompanying drawings will allow a good understanding of what the invention consists of and the manner in which it is implemented. Moreover, various other features of the invention emerge from the additional description made with reference to the accompanying drawings, which illustrate non-limiting embodiments of the invention, in which:

[0032] Figure 1 Schematically shows a beam scanning system according to a first embodiment of the present invention;

[0033] Figure 2 A plane mirror rotatable about two lateral rotation axes mounted on a stage is schematically shown;

[0034] Figure 3 An example of beam scanning in the image focal plane of a microscope objective is shown;

[0035] Figure 4Another example of beam scanning in the object focal plane of a microscope objective is shown;

[0036] Figure 5 An alternative to the first embodiment is shown, in which a beam scanning system is used for transmission measurements;

[0037] Figure 6 Another alternative to the first embodiment is shown, in which the beam scanning system is combined with a spectrometer for transmission spectrum measurement;

[0038] Figure 7 A further alternative to the first embodiment is shown, in which the beam scanning system is combined with a two-dimensional sensor for transmission measurement;

[0039] Figure 8 A further alternative to the first embodiment is shown, in which a beam scanning system is used for both the incident beam and the transmitted beam.

[0040] It should be noted that in these Figures, structural and / or functional elements common to different alternatives may be denoted by the same reference numerals. DETAILED DESCRIPTION

[0041] exist Figure 1 In FIG. 1 , a beam scanning system 100 is shown, which is intended to be inserted between a light source 1 and a microscope objective 5 .

[0042] An (XYZ) orthogonal reference system is shown. Figure 2 The plane is the (YZ) plane.

[0043] As a non-limiting example, the microscope is of the confocal type and comprises a confocal aperture 43. The confocal aperture 43 is arranged in a plane conjugate to the focal plane of the microscope objective 5. For example, the confocal aperture 43 is arranged in a plane at the focus of the lens 42.

[0044] The scanning system of this article includes a first deflecting mirror M0, a first focusing mirror M1, a scanning mirror M2, a second focusing mirror M3 and a second deflecting mirror M4.

[0045] The deflection mirrors M0 and M4 are plane mirrors and are used only for folding the excitation beam while maintaining the optical alignment between the excitation beam 10 and the microscope objective 5 along the main optical axis 11 of the microscope.

[0046] The advantage is that Figure 1The beam scanning system 100 forms an optional module between the diaphragm 2 and the microscope objective 5 that can be inserted into and / or removed from the confocal section of the microscope. The excitation light beam 10 is collimated and propagates in a direction parallel to the Z axis. It is particularly advantageous that the insertion or removal of the optional module into or from the confocal section of the microscope does not modify the optical adjustment of the confocal microscope. The optional module can be inserted or removed as needed without modifying the optical alignment of the light source 1, the confocal aperture 43 and the objective 5.

[0047] The first deflecting mirror M0 receives the collimated excitation light beam 10 and reflects the excitation light beam 10 toward the first focusing mirror M1. For example, the first deflecting mirror M0 has a size of at least 15 mm×15 mm and a thickness of 6 mm.

[0048] Advantageously, the first deflecting mirror M0 is mounted on a conventional point-line-plane bracket by means of adjustment screws on the front face of the approach mirror M0. For fine-tuning, an adjustment range of + / -1 degree is sufficient on two rotation axes (for example, one rotation axis is parallel to the X-axis and the other rotation axis is perpendicular to the X-axis). Advantageously, the first deflecting mirror M0 can be adjusted in position at a depth of + / -1 mm along the normal of the deflecting mirror M0. The angle of incidence of the excitation light beam 10 relative to the normal of the M0 mirror is preferably adjustable in a range between 51 degrees and 57 degrees (for example, a value of approximately 54 degrees). After reflection on the deflecting mirror M0, the excitation light beam 10 propagates along the optical axis 110.

