Line scanning second harmonic imaging device

Abstract

The utility model discloses a line scanning second harmonic imaging device, which comprises a line light spot generation module, a vertical light incidence assembly and a scanning imaging module, and is characterized in that the vertical light incidence assembly is arranged in the line light spot generation module; a linear light spot generated in the linear light spot generation module is emitted to the surface of a sample through the vertical light incidence assembly to excite a second harmonic signal; and the second harmonic signal enters the scanning imaging module through the vertical light incidence assembly. The linear light spot generation module converts a light beam emitted by the laser light source into a linear light spot through an optical element, and the linear light spot covers a large area, so that a second harmonic signal on a line can be obtained at a time during scanning instead of point-by-point scanning, the scanning speed is remarkably improved, and the linear light spot generation module is particularly suitable for rapid imaging of a large-area sample; the scanning imaging module scans a sample line by line in a line scanning mode, so that the splicing work of spot scanning data is reduced, and the comprehensive efficiency and resolution of scanning are improved.

Description

TECHNICAL FIELD

[0001] The utility model belongs to the field of spectral imaging, especially relates to a linear laser scanning second harmonic spectral imaging device. BACKGROUND

[0002] Second harmonic generation (SHG) is a nonlinear optical technique that analyzes the second harmonic signal of light on the surface of a substance to study the structure, symmetry, and electronic properties of the substance. Its basic principle is nonlinear optical effect. Under certain conditions, when high-intensity incident light interacts with a substance, the electric polarization intensity of the substance is not only linearly related to the electric field intensity, but also produces higher-order responses, especially the second harmonic response. SHG is a frequency doubling phenomenon, that is, two incident photons interact to produce a photon with twice the frequency. This effect usually occurs only in media with no anti-symmetry, commonly found in structures such as crystals, interfaces, and surfaces that lack central symmetry.

[0003] In the field of biological imaging, SHG spectral technology also has unique advantages. Unlike fluorescence imaging, SHG does not require labeling and can directly image non-centrally symmetric molecular structures such as collagen in biological tissues, with high resolution and low light damage. Therefore, SHG has gradually attracted attention in biomedical research and has been used to observe structural information in living tissues, such as analyzing collagen fiber distribution in skin or soft tissue and changes in tumor microenvironment.

[0004] In Chinese patent document no. CN104330398A, a multi-mode nonlinear optical microscopic imaging method and device are disclosed. The device mainly consists of a laser system, an optical scanning microscope, a nonlinear optical signal detection and acquisition system, etc. It can work in single laser beam and double laser beam modes, and can realize various modes of nonlinear optical microscopic imaging on ex vivo biological tissues and living cells, such as two-photon excitation fluorescence (TPEF) imaging, multi-photon high harmonic (such as second harmonic SHG, third harmonic THG, etc.) scattering imaging, and coherent Raman scattering (such as anti-Stokes CARS) microscopic imaging. Thus, various nonlinear specific optical signals of biological tissue samples can be obtained in situ, providing an important basis for optical diagnosis and in-depth analysis of samples.

[0005] In the above technical solution, although the microscopic imaging technology can be coupled in one platform or system, that is, the target object can be imaged in multiple modes by the nonlinear optical microscopic imaging on the same platform or system, thereby realizing in-situ characterization and acquisition of multiple specific information on the target object, the excitation light source used is still a point light source, which limits the excitation area of the second harmonic spectrum to the position of the laser spot. Therefore, only a single component of the sample material can be collected during testing, and if the laser spot irradiation position is a plurality of materials, the result will lose analyzability. Therefore, especially when facing a large-area non-single-component sample, the method can not accurately analyze the sample components. Even if the second harmonic spectrum imaging technology is used, the comprehensive efficiency and resolution of single-point scanning are relatively low.

