Laser scanning second harmonic microscopy system and second harmonic test method

Abstract

The present application provides a laser scanning second harmonic microscopy system and a second harmonic testing method. The system includes: a laser emission module, including a femtosecond laser and a first half-wave plate arranged in sequence, the first half-wave plate can rotate along the optical axis of the femtosecond pulse laser; a sample placement module is used to place the sample; a beam adjustment module is arranged between the laser emission module and the sample placement module, including a galvanometer group and a dichroic mirror arranged in sequence, the galvanometer group is connected to the optical path of the first half-wave plate; a signal receiving module, including a first Glan Taylor prism and a photomultiplier tube PMT photodetector arranged in sequence, the first Glan Taylor prism is connected to the optical path of the dichroic mirror, and the first Glan Taylor prism can rotate along the optical axis of the second harmonic light. In this way, the incident laser is directly deflected by the galvanometer group, and continuous multiple measurements can maintain a small deviation in the measurement area and improve the accuracy of the measurement data. And the scanning positioning accuracy is higher, and a second harmonic spectrum image with a higher resolution can be obtained.

Description

Technical Field

[0001] The present application belongs to the field of second harmonic technology, and in particular relates to a laser scanning second harmonic microscopy system and a second harmonic testing method. Background Art

[0002] Ferroelectric materials refer to materials with ferroelectric properties, in which the centers of positive and negative charges in the crystal structure do not coincide, resulting in spontaneous polarization even without an applied electric field. Second Harmonic Generation (SHG) is an emerging method for characterizing ferroelectric materials. For ferroelectric materials, the central symmetry of the crystal is broken, which means that they must have a second harmonic effect. Therefore, this method can accurately capture the local polarity changes of the sample. At present, when measuring sample data, the second harmonic microscopy system often needs to change the measurement position by moving the sample, which makes it more affected by the environment, the accuracy of the measurement data is low, and the precision is controlled by mechanical limits. It is often difficult to obtain a high-resolution second harmonic spectrum image. Summary of the invention

[0003] The embodiments of the present application provide a laser scanning second harmonic microscopy system and a second harmonic testing method to solve the technical problems that the accuracy of measurement data is low and it is often difficult to obtain a second harmonic spectrum image with a higher resolution.

[0004] In a first aspect, an embodiment of the present application provides a laser scanning second harmonic generation microscopy system, the system comprising:

[0005] A laser emission module, comprising a femtosecond laser and a first half-wave plate which are arranged in sequence, wherein the femtosecond laser is used to emit a femtosecond pulse laser, and the first half-wave plate can rotate along the optical axis of the femtosecond pulse laser to adjust the polarization angle of the incident light;

[0006] A sample placement module, used for placing samples;

[0007] A beam adjustment module is arranged between the laser emission module and the sample placement module, the beam adjustment module comprises a galvanometer group and a dichroic mirror arranged in sequence, the galvanometer group is connected to the first half-wave plate optical path, the galvanometer group is used to adjust the position where the femtosecond pulse laser is focused to the sample surface, the femtosecond pulse laser is focused to the sample surface after being reflected by the dichroic mirror to generate second harmonic light;

[0008] The signal receiving module comprises a first Glan-Taylor prism and a photomultiplier tube (PMT) photodetector which are arranged in sequence. The first Glan-Taylor prism is connected to the optical path of the dichroic mirror. The second harmonic light is transmitted through the dichroic mirror and then enters the first Glan-Taylor prism. The first Glan-Taylor prism can rotate along the optical axis of the second harmonic light to adjust the polarization angle of the second harmonic.

[0009] In a second aspect, an embodiment of the present application provides a second harmonic testing method, the method comprising:

[0010] When the sample is fixed on the sample placement module, adjusting the angles of the first half-wave plate and the first Glan Taylor prism to determine M groups of polarization angles, wherein the polarization angles include an incident light polarization angle and a second harmonic polarization angle, and M is an integer greater than 1;

[0011] Under each group of polarization angles, adjusting the deflection angle of the galvanometer group to scan K second harmonic signal intensities corresponding to K positions on the sample surface, to obtain M groups of the K second harmonic signal intensities, where K is an integer greater than 1;

[0012] Determine, according to the M groups of the K second harmonic signal intensities, the M second harmonic signal intensities of the target position under the M groups of polarization angles, wherein the target position is any one of the K positions;

[0013] Perform spectrum fitting on the M second harmonic signal intensities of the target position, determine the fitting function and fitting result of the target position, and draw a second harmonic spectrum image of the target position.

[0014] The laser scanning second harmonic microscopy system and the second harmonic testing method of the embodiment of the present application, wherein the laser scanning second harmonic microscopy system may include a laser emission module, a beam adjustment module, a sample placement module and a signal receiving module; wherein the laser emission module includes a femtosecond laser and a first half-wave plate arranged in sequence, the femtosecond laser is used to emit a femtosecond pulse laser, and the first half-wave plate can rotate along the optical axis of the femtosecond pulse laser to adjust the polarization angle of the incident light; the sample placement module is used to place the sample; the beam adjustment module is arranged between the laser emission module and the sample placement module, The invention comprises a galvanometer group and a dichroic mirror arranged in sequence, wherein the galvanometer group is connected to the optical path of a first half-wave plate and is used for adjusting the position where a femtosecond pulse laser is focused on the surface of a sample, and the femtosecond pulse laser is focused on the surface of the sample after being reflected by the dichroic mirror to generate second harmonic light; the signal receiving module comprises a first Glan Taylor prism and a photomultiplier tube PMT photodetector arranged in sequence, wherein the first Glan Taylor prism is connected to the optical path of the dichroic mirror, and the second harmonic light is incident on the first Glan Taylor prism after being transmitted by the dichroic mirror, and the first Glan Taylor prism can rotate along the optical axis of the second harmonic light and is used for adjusting the polarization angle of the second harmonic.