[0049] The first focusing mirror M1 is preferably a complex curved mirror or a toroidal mirror. The first focusing mirror M1 has a first radius of curvature r1 around the Z axis and a second radius of curvature r2 around the X axis. The first focusing mirror M1 is, for example, rectangular, and the size of the first focusing mirror M1 is at least 15 mm×10 mm and the thickness is 6 mm. The incident angle of the excitation light beam 10 relative to the normal of the first focusing mirror M1 is about 11 degrees. The first focusing mirror M1 actually focuses the excitation light beam 10 to a focus in an intermediate focal plane 20 arranged perpendicular to the optical axis. In addition, the complex curved shape of the first focusing mirror M1 is calculated, and the spherical aberration in the focal plane 6 caused by the mirror M1 and the mirror M3 is corrected accordingly.

[0050] Advantageously, the first focusing mirror M1 can be mounted on a conventional point-line-plane mount by means of an adjustment screw close to the rear of the mirror M1. For fine adjustments, an adjustment range of + / -1 degree on two rotation axes (e.g., one rotation axis parallel to the X-axis and the other rotation axis perpendicular to the X-axis) is sufficient. Advantageously, fine adjustments to a depth of + / -1.5 mm along the normal to the mirror are also available. Furthermore, it is desirable that rotation of the mirror M1 is available so as to coarsely and / or finely adjust the angle of incidence of the excitation beam on the mirror M1 to a value in the range of approximately 8 degrees to 14 degrees relative to the optical axis 110.

[0051] According to an alternative, the first focusing mirror M1 is mounted to a translation rail 31 oriented according to an axis (in the YZ plane) perpendicular to the optical axis 11 (in other words, oriented along the Y axis). The movement of the first focusing mirror M1 on this rail 31 makes it possible to adjust the distance between the first focusing mirror M1 and the main optical axis 11 of the microscope. As a non-limiting example, a rough adjustment of the depth of the mirror M1 can adjust the position of the first focusing mirror M1 to a distance of about 50 mm (-17 mm / +33 mm) from the main optical axis 11 of the microscope.

[0052] A single scanning mirror M2 is arranged on the optical path of the excitation light beam 10 between the first focusing mirror M1 and the intermediate focal plane 20 .

[0053] Therefore, the first focusing mirror M1 receives the collimated excitation light beam 10 and reflects the excitation light beam 10 toward the scanning mirror M2 while focusing the excitation light beam 10 to a focus 21 in the intermediate focal plane 20 .

[0054] Figure 2 An example of a scanning mirror M2 with two lateral rotation axes 325, 326 mounted on a stage 32 is shown. The scanning mirror M2 is a plane mirror. The two rotation axes 325, 326 are in the plane of the scanning mirror M2. For example, one rotation axis 325 is parallel to the X-axis and the other rotation axis 326 is approximately parallel to the Z-axis. Actuators 321, 322, 323, and 324 allow the mirror M2 to rotate around the rotation axes 325 and / or 326. According to a preferred embodiment, actuators 321, 322, 323, and 324 are piezoelectric or voice coil type actuators.

[0055] The incident angle of the excitation light beam 10 on the scanning mirror M2 is preferably less than or equal to 15 degrees (eg, approximately 8 degrees).

[0056] The rotation of the mirror M2 about the rotation axis 325 parallel to the X axis actually moves the focus 21 in the intermediate focal plane 20 transversely to the X axis towards another focus 22, 23, 24 or 25. In a first approximation, the focus 21, the focus 22, the focus 23, the focus 24 and the focus 25 are located in the intermediate focal plane 20. Similarly, the rotation of the mirror M2 about the rotation axis 326 parallel to the Z axis actually moves the focus 21 in the intermediate focal plane 20 transversely to the Z axis. In this way, the scanning mirror M2 can move the focus 21 in the intermediate focal plane 20 in two directions transverse to the intermediate focal plane. The first focusing mirror M1 is suitable for correcting the spherical aberration caused by the mirror M1 and the mirror M3.