[0006] Therefore, in order to improve the application potential of the second harmonic spectrum imaging when facing complex sample materials, it is necessary to develop a new linear scanning second harmonic imaging device, which can improve the application potential of the second harmonic spectrum imaging when the device faces complex sample materials, and further improve the comprehensive efficiency and resolution of scanning. The utility model discloses

[0007] The utility model wants to solve the technical problem to provide a kind of linear scanning second harmonic imaging device based on line laser spot generation and further improve the comprehensive efficiency and resolution of scanning.

[0008] To solve the above technical problems, the technical scheme adopted by the utility model is: a kind of linear scanning second harmonic imaging device, including line spot generation module, vertical light component and scanning imaging module, the vertical light component is provided in the line spot generation module;Line spot generated in the line spot generation module is shot to sample surface by the vertical light component, and excites second harmonic signal;The second harmonic signal enters the scanning imaging module by the vertical light component.

[0009] The line spot generation module converts the light beam emitted by the laser light source into a linear spot by optical elements. Since the linear spot covers a large area, the second harmonic signal on a line can be acquired at one time during scanning, rather than point-by-point scanning, which significantly improves the scanning speed and is particularly suitable for fast imaging of large-area samples. The vertical light component is responsible for scanning the linear spot to the sample surface in the vertical direction and ensuring that the excitation light can uniformly irradiate the sample surface. The scanning imaging module scans the sample line by line in a line scanning mode. By constantly changing the position of the laser spot on the sample surface and simultaneously detecting the second harmonic signal, an imaging data is finally formed. The imaging mode of the linear spot scanning a line at one time makes the information of each frame of image more complete, reduces the splicing work of point scanning data, and therefore the data processing steps are simpler, which helps to more efficiently analyze the imaging result and improves the comprehensive efficiency and resolution of scanning.

[0010] Preferably, the line spot generating module comprises a laser providing an excitation light source, an output end of the laser is connected with a beam expander lens group, a DOE diffractive element is arranged behind the beam expander lens group, the DOE diffractive element is installed on a rotatable adjusting frame, and an adjustable line diaphragm is further arranged on the rotatable adjusting frame.

[0011] The laser is used as the light source to provide a high-power, monochromatic and coherent laser beam, and the laser part uses a modular design, so that different lasers can be easily switched without considering recalibration of the optical path, thereby greatly improving the use scenario coverage of the device. The laser output beam usually has a certain spot size and divergence angle, and if it is directly used for line spot generation, it will affect the subsequent imaging effect. Therefore, the laser output end is connected to a beam expander lens group to expand and collimate the laser beam to meet the requirements of the subsequent diffractive optical element on the beam width and shape. The DOE diffractive element is used to convert the circular spot of the laser beam into a linear spot. The line diaphragm itself can adjust the diaphragm size, and can also be used in combination with the DOE diffractive element. The shape and size of the final line scanning spot are adjusted by adjusting the rotation angle of the line spot, the size of the line diaphragm, and the rotation angle of the line diaphragm in three dimensions. The modular design improves the stability and overall efficiency of scanning.

[0012] Preferably, the beam expander lens group comprises a beam expander plano-concave lens and a beam expander plano-convex lens, the beam expander plano-concave lens is located in front of the laser beam emitted by the laser, the beam expander plano-convex lens is arranged behind the beam expander plano-concave lens, and the focal points of the beam expander plano-concave lens and the beam expander plano-convex lens coincide.

[0013] The design of the beam expander lens group can ensure that the laser beam has a suitable spot size for diffraction and can provide suitable excitation conditions for subsequent second harmonic signal generation. The design of the combined lens can not only effectively expand the beam, but also maintain the quality and collimation of the beam. By adjusting the distance between the two lenses, the expansion degree of the beam and the size of the spot can be controlled to provide a stable light source for subsequent diffractive optical processing.

[0014] Preferably, the vertical light inlet assembly comprises at least one two-grade switch, and the two-grade switch is provided with a dichroic mirror.

[0015] Preferably, the two-grade switch comprises a light inlet grade and an empty grade, the light inlet grade is provided with a dichroic mirror, is used for reflecting the line spot downward, and the second harmonic signal is collected upward; and the empty grade is used for blocking the line spot and the second harmonic signal.