[0015] In this way, different positions on the sample surface can be directly scanned by adjusting the galvanometer group without moving the sample for scanning. On the one hand, by adjusting the galvanometer group for continuous multiple measurements, the deviation of the measurement area can be kept small, thereby effectively improving the accuracy of the measurement data. On the other hand, when scanning different positions on the sample surface, it is not limited by mechanical limits, and the positioning accuracy of the scanning point is higher, thereby obtaining a second harmonic spectrum image with higher resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 is a schematic structural diagram of a laser scanning second harmonic microscopy system provided by an embodiment of the present application;

[0018] Figure 2 It is a flow chart of a second harmonic testing method provided in another embodiment of the present application.

[0019] Reference numerals:

[0020] 1. Laser emission module; 11. Femtosecond laser; 12. First half-wave plate; 13. Second half-wave plate; 14. Second Glan-Taylor prism;

[0021] 2. Sample placement module;

[0022] 3. beam adjustment module; 31. galvanometer group; 32. dichroic mirror; 33. scanning lens; 34. sleeve lens; 35. objective lens; 36. first bandpass filter;

[0023] 4. Signal receiving module; 41. First Glan Taylor prism; 42. PMT photodetector; 43. First beam splitter; 44. Second beam splitter; 45. Illumination assembly; 46. CCD camera; 47. Second bandpass filter;

[0024] 5. Reflection module; 51. First reflector; 52. Second reflector; 53. Third reflector. DETAILED DESCRIPTION

[0025] The features and exemplary embodiments of various aspects of the present application will be described in detail below. In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only intended to explain the present application, rather than to limit the present application. For those skilled in the art, the present application can be implemented without the need for some of these specific details. The following description of the embodiments is only to provide a better understanding of the present application by illustrating the examples of the present application.

[0026] It should be noted that the terms "upper", "lower", "left", "right", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on this patent. The terms "first" and "second" are only used for the convenience of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features. "Multiple" means two or more, unless otherwise clearly and specifically defined. In addition, terms such as "horizontal", "vertical", and "overhanging" do not mean that the components are required to be absolutely horizontal or overhanging, but can be slightly tilted. For example, "horizontal" only means that its direction is more horizontal than "vertical", and does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0027] It should also be noted that, unless otherwise clearly specified and limited, the terms "set", "install", "connect", and "connect" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two elements. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0028] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the statement "include..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0029] In order to illustrate the technical solution described in this application, a detailed description is given below in conjunction with specific drawings and embodiments.

[0030] like Figure 1 As shown, the embodiment of the present application provides a laser scanning second harmonic microscopy system, which may include:

[0031] The laser emission module 1 comprises a femtosecond laser 11 and a first half-wave plate 12 which are arranged in sequence, wherein the femtosecond laser 11 is used to emit a femtosecond pulse laser, and the first half-wave plate 12 can rotate along the optical axis of the femtosecond pulse laser to adjust the polarization angle of the incident light;

[0032] Sample placement module 2, used for placing samples;

[0033] The beam adjustment module 3 is arranged between the laser emission module 1 and the sample placement module 2. The beam adjustment module 3 includes a galvanometer group 31 and a dichroic mirror 32 which are arranged in sequence. The galvanometer group 31 is connected to the first half-wave plate 12 in an optical path. The galvanometer group 31 is used to adjust the position where the femtosecond pulse laser is focused to the sample surface. The femtosecond pulse laser is focused to the sample surface after being reflected by the dichroic mirror 32 to generate second harmonic light.

[0034] The signal receiving module 4 includes a first Glan-Taylor prism 41 and a photomultiplier tube (PMT) photodetector 42 arranged in sequence. The first Glan-Taylor prism 41 is connected to the optical path of the dichroic mirror 32. The second harmonic light is transmitted through the dichroic mirror 32 and then enters the first Glan-Taylor prism 41. The first Glan-Taylor prism 41 can rotate along the optical axis of the second harmonic light to adjust the polarization angle of the second harmonic.

[0035] like Figure 1 As shown, after the femtosecond laser 11 emits a femtosecond pulse laser, the femtosecond pulse laser is emitted to the first half-wave plate 12. The first half-wave plate 12 can rotate along the optical axis of the femtosecond pulse laser. At this time, the polarization angle of the incident light of the femtosecond pulse laser can be changed by rotating the first half-wave plate 12. The calculation formula of the incident light polarization angle can be shown as formula (1):

[0036]

[0037] in, It can be the polarization angle of the incident light, and θ1 can be the angle of the first half-wave plate 12. For example, θ1 can be the angle between the first half-wave plate 12 and the X-axis in the laboratory coordinate system.

[0038] It is understandable that the femtosecond laser 11 and the first half-wave plate 12 can be selected according to actual measurement requirements, and are not specifically limited here. For example, in this embodiment, a titanium sapphire femtosecond laser 11 with a wavelength of 800nm ​​can be used, with a pulse width of 35fs, a repetition frequency of 80MHz, and a total power of 300mW. The first half-wave plate 12 can be a zero-order quartz half-wave plate.

[0039] After the polarization angle of the incident light of the femtosecond pulse laser is adjusted by the first half-wave plate 12, it can be incident on the galvanometer group 31, and after the direction is adjusted by the galvanometer group 31, it can be directed to the dichroic mirror 32. Among them, the galvanometer group 31 can be controlled by a computer and can be deflected in two different directions. Its deflection angle can be selected according to actual needs. For example, in this embodiment, the maximum deflection angle of the galvanometer group 31 can be 7.5°, which corresponds to a deflection angle of 15° for the femtosecond pulse laser. The control voltage of the computer can be 1.33V / °. Then, after the deflection angle of the galvanometer group 31 is converted by the focal length, the relationship between it and the spot offset can be shown as formula (2):

[0040] D=Cβ (2)

[0041] Wherein, D is the size of the center position of the light spot offset, C is the proportional coefficient, which may be 25.5 μm / °, and β is the deflection angle of the galvanometer group 31 .

