A deep-sea microscopic raman imaging system and method

Abstract

This invention relates to a micro-Raman imaging system, specifically a deep-sea micro-Raman imaging system and method. It addresses the problems of existing micro-Raman imaging systems in deep-sea particulate matter detection, which cannot simultaneously achieve high resolution, a large field of view optical images, and accurate Raman signals, and whose resolution and magnification are limited by the increased working distance of the microscope array. The system of this invention includes a microscopic module composed of a low-magnification objective lens and a high-magnification objective lens that can be switched via an XY linear translation stage; a dichroic mirror disposed between the microscopic module and the microscopic imaging module; a microscopic imaging module composed of an imaging relay mirror and a first camera sequentially arranged on the transmission path of the dichroic mirror; a beam expander and a beam splitter sequentially arranged on the reflection path of the dichroic mirror; a Raman coupler disposed on the transmission path of the beam splitter; and a laser source disposed on the reflection path of the beam splitter. The Raman coupler is used to couple the Raman signal light to the focusing surface of the Raman spectrometer to achieve Raman spectral imaging.

Description

Technical Field

[0001] This invention relates to micro Raman imaging systems, specifically to a deep-sea micro Raman imaging system and method. Background Technology

[0002] Marine ecological monitoring plays a crucial role in the development and protection of the ocean and is receiving increasing attention. Deep-sea pollutants, such as marine microplastics, pose a significant threat to the ecosystem. Real-time monitoring of these pollutants helps researchers develop protection plans, and micro-Raman imaging systems are an important monitoring tool. Micro-Raman imaging technology combines Raman spectroscopy imaging systems and optical microscopy imaging systems. It allows for rapid identification of minute objects, accurate qualitative analysis of morphology and composition, and quantitative detection of abundance. This offers significant advantages over previous single imaging techniques. Current micro-Raman imaging systems require the microscope group in the optical microscopy imaging system to capture the target's position during target detection. Then, a laser beam emitted from the laser source in the Raman spectroscopy imaging system excites Raman signal light on the target. This Raman signal light passes sequentially through the microscope group and various mirror groups in the Raman spectroscopy imaging system before finally focusing onto the Raman spectrometer to achieve Raman spectroscopy imaging. However, existing micro-Raman imaging systems still have the following problems in use:

[0003] 1) When detecting particulate matter in the deep sea, to obtain the abundance of the sample and quickly locate the local particulate matter sample, a large field of view and low magnification optical microscope group is required. However, to perform accurate morphology analysis of local particulate matter and obtain high-resolution optical images, a high-magnification optical microscope group is required. Existing micro Raman imaging systems require large field of view imaging for rapid particulate matter location, and their optical microscope groups generally have low magnification. This limits the imaging resolution and makes it impossible to obtain high-resolution optical images of the sample area. This will prevent the Raman spectroscopy imaging system from performing accurate scanning imaging and thus from obtaining accurate Raman signals.

[0004] 2) When using micro Raman imaging systems in the deep sea, pressure-resistant windows are often required to ensure that the pressure of the internal system is within a reasonable range. As the water depth increases, the thickness of the pressure-resistant window also needs to be increased. The optical microscopic imaging system in the existing micro Raman imaging system only has one microscope group. In order to adapt to the fact that the thickness of the pressure-resistant window increases with depth, the working distance of the microscope group will also increase. Since the working distance is inversely proportional to the resolution and magnification of the microscope group, the resolution and magnification of the microscope group are limited. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problems of existing micro Raman imaging systems when detecting particulate matter in the deep sea, which are unable to simultaneously obtain high-resolution, large-field-of-view optical images and accurate Raman signals, and the increased working distance of the microscope group leads to limitations in resolution and magnification. The invention provides a deep-sea micro Raman imaging system and method.

[0006] To address the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0007] A deep-sea micro Raman imaging system, characterized in that it includes a micro module, a micro imaging module, a separation module, and a Raman imaging module;

[0008] The microscopic module includes a pressure-resistant window set at the observation position. A low-power objective and a high-power objective are set inside the pressure-resistant window. Both the low-power objective and the high-power objective are mounted on an XY linear translation stage. The movement direction of the XY linear translation stage is perpendicular to the optical path direction, and it is used to switch between the high-power objective and the low-power objective in the optical path.

[0009] The separation module includes a dichroic mirror, which is located in the optical path between the microscopic module and the microscopic imaging module. The dichroic mirror is used to separate the microscopic imaging signal light and the Raman signal light. The microscopic imaging signal light is transmitted light, and the Raman signal light is reflected light.

[0010] The imaging module includes an imaging relay mirror and a first camera, which are sequentially arranged in the transmission light path of the dichroic mirror.

[0011] The Raman imaging module includes a laser source, a beam expander, a beam splitter, and a Raman coupler.

[0012] The beam expander and beam splitter are sequentially arranged in the reflected light path of the dichroic mirror;

[0013] The Raman coupler is positioned on the transmission path of the beam splitter, while the laser source is positioned on the reflection path of the beam splitter. The Raman coupler is used to couple the Raman signal light to the focusing surface of the Raman spectrometer to achieve Raman spectral imaging.