[0057] exist Figure 1In the example shown, when mirror M2 focuses the light beam to focal point 21, the incident light beam propagates along optical axis 111. Similarly, when mirror M2 focuses the light beam to focal point 22, focal point 23, focal point 24, or focal point 25, respectively, the incident light beam propagates along optical axis 112, optical axis 113, optical axis 114, or optical axis 115, respectively.

[0058] During the scanning period of the mirror M2, the rotation amplitude about each rotation axis is approximately 6 degrees.

[0059] The second focusing mirror M3 is preferably a spherical mirror. Figure 1 In the example shown, the second focusing mirror M3 has a radius of curvature of 300 mm. The position and size of the second focusing mirror M3 are determined according to the aperture of the microscope objective 5 and the scanning range of the mirror M2 to limit the vignetting effect of the light beam on the edge of the mirror M3. The incident angle of the excitation light beam 10 relative to the normal of the second focusing mirror M3 is about 6 degrees. The second focusing mirror M3 can collect the incident light beam reflected by the scanning mirror M2, regardless of the position of the focus in the intermediate focal plane during the scanning of the scanning mirror M2. The second focusing mirror M3 reflects the incident light beam toward the second deflection mirror M4 and / or the microscope objective 5. Alternatively, the second focusing mirror M3 is a complex curved mirror. In this case, the mirror M1 corrects a part of the optical aberrations, and the second focusing mirror M3 is suitable for correcting another part of the optical aberrations.

[0060] Advantageously, the second focusing mirror M3 can be mounted on a conventional point-line-plane mount by means of an adjustment screw close to the rear of the mirror M3. For fine adjustments, an adjustment range of + / -1 degree on two rotation axes (e.g., one rotation axis is parallel to the X-axis and the other rotation axis is perpendicular to the X-axis) is sufficient. Advantageously, a fine adjustment of depth + / -1.5 mm is also available. Furthermore, it is desirable that rotation of the mirror M3 is available so as to coarsely and / or finely adjust the angle of incidence of the excitation beam on the mirror M3 to a value in the range of approximately 5.75 degrees to 7.5 degrees relative to the optical axis 110.

[0061] According to an alternative, the second focusing mirror M3 is mounted on a translation rail 33. The movement of the second focusing mirror M3 on this rail 33 makes it possible to adjust the distance between the second focusing mirror M3 and the main optical axis 11 of the microscope. As a non-limiting example, a rough adjustment of the depth of the mirror M3 can adjust the position of the second focusing mirror M3 to a distance of about 50 mm from the main optical axis 11 of the microscope.

[0062] The second focusing mirror M3 receives an incident light beam focused to point 21, 22, 23, 24 or 25 of the intermediate focal plane and reflects the light beam toward the second deflecting mirror M4, or directly toward the microscope objective 5 if the deflecting mirror M4 is not used.

[0063] The second focusing mirror M3 can bring the image of the scanning mirror M2 back to the pupil of the microscope objective 5. Therefore, the second focusing mirror M3 limits the vignetting effect of the microscope objective 5 when the direction of the scanning mirror M2 changes.

[0064] exist Figure 1 In the example shown, the second deflection mirror M4 receives the excitation light beam and reflects it toward the microscope objective 5. By way of example, the second deflection mirror M4 has a rectangular shape of 30 mm×45 mm and a thickness of 6 mm.

[0065] Advantageously, the second deflecting mirror M4 can be mounted on a conventional point-line-plane support by means of adjustment screws on the front face of the approach mirror M4. For fine adjustments, an adjustment range of + / -1 degree on two rotation axes (for example, one rotation axis is parallel to the X-axis and the other rotation axis is perpendicular to the X-axis) is sufficient. Advantageously, the second deflecting mirror M4 can be adjusted in position at a depth of + / -1 mm along the normal to the deflecting mirror M4. Depending on the distance between the deflecting mirror M4 and the microscope objective 5, the angle of incidence of the excitation light beam relative to the normal to the mirror M4 is variable within a range of several degrees as a function of the scanning of the mirror M2. After reflection on the second deflecting mirror M4, the excitation light beam is propagated at a variable angle relative to the main optical axis 11 of the microscope objective 5, depending on the inclination angle of the scanning mirror M2.