[0042] In addition, the transmission band cut-off wavelength and the starting wavelength of the dichroic mirror 32 can also be selected according to the actual measurement requirements and are related to the wavelength of the femtosecond pulse laser. For example, in this embodiment, the transmission band cut-off wavelength of the dichroic mirror 32 can be 740nm, and the starting wavelength can be 940nm. In this way, the femtosecond pulse laser of about 800nm ​​cannot be transmitted by the dichroic mirror 32, and then is reflected by the dichroic mirror 32 and emitted to the sample surface placed on the sample placement module 2. The position of the light spot of the femtosecond pulse laser on the sample surface can indicate the position being measured at this time.

[0043] It is understandable that after a femtosecond pulse laser of about 800 nm is irradiated onto the sample surface, the wavelength of the second harmonic light generated by the sample is usually about 400 nm, that is, the second harmonic light can be transmitted by the dichroic mirror 32 at this time.

[0044] After passing through the dichroic mirror 32, the second harmonic light can be incident on the first Glan-Taylor prism 41, and the first Glan-Taylor prism 41 can be rotated along the optical axis of the second harmonic light. For example, since the second harmonic light generated by the sample is usually in a mixed polarization state, the first Glan-Taylor prism 41 can be rotated at this time to freely select the second harmonic polarization angle α according to the measurement requirements, that is, the component of the second harmonic light matching the second harmonic polarization angle α can pass through the first Glan-Taylor prism 41, and then can be collected by the PMT photodetector 42.

[0045] It can be understood that when the first Glan-Taylor prism 41 only allows the second harmonic light polarized in a certain direction to pass through, and in a state different from that in a direction by 90°, the components of the collected second harmonic light are orthogonal to each other.

[0046] Thus, when measuring sample data, the first half-wave plate 12 and the first Glan Taylor prism 41 can be adjusted to change the incident light polarization angle to The measured second harmonic polarization angle α is set to a determined value. At this time, the light spot can be focused to different positions on the sample surface by adjusting the deflection angle of the galvanometer group 31, and the second harmonic signal intensity at the position is measured until the scanning of each point on the sample is completed. The whole process does not require moving the sample for scanning. On the one hand, by adjusting the galvanometer group 31 for continuous multiple measurements, the small deviation of the measurement area can be maintained, thereby effectively improving the accuracy of the measurement data. On the other hand, when scanning different positions on the sample surface, the deflection angle of the galvanometer group 31 can be accurately controlled by the computer for precise positioning, which is not limited by mechanical limits, and the positioning accuracy of the scanning point is higher, thereby obtaining a second harmonic spectrum image with a higher resolution.

[0047] Optionally, in some embodiments, the laser emission module 1 may further include:

[0048] A second half-wave plate 13 is connected to the optical path of the femtosecond laser 11, and the second half-wave plate 13 can rotate along the optical axis of the femtosecond pulse laser;

[0049] The second Glan Taylor prism 14 is optically connected to the second half-wave plate 13 and the first half-wave plate 12.

[0050] The second half-wave plate 13 and the second Glan-Taylor prism 14 are used to adjust the intensity of incident light.

[0051] like Figure 1 As shown, after the femtosecond laser 11 emits a femtosecond pulse laser, the incident light may first pass through the second half-wave plate 13 and the second Glan-Taylor prism 14 in sequence, and then pass through the first half-wave plate 12 to adjust the polarization angle of the incident light.

[0052] For example, the femtosecond pulse laser emitted by the femtosecond laser 11 may be linearly polarized light in the vertical direction. The polarization direction of the incident light may be changed after passing through the second half-wave plate 13. At this time, the second Glan Taylor prism 14 may be configured to allow only horizontally polarized light to pass through. Therefore, the incident light intensity may be changed by rotating the second half-wave plate 13. The calculation formula of the incident light intensity may be as shown in formula (3):

[0053] I=I0cos 2 (θ2) (3)

[0054] Among them, I0 is the original laser energy, which can be about 300mW, and θ2 can be the angle of the second half-wave plate 13. For example, when a reflector placed at 45° is provided behind the laser emission module 1, θ2 can be the angle between the second half-wave plate 13 and the straight line at 45° to the X-axis in the laboratory coordinate system.

[0055] It is understandable that the second half-wave plate 13 may also be selected according to actual measurement requirements. For example, in this embodiment, the second half-wave plate 13 may also be a zero-order quartz half-wave plate.

[0056] In this way, the intensity of the incident light can be adjusted by the second half-wave plate 13 and the second Glan Taylor prism 14, thereby enriching the measurement conditions of the laser scanning second harmonic microscopy system and meeting various measurement requirements.

[0057] Optionally, in some embodiments, the laser scanning second harmonic generation microscopy system further comprises:

[0058] The reflection module 5 includes at least one reflection mirror, and the reflection module 5 is arranged between the first half-wave plate 12 and the galvanometer mirror group 31 .

[0059] In this embodiment, a reflection module 5 may be provided between the first half-wave plate 12 and the galvanometer group 31 to adjust the propagation path of the femtosecond pulse laser to adapt the beam adjustment module 3, so that the final femtosecond pulse laser can be smoothly directed to the sample surface. The reflection module 5 may include at least one reflector, and the specific number, placement position, and placement direction of the reflector may be set according to actual needs, so that the femtosecond pulse laser passing through the first half-wave plate 12 can be reflected and directed into the galvanometer group 31, which is not specifically limited here.

[0060] For example, Figure 1 As shown, the reflection module 5 may include a first reflection mirror 51, a second reflection mirror 52 and a third reflection mirror 53, wherein the first reflection mirror 51, the second reflection mirror 52 and the third reflection mirror 53 may all be laser reflection mirrors and are placed at 45° to the optical axis of the femtosecond pulse laser. The femtosecond pulse laser passing through the first half-wave plate 12 may be reflected by the first reflection mirror 51 to the second reflection mirror 52, and then reflected by the second reflection mirror 52 to the third reflection mirror 53, and finally reflected by the third reflection mirror 53 to enter the galvanometer group 31.