[0016] The basic function of the two-grade switch is to adjust the light path through two working states, so that the light beam can propagate along different paths; the vertical light entry assembly can switch between the light entry grade and the empty grade through the combination of the two-grade switch and the dichroic mirror, so as to ensure the effective separation and accurate collection of the laser beam and the second harmonic signal. The design not only improves the flexibility of the system, but also enhances the quality and reliability of the signal.

[0017] Preferably, the scanning imaging module comprises a band-pass filter, the second harmonic signal enters the band-pass filter through the vertical light entry assembly and is transmitted to the spectrometer, and the spectrometer is connected with a CCD camera.

[0018] The band-pass filter is an important optical element in the scanning imaging module, which mainly functions to selectively transmit light within a specific wavelength range and block other unnecessary wavelengths; the spectrometer is responsible for spectral analysis of the transmitted light signal; the CCD camera is the core imaging device in the scanning imaging module, which is mainly used to capture the second harmonic signal image transmitted from the spectrometer, and has high sensitivity and low noise characteristics, which can effectively record weak second harmonic signals; the scanning imaging module realizes accurate filtering, analysis and imaging of the signal through the cooperation of the band-pass filter, the spectrometer and the CCD camera.

[0019] Preferably, the CCD camera receives the second harmonic signal processed by the spectrometer, and the CCD camera is connected with a computer system to display the collected image in real time.

[0020] The CCD camera receives and converts the second harmonic signal processed by the spectrometer to generate high-quality images, and is connected with a computer system to realize real-time display and image processing. The computer system provides functions such as automatic splicing, color coding and intensity analysis of imaging data through special software, so that the generated image can accurately reflect the optical and structural characteristics of the sample.

[0021] Preferably, an electrically controlled scanning table is further arranged below the line light spot generating module and the scanning imaging module, and the sample to be scanned is placed on the electrically controlled scanning table.

[0022] Preferably, the electrically controlled scanning table comprises a translation table, the translation table comprises an X-axis translation table, a Y-axis translation table and a Z-axis translation table, and a fixing device is arranged on the translation table.

[0023] Preferably, the fixing device is a vacuum adsorption cavity, and the vacuum adsorption cavity is connected with a vacuum pump.

[0024] The motorized scanning stage is designed to ensure stability during movement, preventing vibration from interfering with signal acquisition. The fixing mechanism on the translation stage allows for flexible selection of the fixing method based on the sample shape and characteristics, ensuring target stability during scanning and preventing damage to fragile samples. The electrically controlled scanning stage design ensures the line laser is perpendicular to the scanning direction, enabling large-area sample coverage imaging through stage movement, significantly improving imaging efficiency compared to traditional point-by-point scanning methods. Furthermore, the system employs a modular design, compatible with various optical devices, allowing users to flexibly expand functionality according to experimental needs. This scanning stage exhibits extremely low noise and vibration during operation, making it suitable for second harmonic imaging, and demonstrating exceptional performance, particularly in imaging biological tissues and low-dimensional materials. Attached Figure Description

[0025] The following is a detailed description of the embodiments of this utility model in conjunction with the accompanying drawings:

[0026] Figure 1 This is a schematic diagram of the overall design of this utility model;

[0027] Figure 2 for Figure 1 Schematic diagram of the centerline spot generation module and the scanning imaging module;

[0028] Figure 3 A top view of the line spot generation module;

[0029] Figure 4 for Figure 3 Top view of the vertical light-incident component;

[0030] Figure 5 This is a top view of the scanning imaging module;

[0031] Figure 6 This is a frontal view of the motorized scanning stage.