[0066] exist Figure 1 and Figure 3 In the first embodiment shown, the second focusing mirror M3 is arranged at a predetermined distance from the intermediate focal plane 20 to receive an incident light beam focused at a point 21, 22, 23, 24 or 25 of the intermediate focal plane and form a collimated light beam. Therefore, the microscope objective 5 receives a collimated light beam with a variable inclination according to the scanning of the mirror M2 around one or two rotation axes, and the variable inclination is a function of the scanning of the mirror M2 around one or two rotation axes transverse to the optical axis 11 of the microscope objective. The second focusing mirror M3 collimates the light beam from a point in the intermediate focal plane 20. The second deflection mirror reflects the collimated light beam toward the microscope objective 5. Therefore, at the objective focus 51 of the microscope objective 5, the incident light beam remains collimated (see Figure 3 ). The object focal plane 51 is also referred to as the back focal plane. The microscope objective 5 focuses the incident light beam 10 to a point 62 of the image focal plane 52 of the microscope objective 5. The image focal plane 52 is also referred to as the front focal plane. Therefore, the angle of the scanning mirror M2 is tilted so that the point 62 in the image focal plane 52 of the microscope objective 5 can be moved in the X and / or Y direction. Therefore, an area of ​​the image focal plane 52 of the microscope can be scanned by changing the tilt of a single scanning mirror M2, while the other mirrors M0, M1, M2 and M3 remain fixed.

[0067] In the first embodiment, the first focusing mirror M1 and the second focusing mirror M3 form an afocal optical system.

[0068] In short, in a first embodiment, the excitation beam movement system 100 can move the focus 62 in the front focal plane 52 along two spatial directions (X, Y) transverse to the optical axis 11 of the microscope objective.

[0069] The incident light beam 10 may be used as an excitation light beam to generate a light beam by backscattering, transmission or reflection.

[0070] A particularly interesting application involves fluorescence or Raman scattering. In this case, the light source 1 is advantageously a laser source.

[0071] In the configuration for Raman backscattering measurements, the microscope objective 5 collects a backscattered or reflected light beam 30 formed by backscattering or reflection of the excitation light beam 10 at a point 62 of the sample 6. The collected light beam 30 follows the reverse path of the excitation light beam by successive reflections on mirrors M4, M3, M2, M1 and M0. An optical component 41 of the semi-reflective plate or dichroic plate type can separate the excitation light beam 10 from the backscattered or reflected light beam 30. In the case of a confocal microscope, the collected light beam 30 passes through a confocal aperture 43.

[0072] Optical components (eg, lens 42 ) focus the backscattered or reflected beam 30 onto an input slit of a spectrometer 44 .

[0073] Thus, the beam scanning system 100 can convert a confocal microscope into a beam scanning fluorescence or Raman microscope without modifying the optical settings of the confocal microscope.

[0074] In other applications of the first embodiment, such as Figures 5 to 8 As shown, the transmission measurement is performed in conjunction with the same excitation beam movement system 100 described above. Figures 5 to 8 The beam scanning system 100 arranged on the optical path of the excitation beam 10 is not shown.

[0075] According to the first alternative, if Figure 5 As shown, the further objective 15 is used to collect the transmitted light beam 70. The movement system 100 can move the focus 62 in the front focal plane 52 of the microscope objective in two directions transverse to the optical axis 11 of the microscope objective. In this alternative, the further objective 15 only collects the signal from the point 60 at the center of the field of view of the objective 15 on the optical axis 11, regardless of the position of the excitation point, in other words, regardless of the (X, Y) position of the focus 62. The objective 15 focuses the transmitted light beam 70 on the spectrometer 44 or directly on the detector.