[0061] Optionally, in some embodiments, the beam adjustment module 3 may further include:

[0062] The focusing assembly includes a scanning lens 33 , a sleeve lens 34 and an objective lens 35 . The scanning lens 33 and the sleeve lens 34 are sequentially arranged between the galvanometer group 31 and the dichroic mirror 32 , and the objective lens 35 is arranged between the dichroic mirror 32 and the sample placement module 2 .

[0063] like Figure 1 As shown, the beam adjustment module 3 may also include a focusing assembly, which may include a scanning lens 33, a sleeve lens 34 and an objective lens 35. After the femtosecond pulse laser is adjusted in direction by the galvanometer group 31, it passes through the scanning lens 33 and the sleeve lens 34 in sequence, and then is reflected by the dichroic mirror 32, and then is emitted to the sample surface through the objective lens 35, and the focus is a certain point on the sample plane. In this way, using the focusing assembly composed of the scanning lens 33, the sleeve lens 34 and the objective lens 35, for the light beams with different deflection angles emitted by the galvanometer, the light emitted by the objective lens 35 is approximately perpendicular to the sample surface, so that the measurement conditions of each position of the sample in the second harmonic imaging can be effectively guaranteed to be consistent, thereby further improving the accuracy of the second harmonic imaging.

[0064] It is understandable that the objective lens 35 can be selected according to the focusing requirements. For example, in this embodiment, the objective lens 35 can be a long working distance objective lens 35, whose working distance can be 13 mm and magnification can be 50×.

[0065] Optionally, in some embodiments, the focusing assembly further includes a first bandpass filter 36 , which is disposed between the tube lens 34 and the dichroic mirror 32 .

[0066] like Figure 1 As shown, a first bandpass filter 36 can be provided between the sleeve lens 34 and the dichroic mirror 32, and the first bandpass filter 36 can be used to filter out femtosecond pulse lasers whose wavelengths do not meet the conditions. For example, the central wavelength transmitted by the first bandpass filter 36 can be 800nm, and the half-height width can be 40nm. Then the femtosecond pulse laser within the wavelength range can pass through the first bandpass filter 36, and then can be reflected by the dichroic mirror 32 and enter the objective lens 35, without meeting the full cutoff of the wavelength range. It can be understood that the central wavelength and half-height width can be selected according to actual conditions, and are not limited here.

[0067] Optionally, in some embodiments, the focusing assembly further includes a stepper motor, which is connected to the objective lens 35 and is used to drive the objective lens 35 to move in a direction toward the sample placement module 2. In this way, the objective lens 35 can be driven away from or close to the sample by the stepper motor, thereby adjusting the depth of the focus of the femtosecond pulse laser on the sample. Thus, the second harmonic signal intensity at different depths of the sample can be measured.

[0068] Optionally, in some embodiments, the signal receiving module 4 further includes:

[0069] A first beam splitter 43, disposed between the dichroic mirror 32 and the first Glan Taylor prism 41;

[0070] a second beam splitter 44, connected to the optical path of the second beam splitter 44;

[0071] The lighting assembly 45 is optically connected to the second beam splitter 44;

[0072] A charge coupled device (CCD) camera 46 is optically connected to the second beam splitter 44 .

[0073] like Figure 1 As shown, in this embodiment, the signal receiving module may further include a first beam splitter 43, a second beam splitter 44, an illumination assembly 45 and a CCD camera 46. For example, the second harmonic light generated by the sample passes through the dichroic mirror 32 and is split into two beams by the first beam splitter 43, wherein the reflected light may be incident on the first Glan Taylor prism 41, and after passing through the first Glan Taylor prism 41, it may be collected by the PMT photodetector 42. The transmitted light may be split into two beams by the second beam splitter 44 again, wherein one beam of reflected light is illuminated by the illumination assembly 45, and then may be captured by the CCD camera 46 together with the other beam of transmitted light through the second beam splitter 44 to obtain a sample surface image as a basis for observing the sample surface.

[0074] It is understandable that the first beam splitter 43 and the second beam splitter 44 can be selected according to actual needs. For example, in this embodiment, the first beam splitter 43 can be a 70:30 beam splitter, and the second beam splitter 44 can be a 50:50 beam splitter. In some examples, the second beam splitter 44 can also be replaced by a dichroic mirror.

[0075] Optionally, in some embodiments, the signal receiving module 4 further includes:

[0076] The second bandpass filter 47 is disposed between the dichroic mirror 32 and the first Glan-Taylor prism 41 .

[0077] like Figure 1 As shown, a second bandpass filter 47 can be provided between the dichroic mirror 32 and the first Glan-Taylor prism 41, and the second bandpass filter 47 can be used to filter out the second harmonic light whose wavelength does not meet the conditions. For example, the central wavelength transmitted by the second bandpass filter 47 can be 400nm, and the half-height width can be 10nm. Then the second harmonic light within the wavelength range can pass through the second bandpass filter 47, and then can be injected into the first Glan-Taylor prism 41, without meeting the full cutoff of the wavelength range. It can be understood that the central wavelength and half-height width can be selected according to actual conditions, and are not limited here.

[0078] It can be understood that, when the signal receiving module 4 also includes a first beam splitter 43 arranged between the dichroic mirror 32 and the first Glan Taylor prism 41, the second bandpass filter 47 can also be arranged between the first beam splitter 43 and the first Glan Taylor prism 41, so that the reflected light of the first beam splitter 43 can first pass through the second bandpass filter 47 and then enter the first Glan Taylor prism 41.

[0079] Optionally, in some embodiments, the sample placement module 2 includes:

[0080] The first placement component includes at least one of a three-axis translation table, a tilting table and a rotating table, wherein the three-axis translation table is used to adjust the displacement of the sample, the tilting table is used to adjust the inclination of the sample, and the rotating table is used to adjust the placement angle of the sample.