[0032] Among them: 1-line spot imaging module, 2-vertical incident light assembly, 3-scanning imaging module, 4-laser, 5-beam expander lens group, 6-DOE diffraction element, 7-rotatable adjustment frame, 8-adjustable line aperture, 9-beam expander plano-concave lens, 10-beam expander plano-convex lens, 11-two-position switch, 12-dichroic mirror, 13-incident light position, 14-neutral position, 15-bandpass filter, 16-spectrometer, 17-CCD camera, 18-electronically controlled scanning stage, 19-translation stage, 20-X-axis translation stage, 21-Y-axis translation stage, 22-Z-axis translation stage, 23-vacuum adsorption cavity, 24-vacuum pump, 25-objective lens. Detailed Implementation

[0033] As attached Figures 1-6As shown, the line scanning second harmonic imaging device of the embodiment comprises a line light spot generating module 1, a vertical light entry assembly 2 and a scanning imaging module 3, and the vertical light entry assembly 2 is arranged in the line light spot generating module 1; the line light spot generated in the line light spot generating module 1 is emitted to the sample surface through the vertical light entry assembly 2 to excite a second harmonic signal; and the second harmonic signal enters the scanning imaging module 3 through the vertical light entry assembly 2.

[0034] The line light spot generating module 1 comprises a laser 4 for providing an excitation light source, the output end of the laser 4 is connected with an expansion lens group 5, a DOE diffraction element 6 is arranged behind the expansion lens group 5, and the DOE diffraction element 6 is installed on a rotatable adjusting frame 7, and an adjustable line diaphragm 8 is also arranged on the rotatable adjusting frame 7.

[0035] As shown in particular, Figure 2 , Figure 3 The DOE diffraction element 6 is an important component for generating a line light spot, and the DOE diffraction element 6 is installed on a 30mm cage structure rotatable 1 inch rotatable adjusting frame 7, and the generation angle of the line light spot can be adjusted by rotation; the adjustable line diaphragm 8 is behind the DOE diffraction element 6, and the adjustable line diaphragm 8 is also installed on the rotatable adjusting frame 7, and the adjustable line diaphragm 8 can adjust the size of the diaphragm itself, and can also be used in combination with the DOE diffraction element 6, and the shape and size of the final line scanning light spot are adjusted by adjusting the rotation angle of the line light spot, the size of the line diaphragm and the rotation angle of the adjustable line diaphragm 8.

[0036] The expansion lens group 5 comprises an expansion flat concave lens 9 and an expansion flat convex lens 10, the expansion flat concave lens 9 is located at the front end of the laser beam emitted by the laser 4, the expansion flat convex lens 10 is arranged behind the expansion flat concave lens 9, and the focal points of the expansion flat concave lens 9 and the expansion flat convex lens 10 coincide.

[0037] As shown in particular, Figure 3 The expansion lens group 5 is composed of the expansion flat concave lens 9 and the expansion flat convex lens 10, wherein the focal length of the expansion flat concave lens 9 is-20mm, the focal length of the expansion flat convex lens 10 is 100mm, and the focal points of the two lenses are adjusted to coincide during installation to achieve the best expansion effect.

[0038] The vertical light entry assembly 2 at least comprises a two-grade switch 11, the two-grade switch 11 is provided with a dichroic mirror 12, and the two-grade switch 11 comprises a light entry grade 13 and a blank grade 14, the light entry grade 13 is provided with the dichroic mirror 12, is used for reflecting the line light spot downward, and the second harmonic signal is collected upward; and the blank grade 14 is used for blocking the line light spot and the second harmonic signal.

[0039] As shown in particular, Figure 4As shown, the light entry slot 13 is provided with a dichroic mirror 12, which can reflect the linear light spot downward and focus it on the sample through the objective lens 25, while the excited second harmonic signal is collected upward and passes through the dichroic mirror 12 to the scanning imaging module 3; the other slot is empty, which can block the laser spot and interrupt the second harmonic scanning imaging. In this embodiment, the vertical light entry assembly is also modularly designed, which can be matched with the wavelength switching of the excitation light source to correspond to the dichroic mirror 12.

[0040] The scanning imaging module 3 includes a band-pass filter 15, and the second harmonic signal enters the spectrometer 16 through the band-pass filter 15 after passing through the vertical light entry assembly 2. The spectrometer 16 is connected with a CCD camera 17, which receives the second harmonic signal processed by the spectrometer 16. The CCD camera 17 is connected with a computer system, which displays the collected image in real time.