[0076] This first alternative can be applied, for example, to measure the propagation of a light beam in a planar waveguide. To this end, the waveguide is placed in the plane of the sample 6, one end of the waveguide is excited at an excitation point 62 away from the optical axis, and the signal is collected by transmission at the other end of the waveguide located on the optical axis 11.

[0077] Another application of this first alternative consists in measuring the entire transmitted beam 70 in the field of view of the objective 5 by rapidly scanning the sample surface and collecting the integrity of the transmitted beam 70 in order to integrate information over the entire volume or surface of the sample.

[0078] According to the second alternative, if Figure 6 As shown, another objective lens 15 is used to collect the transmitted light beam 72 from any focal point 62. In a specific example of the application of this second alternative, the excitation point 62 is moved in a single direction (e.g., X or Y) in the sample plane, and the transmitted light beam 72 is collected along the length of the input slot of the spectrometer 44 according to the position of the excitation point 62 along the scanning direction.

[0079] According to the third alternative, if Figure 7 As shown, another objective lens 15 is used to collect the transmitted light beam 72 from the excitation point 62 and to form an image of the transmitted light beam 72 on the two-dimensional matrix sensor 45. By moving the excitation point 62 on the sample in the XY plane, an image of the sample is formed by transmission according to the excitation point 62. This alternative is particularly suitable for confocal microscopy intensity measurements, for example, by performing transmission measurements on the sample at each point, or for hyperspectral microscopy measurements using a hyperspectral camera. In order to obtain spectral information, it is recommended to add a filter in order to select a specific spectral band. The filter can have a fixed spectral band and be replaced by another filter to change the spectral range. As an alternative, the filter is a tunable bandpass filter.

[0080] According to the fourth alternative, if Figure 8 As shown, a second beam scanning system 102 is used, which is arranged on the optical path of the transmitted beam 72, which is collimated by another objective lens 15. The second beam scanning system 102 includes optical components M4, optical components M3, optical components M2, optical components M1 and optical components M0 (similar to the optical components of the beam scanning system 100 arranged on the optical path of the excitation beam 10). In addition, the two scanning mirrors M2 of the two beam scanning systems 100 and 102 are synchronized. At the output end of the second beam scanning system 102, the transmitted beam 80 has a fixed direction, regardless of the position of the excitation point 62 in the XY plane of the sample. The transmitted beam 80 is then sent to a spectrometer or a detector.

[0081] exist Figure 4In the second embodiment shown, the second focusing mirror M3 is arranged at a predetermined distance from the intermediate focal plane 20 so as to receive the excitation light beam 10 focused at a point 21, 22, 23, 24 or 25 of the intermediate focal plane 20 and form a light beam focused at a point 61 in the object focal plane 51 of the microscope objective 5. The variable inclination around the two rotation axes of the scanning mirror M2 can modify the XY position of the point 61 in the object focal plane 51. Therefore, the microscope objective 5 receives the focused light beam and forms a collimated light beam in the image focal plane 52, which has a variable inclination as a function of the scanning of the mirror M2 around one or two rotation axes transverse to the optical axis 11 of the microscope objective. In other words, the second focusing mirror M3 causes the image of the intermediate focal plane 20 to be formed in the object focal plane 51 of the microscope objective 5. The second deflection mirror M4 reflects the collimated light beam toward the microscope objective 5. Therefore, in the object focal plane 51 of the microscope objective 5, the incident light beam is focused (see Figure 4 ). The microscope objective 5 collimates the incident light beam 10 in the image focal plane 52 of the microscope objective 5. Therefore, the angular tilt of the scanning mirror M2 can adjust and / or modify the tilt angle α of the excitation light beam 10 in the image focal plane 52 of the microscope objective 5 relative to the two rotation axes transverse to the main optical axis 11.

[0082] Therefore, the tilt angle α of the excitation beam in the image focal plane 52 of the microscope can be adjusted by adjusting the tilt of a single scanning mirror M2, while the other mirrors M0, M1, M2 and M3 remain fixed. The microscope objective 5 collects the beam 30 reflected or backscattered on the sample 6.