[0081] In this embodiment, the sample placement module 2 may include a first placement component, and the first placement component is used to adjust the position of the sample, wherein the first placement component may include at least one of a three-axis translation stage, a tilt stage, and a rotation stage.

[0082] For example, the travel of the three-axis translation stage can be 25.4mm, the step length can be 200nm, and the three-axis translation stage can be controlled by a motor and can be used to adjust the sample position over a large range. The maximum tilt angle of the tilt stage can be ±5°, which can be adjusted manually and can be used for samples that need to be leveled. The rotation stage can rotate arbitrarily within the range of 360° and can be adjusted manually, which can be used to adjust the placement angle of the sample. In other words, it can be used to select the surface of the sample to be measured.

[0083] Optionally, in some embodiments, the sample placement module 2 further includes:

[0084] The second placement component can be used in conjunction with the first placement component. The second placement component includes N temperature controllers, and the N temperature controllers correspond to the N preset temperature intervals one by one.

[0085] Among them, the target temperature controller is used to test samples within the target temperature range, the target temperature controller is any temperature controller among N temperature controllers, the target temperature range is the temperature range corresponding to the target temperature controller among N preset temperature ranges, and N is a positive integer.

[0086] In this embodiment, the sample placement module 2 may further include a second placement component, which may include N temperature controllers for carrying samples of different temperatures. For example, the second placement component may include a low temperature thermostat, which may achieve measurement in the temperature range of 4K to 500K, and may also include a high temperature heating stage, which may achieve measurement in the temperature range of 30°C to 800°C.

[0087] The second placement component can be used in conjunction with the first placement component. In other words, the temperature controller in the second placement component can be combined with the first placement component at will. For example, the combination of the rotating table and the high-temperature heating table can adjust the placement angle of the sample within the temperature range of 30°C to 800°C.

[0088] It is understandable that the existing second harmonic test methods mainly include single-point polarization test and microscopic test, which can obtain the average polarization direction information and structural distribution information of the sample respectively. It is often impossible to combine the two types of information, resulting in the tester not being able to obtain complete information in one test, and it is necessary to combine multiple measurement results to infer the nanostructure of the material. This process requires the application of a lot of knowledge about the material itself and the second harmonic principle, which increases the difficulty of characterizing the material properties. At the same time, the inference process itself will inevitably produce a priori bias and errors, resulting in poor reliability of the second harmonic test method.

[0089] Based on this, the embodiment of the present application further provides a second harmonic testing method using the above-mentioned laser scanning second harmonic microscopy system, which may include the following steps:

[0090] Step 201, when the sample is fixed on the sample placement module, adjust the angles of the first half-wave plate and the first Glan Taylor prism to determine M groups of polarization angles, where the polarization angles include the incident light polarization angle and the second harmonic polarization angle, and M is an integer greater than 1;

[0091] Step 202, at each set of polarization angles, adjusting the deflection angle of the galvanometer group to scan K second harmonic signal intensities corresponding to K positions on the sample surface, to obtain M sets of K second harmonic signal intensities, where K is an integer greater than 1;

[0092] Step 203, determining M second harmonic signal intensities of a target position under M groups of polarization angles according to M groups of K second harmonic signal intensities, wherein the target position is any position among the K positions;

[0093] Step 204 , performing spectrum fitting on the M second harmonic signal intensities at the target position, determining the fitting function and fitting result at the target position, and drawing a second harmonic spectrum image at the target position.

[0094] In the embodiment of the present application, the sample can be first fixed on the sample placement module, and then the angles of the first half-wave plate and the first Glan Taylor prism can be adjusted to change the polarization angle of the incident light to The measured second harmonic polarization angle α is set to a certain value. The deflection angle of the galvanometer group is controlled to focus the light spot on different positions on the sample surface, and the second harmonic signal intensity at the position is measured until the K positions on the sample are scanned to obtain K second harmonic signal intensities corresponding to the K positions.

[0095] Reset the incident light polarization angle and the measured second harmonic polarization angle α, repeating the above scanning measurement steps until M groups of K second harmonic signal intensities under M groups of polarization angles are obtained.

[0096] The M groups of K second harmonic signal intensity data are reorganized into spectrum data at different polarization angles corresponding to each position (i.e., M second harmonic signal intensities at the target position at M groups of polarization angles). The M second harmonic signal intensities at the target position are spectrally fitted to determine the fitting function and fitting result of the target position, and then the fitting result of the target position can be remapped into a second harmonic spectrum image. The distribution of different structures of the sample in space can be determined based on the second harmonic spectrum images at K positions.

[0097] The embodiment of the present application can directly obtain the spectral information of each position of the sample, and then directly determine the distribution of different structures of the sample in space based on the spectral information of each position. Compared with the existing method, it has a larger amount of information, can accurately characterize the nanoscale structural distribution in the sample, and has higher reliability.

[0098] Optionally, in some embodiments, after the above step 204, the second harmonic testing method may further include the following steps:

[0099] Perform matrix decomposition on K second harmonic spectrum images at K positions to determine the structural components of the sample.

[0100] In this embodiment, K second harmonic spectrum images at K positions can be matrix decomposed to determine the proportion of different structures at each position, and then the structural composition of the sample can be determined. Based on this, an image of the structural proportion can be redrawn to infer the nano-local structural characteristics of the sample.

[0101] Optionally, in some embodiments, the above step 202 may include the following steps:

[0102] At each set of polarization angles, adjusting the galvanometer group to an ideal deflection angle to scan the sample surface to obtain the first and second harmonic signal intensities, wherein the ideal deflection angle is determined based on a preset scanning position on the sample surface;

[0103] Record the actual deflection angle of the galvanometer group during the scanning process;

[0104] determining a first position of the sample surface according to the actual deflection angle;

[0105] The first second harmonic signal strength is determined as the second harmonic signal strength corresponding to a first position, where the first position is any position among the K positions.