[0041] As shown in the specific embodiment, Figure 5 As shown, the scanning imaging module 3 is located above the linear light spot generation module 1 and is connected with the vertical light entry assembly 2 in the linear light spot generation module 1. The second harmonic signal generated by the linear light spot scanning sample passes through the dichroic mirror 12 in the vertical light entry assembly 2 and reaches the scanning imaging module 3. The second harmonic signal filtered by the band-pass filter 15 enters the spectrometer 16 after coupling processing. The CCD camera 17 is connected with the spectrometer 16. The second harmonic signal is split by the spectrometer 16 and transmitted to the CCD camera 17 through an optimized light path for signal capture and image collection. The CCD camera 17 is connected to the computer system. The computer software can display the collected image in real time and enhance the signal.

[0042] A motorized scanning stage 18 is further arranged below the linear light spot generation module 1 and the scanning imaging module 3. The sample to be scanned is placed on the motorized scanning stage 18. The motorized scanning stage 18 includes a translation stage 19, which includes an X-axis translation stage 20, a Y-axis translation stage 21 and a Z-axis translation stage 22. The translation stage 19 is provided with a fixing device.

[0043] As shown in the specific embodiment, Figure 6 The fixing device is a vacuum suction cavity 23 connected with a vacuum pump 24. The motorized scanning stage 18 adopts a track-driven design, which controls the track movement through a high-precision stepping motor or a servo motor to realize sub-micron or even nanometer level movement precision. The control system of the motorized scanning stage 18 combines a precision encoder and a displacement sensor to monitor the stage position in real time and realize parameter setting and scanning path planning through software, which supports large-scale and high-speed scanning and is suitable for the second harmonic imaging needs of various samples. The linear laser is perpendicular to the scanning direction, and the movement of the translation stage 19 completes the coverage imaging of a large-area sample.

[0044] In actual use, the flat substrate sample is placed on the motorized scanning stage module 18, and the vacuum suction cavity 23 method is selected for fixation. The vacuum pump 24 is turned on, and the sample is flatly adsorbed on the stage. The software is opened, the Z-axis translation stage 22 of the motorized scanning stage is adjusted, and the optimal coupling distance between the sample surface and the linear laser spot is ensured. Then, the X-axis translation stage 20 and the Y-axis translation stage 21 of the motorized scanning stage are adjusted to adjust the position of the sample, so that the linear spot is located at the starting position of the planned scanning area. The laser 4 in the linear spot generation module 1 is turned on.

[0045] It is important to note that this embodiment uses a femtosecond pulsed laser 4 with a wavelength of 1064 nm, a pulse width of 200 fs, and a repetition frequency of 80 MHz, which can generate a sufficiently strong second harmonic signal on the sample surface. By adjusting the relative positions of the beam-expanding plano-concave lens 9 and the beam-expanding plano-convex lens 10, their focal points are made to coincide, achieving the optimal beam expansion effect. At this point, the laser beam is collimated. Then, the rotation angle of the cage-like rotatable adjustment frame 7 equipped with the DOE diffraction element 6, the rotation angle of the cage-like rotatable adjustment frame 7 equipped with the adjustable line aperture 8, and the aperture size of the adjustable line aperture 8 are adjusted. Finally, the line beam is reflected downwards by the two-stage switch 11 and uniformly illuminates the surface of the sample placed on the motorized scanning stage 18. The entire experimental process is parameter-set in the software, which provides precise adjustment functions for parameters such as scanning range, step size, and scanning speed. To cover the entire sample surface, the scanning range is typically set to the size of the entire sample or the main sample area, with a step size usually set to one-hundredth of the single-axis distance of the scanning area to ensure high-resolution images. The scanning speed is also set to an appropriate value to balance accuracy and efficiency. After all parameters are set, the device is started, and the motorized scanning stage 18 begins precise stepping along a direction perpendicular to the line spot, while the line spot remains stationary, illuminating the sample surface. During scanning, when the line spot excites the sample, the second harmonic signal generated on the sample surface is transmitted upwards and enters the collection optical path of the scanning imaging module 3. The high numerical aperture objective lens 25 in the collection optical path efficiently focuses these signals while removing excitation and stray light through the bandpass filter 15, allowing only the second harmonic signal of the target wavelength band to pass through. Subsequently, the signal is transmitted to the spectrometer 16, which performs spectral processing and guides the spectralized signal to the high-sensitivity CCD camera 17 for image capture. With its excellent low-noise performance and high signal-to-noise ratio, the CCD camera 17 accurately records the spatial distribution and intensity of the second harmonic signal. Throughout the scanning process, each step of the motorized scanning stage 18 corresponds to one frame of image acquired by the CCD camera 17. Computer software stitches these images together in real time, gradually generating a complete image covering the entire sample surface. After imaging is complete, the stitched final image can be viewed directly in the software interface, and the signal intensity of the region of interest can be analyzed. The software also provides various data processing functions, such as color encoding of the image, analysis of signal strength distribution, and extraction of structural features from specific regions of the image.