[0083] In the second embodiment, the first focusing mirror M1 , the second focusing mirror M3 and the microscope objective 5 form an afocal optical system.

[0084] In a second embodiment, the excitation beam movement system 100 can adjust or scan the tilt angle α of the collimated excitation beam in the front focal plane 52 about two rotation axes transverse to the optical axis 11 of the microscope objective.

[0085] In this second embodiment, the beam diameter at the output of the objective lens 5 depends mainly on the beam diameter at the input of the beam scanning system 100 (e.g., 4 mm), the distance between the mirror M3 and the intermediate focal plane 20 (e.g., about 200 mm), and the objective lens magnification (e.g., 100 times). In this example, the beam diameter at the output of the objective lens 5 is about 20 m, which determines the XY lateral spatial resolution. On the other hand, this allows for extreme axial resolution along the Z axis for TIRF or TIRR mode.

[0086] In the application of the second embodiment, the microscope objective 5 is a total internal reflection fluorescence (TIRF) objective. Similar to the first embodiment, the microscope is therefore suitable for performing total internal reflection fluorescence measurements in the sample 6 at a determined angle of incidence α. In another application, the microscope objective 5 is a total internal reflection Raman (TIRR) objective. Therefore, in order to obtain total internal reflection, the microscope is suitable for performing total internal reflection Raman spectroscopy measurements in the sample 6 at a determined angle of incidence α according to the sample optical index.

[0087] According to another alternative, the tilt of the scanning mirror M2 is controlled in such a way that the tilt angle α of the excitation light beam is adjusted or scanned in the image focal plane 52 of the microscope, and the other mirrors M0, mirror M1, mirror M2 and mirror M3 remain fixed. In another application, the microscope objective 5 collects the light beam reflected on the sample 6. Therefore, the microscope is suitable for performing ellipsometric measurements at a determined incident angle α within an angular range determined according to the numerical aperture of the microscope objective 5 or according to the incident angle α within an angular range determined according to the numerical aperture of the microscope objective 5. For example, for a microscope objective 5 with a numerical aperture of approximately 0.95, the angular range of the angle α extends from approximately 0 degrees to 72 degrees. Advantageously, in ellipsometry applications, the light source 1 is selected from monochromatic or polychromatic light sources for measurements at one or several determined wavelengths, or is selected from broad spectrum light sources for spectral measurements.

[0088] According to a specific embodiment, the deflection mirror M0 and / or the deflection mirror M4 may be omitted. In this case, the optical axis of the incident light beam is generally deviated by the optical system formed by the first focusing mirror M1, the scanning mirror M2 and the second focusing mirror M3.

[0089] According to an alternative to any of the above-described embodiments, the first focusing mirror M1 is replaced by a first lens-based focusing optical system L1 . It is particularly advantageous that the first lens-based focusing optical system L1 preferably corrects spherical aberrations in the intermediate focal plane 20 .

[0090] As an alternative or in addition, the second focusing mirror M3 is replaced by a second lens-based focusing optical system L3 arranged between the scanning mirror M2 and the microscope objective 5. The second lens-based focusing optical system L3 may be used in combination with the mirror M1 or the lens-based optical system L1.

[0091] The mobile system 100 combined with the microscope objective 5 according to the first embodiment can be used to position the measurement point 62 to a determined point of the front focal plane, for example, on the sample 6. The excitation beam forms a light spot 62, which is suitable for movement and precise positioning in the front focal plane of the microscope objective according to the tilt of the scanning mirror M2 around one or two rotation axes. In addition, by the reverse return of light, the mobile system 100 is suitable for confocal collection of reflected or backscattered signals.

[0092] According to a specific embodiment comprising a particle detector system, the position of one or several particles on the sample surface is pre-located, for example based on an image capture and processing system, and a measurement point 62 is automatically positioned on each particle for individual measurement.