[0106] It is understandable that when it is necessary to scan the preset scanning position on the sample surface, the galvanometer group can be controlled to deflect to an ideal deflection angle that matches the preset scanning position. However, the actual deflection angle of the galvanometer group during the scanning process may deviate from the ideal deflection angle to a certain extent. At this time, the actual position of the light spot on the sample surface also deviates from the preset scanning position to a certain extent. At this time, the actual deflection angle of the galvanometer group during the scanning process can be recorded, and the first position of the sample surface can be determined based on the actual deflection angle. The first position is the actual position of the light spot on the sample surface. The first second harmonic signal intensity obtained by scanning is matched with the first position to correct the data offset. In this way, the second harmonic signal intensity corresponding to each position can be made more accurate, further improving the reliability of the second harmonic test.

[0107] In order to further understand the second harmonic testing method provided in the embodiment of the present application, the method will be described below using a specific scenario embodiment.

[0108] For example, the scenario embodiment may include the following steps:

[0109] Polarization measurement: Rotate the first half-wave plate to change the polarization angle of the incident light After the motor stops, wait for an additional 500ms to ensure that the motor is not shaking, then trigger the PMT photodetector to collect data for 1000ms, and use the total number of collected photoelectrons as the measured second harmonic signal strength. The step length is 35 times of step measurement to determine the relationship between the second harmonic signal intensity and the polarization angle of the incident light.

[0110] Anisotropy measurement: Rotate the first half-wave plate to change the polarization angle of the incident light , turn the first Glan Taylor prism to change the second harmonic polarization angle α to 0, wait for the motor to stop, wait for an additional 500ms to ensure that the motor is not shaking, and then trigger the PMT photodetector to collect a 1000ms acquisition. The total number of collected photoelectrons is used as the measured second harmonic signal intensity. , Δα=10° is the step length, and 35 step measurements are performed to determine the relationship between the second harmonic signal intensity and the sample direction.

[0111] Second harmonic imaging: First, perform the measurements described in the above polarization measurement and anisotropy measurement, and rotate the first half-wave plate and the first Glan-Taylor prism to change the polarization angle of the incident light. The second harmonic polarization angle α is the angle of the maximum value in the above polarization measurement and anisotropy measurement results. After waiting for the motor to stop, wait for an additional 500ms to ensure that the motor is not shaking. Generate a sawtooth wave signal and a triangle wave signal to control the galvanometer group to perform continuous deflection movement, so that the light spot scans on the sample, and synchronously collects the PMT photodetector signal. Finally, according to the corresponding relationship between the scanning time and the scanning position, the collected signals are restored to the surface scanning data of the sample surface in sequence.

[0112] Second harmonic spectral imaging: Rotate the first half-wave plate and the first Glan-Taylor prism to change the polarization angle of the incident light and the second harmonic polarization angle α=0, wait for the motor to stop, and wait for an additional 500ms to ensure that the motor is not shaking. Generate a sawtooth wave signal and a triangle wave signal to control the galvanometer group to perform continuous deflection movement, so that the light spot scans on the sample, and synchronously collect the PMT photodetector signal and the actual deflection angle of the galvanometer group.

[0113] According to the corresponding relationship between scanning time and scanning position, the collected signals are restored to the surface scanning data of the sample in sequence. The actual deflection angle of the galvanometer group is converted into the actual position of the light spot to correct the data offset. , Δα=10° is the step length, and 35 step measurements are performed. The result is three-dimensional data of (36×n×n). The data is reorganized into (n×n×36) to obtain the anisotropic spectrum data of each position.

[0114] For the data at each position, the second harmonic is fitted as follows:

[0115] For a sample placed along the

[100] direction, the second harmonic coupling coefficient tensor has the following form:

[0116]

[0117] Therefore, the outgoing second harmonic polarization vector has the following form:

[0118]

[0119] Therefore, the measured second harmonic signal strength is:

[0120]

[0121]

[0122] I∝(P x cosα+P y sinα) 2 +C

[0123] If there are two coexisting structures in the sample in the measured area, the proportion of one structure can be represented by F1, and the proportion of the other structure can be 1-F1. In this case, the measured second harmonic signal intensity can be expressed as:

[0124]

[0125] In order to ensure the consistency of fitting at each position, during the fitting process, the d ij The F1 at each position is equal, but different F1 is allowed to reflect the different structural compositions at different positions.

[0126] Each F1 obtained by fitting has a one-to-one correspondence with each position, based on which the image of the structural ratio can be redrawn, thereby inferring the nano-local structural characteristics of the sample.

[0127] In some examples, when the laser scanning second harmonic microscopy system includes an objective lens and a stepper motor, and the stepper motor can drive the objective lens to move in the direction toward the sample placement module, before performing the second harmonic test, the stepper motor can also be controlled to drive the objective lens to move in the direction toward the sample placement module for focusing. In other words, by adjusting the distance between the objective lens and the sample placement module, the focus of the objective lens is on the upper surface of the sample. For example, the sample surface can be microscopically observed by a CCD camera, and the stepper motor can be operated synchronously. It can be understood that when the characteristic contour of the sample surface is clear, it means that the focus is located near the upper surface of the sample, and the depth of the current test can be considered to be "0" at this time. For most samples, when the focus is located near the upper surface of the sample, the second harmonic signal intensity of the sample is high, which is beneficial to improve the signal-to-noise ratio of the test.