[0046] The above-described specific embodiments further specifically describe the purposes, technical solutions and beneficial effects of the present application, and it should be understood that the above-described specific embodiments are merely examples of the present application and are not intended to limit the present application; any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A line-scanning second harmonic imaging device, characterized in that, The device includes a line spot generation module, a vertical light incident component, and a scanning imaging module. The line spot generation module is equipped with the vertical light incident component. The line spot generated in the line spot generation module is incident on the sample surface through the vertical light incident component, exciting a second harmonic signal. The second harmonic signal enters the scanning imaging module through the vertical light incident component.

2. The line-scanning second harmonic imaging device according to claim 1, characterized in that, The line spot generation module includes a laser that provides an excitation source. The output end of the laser is connected to a beam expander lens group. A DOE diffraction element is disposed behind the beam expander lens group. The DOE diffraction element is mounted on a rotatable adjustment frame. An adjustable line stop is also disposed on the rotatable adjustment frame.

3. The line-scanning second harmonic imaging device according to claim 2, characterized in that, The beam expanding lens group includes a beam expanding plano-concave lens and a beam expanding plano-convex lens. The beam expanding plano-concave lens is located at the front end of the laser beam emitted by the laser, and the beam expanding plano-convex lens is disposed behind the beam expanding plano-concave lens. The focal points of the beam expanding plano-concave lens and the beam expanding plano-convex lens coincide.

4. The line-scanning second harmonic imaging device according to claim 1, characterized in that, The vertical light incident component includes at least one two-position switcher, which is equipped with a dichroic mirror.

5. The line-scanning second harmonic imaging device according to claim 4, characterized in that, The two-position switch includes an incident light position and an unattended position. The incident light position is equipped with a dichroic mirror to reflect the linear light spot downwards and collect the second harmonic signal upwards. The unattended position is used to block the linear light spot and the second harmonic signal.

6. The line-scanning second harmonic imaging device according to claim 1, characterized in that, The scanning imaging module includes a bandpass filter. The second harmonic signal passes through the vertical light incident component and enters the bandpass filter to be transmitted into the spectrometer. The spectrometer is connected to a CCD camera.

7. The line-scanning second harmonic imaging device according to claim 6, characterized in that, The CCD camera receives the second harmonic signal after it has been processed by the spectrometer. The CCD camera is connected to a computer system and displays the acquired images in real time.

8. The line-scanning second harmonic imaging device according to claim 1, characterized in that, Below the line spot generation module and the scanning imaging module, there is an electronically controlled scanning stage, on which the sample to be scanned is placed.

9. The line-scanning second harmonic imaging device according to claim 8, characterized in that, The electronically controlled scanning stage includes a translation stage, which includes an X-axis translation stage, a Y-axis translation stage, and a Z-axis translation stage. A fixing device is provided on the translation stage.

10. The line-scanning second harmonic imaging device according to claim 9, characterized in that, The fixing device is a vacuum adsorption chamber, which is connected to a vacuum pump.

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