[0093] In the so-called microspot application, the different positions of the measurement spot 62 are mapped on the sample with spatial microscopic resolution according to a series of variable tilt angles (according to one or two directions) of the scanning mirror M2. In this application, each measurement is performed between two consecutive movements. Therefore, the control of the scanning mirror requires multiple starts and stops, which means a relatively high total acquisition duration.

[0094] The mobile system 100 is also adapted to perform a series of measurements at a series of predetermined times during the continuous or nearly continuous movement of the spot on the sample to limit the fall time between two consecutive measurements. This flight measurement method (or SWIFT mode) can obtain spatially resolved measurements faster than using micro-spot mode.

[0095] In another so-called macrospot application, the signal detected during the movement of the spot 62 on the sample is integrated in such a way as to obtain a spatially averaged measurement over a surface of larger size than the laser spot 62. The shape of the macrospot can be predetermined, for example, disk-shaped, ring-shaped, square, rectangular, or a shape suitable for the object under consideration, for example, a biological form to be detected by the imaging system.

[0096] According to another alternative, a series of measurements is obtained by moving the measuring point 62 along a line on the sample and forming an image of this line in the longitudinal direction of the input slot of the spectrometer 44. The spectrometer is equipped with a sensor of the two-dimensional camera type, so that a series of spectra along a line on the sample can be obtained very quickly.

[0097] The moving system 100 in combination with the microscope objective 5 according to the second embodiment is suitable for irradiating the sample with a collimated excitation light beam at a predetermined angle of incidence α in the front focal plane. It is particularly advantageous if the same microscope objective 5 and the moving system 100 are used to collect the light beam formed by the reflection of the excitation light beam on the sample, the reflected light beam forming an angle symmetrical to the angle α with respect to the optical axis 11 of the microscope objective. The moving system 100 according to the second embodiment is applied in total internal reflection fluorescence (TIRF) microspectrometry or total internal reflection Raman (TIRR) microspectrometry or ellipsometry.

[0098] The mobile system 100 according to the second embodiment is further adapted to illuminate the sample with the collimated excitation beam at a variable angle of incidence α in order to determine the angle of incidence α such that a maximum sensitivity for TIRF, TIRR or ellipsometry type measurements can be obtained.

[0099] Of course, various other modifications are possible to the invention within the framework of the attached claims.

Claims

1. A beam scanning microspectroscopy device, comprising: at least one light source (1) adapted to emit an excitation light beam (10); A microscope objective (5) is arranged along the main optical axis (11) of the micro-spectroscopy device, the microscope objective (5) having an object focal plane (51) and an image focal plane (52); a system (100) for moving the excitation light beam along two spatial directions (X, Y) transverse to the optical axis of the light beam, the microscope objective (5) and the moving system (100) being suitable for moving the excitation light beam (10) on the sample; and an optical system (5, 15) being suitable for collecting a light beam (30, 70, 72) formed by reflection, scattering and / or transmission of the excitation light beam (10) on the sample; and a spectral detection system being suitable for receiving the light beam (30, 70, 72) formed by reflection, scattering and / or transmission, characterized in that the excitation light beam moving system (10) comprises: a first focusing optical component (M1, L1) being suitable for receiving the excitation light beam (10) and focusing the excitation light beam (10) to a focus (21) in an intermediate focal plane (20); another focusing optical component (M3 , L3), arranged on the optical path of the light beam between the intermediate focal plane (20) and the microscope objective (5), the other focusing optical component (M3, L3) being suitable for forming an image of the intermediate focal plane (20) in the object focal plane (51) or the image focal plane (52) of the microscope objective (5), respectively; and a single scanning mirror (M2), arranged on the optical path of the excitation light beam between the first focusing optical component (M1, L1) and the intermediate focal plane (20), the scanning mirror (M2) being a plane and mounted on a stage and being rotatable around two lateral rotation axes, the two rotation axes being within the plane of the scanning mirror (M2), the scanning mirror (M2) being suitable for moving the focus (21) in two lateral directions in the intermediate focal plane (20), so as to move the image of the focus (61, 62) in the object focal plane (51) or the image focal plane (52) of the microscope objective (5), respectively, in the two lateral directions.