[0128] In some examples, the distance between the objective lens and the sample placement module can be adjusted to detect data at a certain depth inside the sample. The second harmonic data can be continuously collected only along the depth direction of the sample by fixing the deflection angle of the galvanometer group and the polarization angle of the first half-wave plate and the first Glan Taylor prism, which is called depth scanning. The depth scanning method may include the following steps:

[0129] Step (1): adjust the distance between the objective lens and the sample placement module to the initial distance so that the objective lens focuses near the upper surface of the sample, and the depth is recorded as "0";

[0130] Step (2): adjusting the deflection angle of the galvanometer group so that the light spot is located at the position to be measured of the sample;

[0131] Step (3): adjusting the first half-wave plate and the first Glan Taylor prism so that the polarization angle is in a target polarization state. It can be understood that in the target polarization state, the second harmonic signal intensity of the sample is the highest;

[0132] Step (4): perform a second harmonic acquisition. The acquisition time can be determined according to the accuracy requirement, but is usually not shorter than 100ms.

[0133] Step (5): Control the stepper motor to drive the objective lens to move once toward the direction of the sample placement module. The moving length can be determined according to the needs. Generally speaking, the moving length is greater than or equal to the shortest step length. The minimum step length can be set according to the actual situation. For example, the minimum step length can be 100nm. After moving, you can wait for an additional 500ms to ensure that the motor is not shaking;

[0134] Step (6): Repeat steps (4) and (5) until the maximum depth required for measurement is reached. The maximum depth can be determined according to requirements. Generally speaking, the maximum depth is less than or equal to the working distance of the objective lens to prevent the objective lens from colliding with the sample. For example, the measurement depth range can be 50-1000 μm.

[0135] The present application also provides a second harmonic depth imaging method, which may include the following steps:

[0136] Step A, when the sample is fixed on the sample placement module, the angles of the first half-wave plate and the first Glan Taylor prism are adjusted to the target polarization angle, the target polarization angle includes the target incident light polarization angle and the target second harmonic polarization angle, and the target polarization angle is any polarization angle whose corresponding second harmonic signal intensity meets the preset conditions.

[0137] It is understandable that before performing depth imaging, polarization measurement is usually required to determine the appropriate target polarization angle. For example, multiple second harmonic signal intensities at multiple polarization angles can be measured, and the polarization angle corresponding to the highest second harmonic signal intensity can be determined as the target polarization angle, or any polarization angle corresponding to the second harmonic signal intensity greater than a preset threshold can be determined as the target polarization angle, which is not specifically limited here.

[0138] Step B, adjusting the stepper motor until the focus of the objective lens is located at the depth to be measured inside the sample.

[0139] Step C, adjusting the deflection angle of the galvanometer group to scan the P second harmonic signal intensities corresponding to the P positions on the surface of the sample at the depth to be measured;

[0140] Step D: drawing a second harmonic image of the depth to be measured according to the P second harmonic signal intensities.

[0141] Based on the above-mentioned depth scanning method and depth imaging method, an embodiment of the present application can also provide a three-dimensional reconstruction method, which can repeat the above-mentioned steps B and C according to the depth scanning method to obtain Q second harmonic data corresponding to Q depths, wherein the second harmonic data includes P second harmonic signal intensities.

[0142] According to the second harmonic data corresponding to each of the Q depths, Q average second harmonic signal intensities corresponding to the Q depths are determined.

[0143] For example, for P second harmonic signal intensities corresponding to any depth among the Q depths, the average value thereof is calculated to obtain the average second harmonic signal intensity corresponding to the depth.

[0144] According to the Q average second harmonic signal intensities, second harmonic signal intensities fitting is performed on the Q second harmonic data to obtain Q target second harmonic data.

[0145] It is understandable that as the depth increases, the second harmonic signal intensity collected will be less affected by the depth, and the influence of the depth itself on the second harmonic signal intensity needs to be excluded when performing three-dimensional reconstruction later. Based on this, the second harmonic signal intensity can be fitted with the second harmonic data at different depths. For example, the weight coefficient of the depth can be determined based on the average second harmonic signal intensity at each depth, for example, the weight coefficient can be 1 / average second harmonic signal intensity. The second harmonic signal intensities of the depth P can be multiplied by the weight coefficient of the depth respectively, and the product can be used as P target second harmonic signal intensities (i.e., the target second harmonic data of the depth).

[0146] A three-dimensional reconstructed image is drawn based on Q target second harmonic data.

[0147] It should also be noted that the exemplary embodiments mentioned in this application describe some methods or systems based on a series of steps or devices. However, this application is not limited to the order of the above steps, that is, the steps can be performed in the order mentioned in the embodiment, or in a different order from the embodiment, or several steps can be performed simultaneously.

[0148] Aspects of the present disclosure are described above with reference to the flowchart and / or block diagram of the method, device (system) and program product according to the embodiment of the present disclosure. It should be understood that each box in the flowchart and / or block diagram and the combination of each box in the flowchart and / or block diagram can be implemented by a computer program or instruction. These programs or instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device to produce a machine so that these instructions executed by the processor of the computer or other programmable data processing device enable the implementation of the function / action specified in one or more boxes of the flowchart and / or block diagram. Such a processor can be, but is not limited to, a general-purpose processor, a special-purpose processor, a special application processor, or a field programmable logic circuit. It can also be understood that each box in the block diagram and / or flowchart and the combination of boxes in the block diagram and / or flowchart can also be implemented by dedicated hardware that performs a specified function or action, or can be implemented by a combination of dedicated hardware and computer instructions.

[0149] The above is only a specific implementation of the present application. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, modules and units described above can refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here. It should be understood that the protection scope of the present application is not limited to this. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in this application, and these modifications or replacements should be included in the protection scope of this application.