2. The beam scanning microspectroscopy device according to claim 1, wherein: The first focusing optics (M1, L1) is a toric mirror.

3. The beam scanning microspectroscopy device according to claim 2, wherein: The toric mirror (M1) is suitable for reducing geometrical optical aberrations in the intermediate focal plane (20).

4. The beam scanning microspectroscopy device according to claim 1, wherein: The first focusing optics (M1, L1) comprises at least one lens.

5. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The first focusing optical component (M1, L1) is mounted on a guide rail (31), and the guide rail (31) is suitable for adjusting the distance between the first focusing optical component (M1, L1) and the microscope objective (5).

6. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The further focusing optical component (M3, L3) is a spherical mirror or a toric mirror.

7. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The further focusing optical component (M3, L3) is mounted on a further guide rail (33), and the further guide rail (33) is suitable for adjusting the distance between the further focusing optical component (M3, L3) and the microscope objective (5).

8. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The scanning mirror (M2) is arranged to receive the excitation light beam (10) with an incident angle less than or equal to 15 degrees.

9. The beam scanning micro-spectroscopy device according to claim 1 or 2 further comprises a first plane deflection mirror (M0) and / or another plane deflection mirror (M4), respectively, wherein the first plane deflection mirror (M0) is arranged upstream of the first focusing optical component (M1, L1), and / or the another plane deflection mirror (M4) is arranged between the another focusing optical component (M3, L3) and the microscope objective (5).

10. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The rotatable stage includes a biaxial rotary actuator (25) of the piezoelectric or voice coil type.

11. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The at least one light source (1) comprises one or several sources of the laser source type and / or the light emitting diode type.

12. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The beam scanning microspectroscopy device comprises a confocal aperture (43) arranged upstream of the first focusing optical component (M1, L1) to collimate the excitation light beam (10) incident on the first focusing optical component (M1, L1).

13. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The further focusing optical component (M3, L3) is a lens.

14. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The spectrum detection system includes: a Raman spectrometer, a coherent anti-Stokes Raman spectrometer, a fluorescence spectrometer, a photoluminescence spectrometer, a cathode luminescence spectrometer, a hyperspectral camera, a spectrum filter or a tunable bandpass filter.

15. The beam scanning microspectroscopy device according to claim 1 or 2, wherein: The microscope objective is a total internal reflection objective, and wherein the further focusing optical component (M3, L3) is adapted to form an image of the intermediate focal plane (20) in the object focal plane of the total internal reflection objective so as to angularly move the collimated excitation light beam in the image focal plane (52) and collect the total internal reflection light beam on the sample.

16. A method for beam scanning microspectroscopy, the method comprising the following steps: An excitation light beam (10) is emitted by a light source; the excitation light beam is guided toward a first focusing optical component (M1, L1) and reflected onto a scanning mirror (M2) of a single plane, so that after the excitation light beam is reflected on the scanning mirror (M2), the excitation light beam (10) is focused to a focus (21) in an intermediate focal plane (20); the excitation light beam is guided toward another focusing optical component (M3, L3) and then guided toward a microscope objective (5) so as to form the intermediate focal plane (51) or the image focal plane (52) of the microscope objective (5), respectively. The invention relates to a method for collecting an image of an intermediate focal plane (20) and tilting the scanning mirror (M2) around two lateral rotation axes to move the focus (21) in the intermediate focal plane (20) along two lateral directions, so as to move the image of the focus (61, 62) in the object focal plane (51) or the image focal plane (52) of the microscope objective (5) along the two lateral directions respectively; collecting light beams (30, 70, 72) formed by reflection, scattering and / or transmission of the excitation light beam, and receiving the light beams (30, 70, 72) formed by reflection, scattering and / or transmission on a spectral detection system.

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