Claims

1. A laser scanning second harmonic generation microscopy system, characterized in that: include: A laser emission module (1) comprises a femtosecond laser (11) and a first half-wave plate (12) which are arranged in sequence, wherein the femtosecond laser (11) is used to emit femtosecond pulse laser light, and the first half-wave plate (12) can rotate along the optical axis of the femtosecond pulse laser light to adjust the polarization angle of incident light; A sample placement module (2), used for placing samples; A beam adjustment module (3) is arranged between the laser emission module (1) and the sample placement module (2), the beam adjustment module (3) comprising a galvanometer group (31) and a dichroic mirror (32) arranged in sequence, the galvanometer group (31) being optically connected to the first half-wave plate (12), the galvanometer group (31) being used to adjust the position where the femtosecond pulse laser is focused on the sample surface, the femtosecond pulse laser being focused on the sample surface after being reflected by the dichroic mirror (32) to generate second harmonic light; A signal receiving module (4) comprises a first Glan Taylor prism (41) and a photomultiplier tube (PMT) photodetector (42) arranged in sequence, wherein the first Glan Taylor prism (41) is optically connected to the dichroic mirror (32), the second harmonic light is incident on the first Glan Taylor prism (41) after being transmitted through the dichroic mirror (32), and the first Glan Taylor prism (41) can rotate along the optical axis of the second harmonic light to adjust the second harmonic polarization angle; Among them, by adjusting the angles of the first half-wave plate and the first Glan Taylor prism to adjust the polarization angle of the incident light and the second harmonic polarization angle, M groups of polarization angles are obtained; at each group of the polarization angles, the deflection angle of the galvanometer group is adjusted to scan K second harmonic signal intensities corresponding to K positions on the sample surface, where M and K are integers greater than 1.

2. The system according to claim 1, characterized in that The laser emission module (1) further comprises: A second half-wave plate (13) is connected to the optical path of the femtosecond laser (11), and the second half-wave plate (13) can rotate along the optical axis of the femtosecond pulse laser; A second Glan Taylor prism (14) is optically connected to the second half-wave plate (13) and the first half-wave plate (12). The second half-wave plate (13) and the second Glan-Taylor prism (14) are used to adjust the intensity of incident light.

3. The system according to claim 1, characterized in that The light beam adjustment module (3) further comprises: The focusing assembly comprises a scanning lens (33), a sleeve lens (34) and an objective lens (35); the scanning lens (33) and the sleeve lens (34) are arranged in sequence between the galvanometer group (31) and the dichroic mirror (32); and the objective lens (35) is arranged between the dichroic mirror (32) and the sample placement module (2).

4. The system according to claim 3, characterized in that The focusing assembly further comprises a first bandpass filter (36), wherein the first bandpass filter (36) is arranged between the tube lens (34) and the dichroic mirror (32).

5. The system according to claim 3, characterized in that The focusing assembly further comprises a stepping motor, which is connected to the objective lens (35) and is used to drive the objective lens (35) to move in a direction towards the sample placement module (2).

6. The system according to claim 1, characterized in that The signal receiving module (4) further comprises: A first beam splitter (43) disposed between the dichroic mirror (32) and the first Glan Taylor prism (41); A second beam splitter (44), optically connected to the second beam splitter (44); An illumination assembly (45) optically connected to the second beam splitter (44); A charge coupled device (CCD) camera (46) is optically connected to the second beam splitter (44).

7. The system according to claim 1, characterized in that The signal receiving module (4) further comprises: A second bandpass filter (47) is arranged between the dichroic mirror (32) and the first Glan-Taylor prism (41).

8. The system according to claim 1, characterized in that Also includes: A reflection module (5) comprises at least one reflection mirror, and the reflection module (5) is arranged between the first half-wave plate (12) and the galvanometer group (31).

9. The system according to claim 1, characterized in that The sample placement module (2) comprises: The first placement component includes at least one of a three-axis translation table, a tilt table and a rotation table, wherein the three-axis translation table is used to adjust the displacement of the sample, the tilt table is used to adjust the inclination of the sample, and the rotation table is used to adjust the placement angle of the sample.

10. The system according to claim 9, characterized in that The sample placement module (2) further comprises: a second placement component, the second placement component can be used in conjunction with the first placement component, the second placement component includes N temperature controllers, and the N temperature controllers correspond one-to-one to N preset temperature intervals, Among them, the target temperature controller is used to place samples within the target temperature range, the target temperature controller is any temperature controller among the N temperature controllers, the target temperature range is the temperature range corresponding to the target temperature controller among the N preset temperature ranges, and N is a positive integer.

11. A method for testing second harmonics using the laser scanning second harmonic microscopy system according to any one of claims 1 to 10, characterized in that: include: When the sample is fixed on the sample placement module, adjusting the angles of the first half-wave plate and the first Glan Taylor prism to determine M groups of polarization angles, wherein the polarization angles include an incident light polarization angle and a second harmonic polarization angle, and M is an integer greater than 1; Under each group of polarization angles, adjusting the deflection angle of the galvanometer group to scan K second harmonic signal intensities corresponding to K positions on the sample surface, to obtain M groups of K second harmonic signal intensities, where K is an integer greater than 1; Determine, according to the M groups of the K second harmonic signal intensities, the M second harmonic signal intensities of the target position under the M groups of polarization angles, wherein the target position is any one of the K positions; Perform spectrum fitting on the M second harmonic signal intensities of the target position, determine the fitting function and fitting result of the target position, and draw a second harmonic spectrum image of the target position.

12. The method according to claim 11, characterized in that After drawing the second harmonic spectrum image of the target position, the method further includes: Matrix decomposition is performed on the K second harmonic spectrum images at the K positions to determine the structural components of the sample.

13. The method according to claim 11, characterized in that The method of adjusting the deflection angle of the galvanometer group under each group of polarization angles to scan K second harmonic signal intensities corresponding to K positions on the sample surface to obtain M groups of the K second harmonic signal intensities comprises: Under each set of polarization angles, adjusting the galvanometer group to an ideal deflection angle to scan the sample surface to obtain the first second harmonic signal intensity, wherein the ideal deflection angle is determined based on a preset scanning position of the sample surface; Recording the actual deflection angle of the galvanometer group during the scanning process; determining a first position of the sample surface according to the actual deflection angle; The first second harmonic signal strength is determined as the second harmonic signal strength corresponding to the first position, where the first position is any position among the K positions.

Images