[0014] Furthermore, it also includes a scanning galvanometer, which is disposed in the optical path between the beam expander and the beam splitter.

[0015] Furthermore, the high-magnification objective lens includes a first cemented lens group, a first lens, a second cemented lens group, a third cemented lens group, and a first aperture arranged sequentially along the optical path, wherein all lenses are spherical lenses;

[0016] The imaging relay mirror includes a fifth lens, a sixth lens, a seventh lens, and a sixth cemented lens group arranged sequentially along the optical path;

[0017] The low-magnification objective lens includes a fourth cemented lens group, a fifth cemented lens group, a second lens, a third lens, a fourth lens, and a second aperture arranged sequentially along the optical path, all of which are spherical mirrors; or, the low-magnification objective lens includes a fourteenth lens, a fifteenth lens, a sixteenth lens, a seventeenth lens, a third aperture, an eighteenth lens, a nineteenth lens, a twentieth lens, and a twenty-first lens arranged sequentially along the optical path, all of which are spherical mirrors, and a second camera is arranged in the optical path between the low-magnification objective lens and the dichroic mirror, the second camera also being arranged on the XY linear translation stage;

[0018] The beam expander includes an eighth lens, a ninth lens, and a tenth lens arranged sequentially along the optical path, and all lenses are spherical mirrors.

[0019] The Raman coupling mirror includes an eleventh lens, a twelfth lens, a thirteenth lens, and a seventh cemented lens group arranged sequentially along the optical path.

[0020] Furthermore, a filter is provided between the laser source and the beam splitter; it also includes a piezoelectric ceramic lifting stage, on which the high-magnification objective lens is mounted on an XY linear translation stage via the piezoelectric ceramic lifting stage. The piezoelectric ceramic lifting stage is used to drive the high-magnification objective lens to move along the optical path direction to achieve clear imaging of different focal planes. The XY linear translation stage is model E24-6020, and the piezoelectric ceramic lifting stage is model P115ZS.

[0021] Furthermore, the focal length of the high-magnification objective lens is less than 20mm, and the focal length ratio between the imaging transfer mirror and the high-magnification objective lens is greater than 20.

[0022] Furthermore, the pressure-resistant window is made of sapphire glass, which is scratch-resistant, has high hardness, and is suitable for bearing pressure in the deep sea.

[0023] This invention also provides a deep-sea micro Raman imaging method, which is characterized by including the following steps:

[0024] S1: Switch the low-power objective lens into the optical path via the XY linear translation stage to achieve low-power imaging of the target particles and obtain the abundance and basic position of the target particles.

[0025] S2: The high-power objective lens is switched into the optical path through the XY linear translation stage, so that the microscopic imaging signal light of the target particle is finally focused on the first camera, thereby achieving high-power imaging of the target particle and obtaining the size, shape and precise position of the target particle.

[0026] S3: The laser light source emits a laser beam, which is reflected by a beam splitter, expanded by a beam expander, and reflected by a dichroic mirror before entering the high-power objective lens. Finally, it is focused on the target particle after passing through a pressure-resistant window and excites Raman signal light at different positions on the target particle.

[0027] S4: The Raman signal light excited at different positions of the target particle passes through the pressure-resistant window and the high-magnification objective lens, and is reflected by the dichroic mirror. It is then compressed by the beam expander, coupled by the beam splitter and the Raman coupler to the focusing surface of the Raman spectrometer, thus realizing Raman spectral imaging.

[0028] Furthermore, steps S3 and S4 also include scanning the laser beam and the Raman signal light by scanning the scanning galvanometer to excite the Raman signal of the target at different positions on the object surface.

[0029] Furthermore, in step S1, the low-power objective lens is switched to the optical path, and the microscopic imaging signal light of the target particle is focused onto the first camera or the second camera.

[0030] Furthermore, in step S4, the high-magnification objective lens is moved along the optical path by the piezoelectric ceramic lifting platform to achieve clear Raman spectral imaging of different focal planes, and tomography of the target particles is achieved based on the Raman signal light.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] (1) The deep-sea micro Raman imaging system provided by the present invention uses high-power and low-power objectives for microscopic imaging, and switches between them using an XY linear translation stage. When low-power imaging is needed, the low-power objective is switched to enter the optical path to obtain the abundance and approximate location of the target particles; when high-power imaging is needed, the high-power objective is switched to enter the optical path to obtain the size, shape and precise location of the target particles. It can quickly locate local particle samples with low-power objectives to obtain optical images with a large field of view, and accurately analyze the morphology of local particles with high-power objectives to obtain high-resolution optical images. Furthermore, the high-power objective is used in conjunction with the Raman imaging module to obtain accurate Raman signals. The microscopic module adopts a design with two interchangeable objectives, and the influence of the thickness of the pressure-resistant window on the resolution and magnification of the microscope group is also eliminated.

[0033] (2) In a deep-sea micro Raman imaging system provided by the present invention, the high-magnification objective lens is set on the XY linear translation stage by a piezoelectric ceramic lifting stage. The piezoelectric ceramic lifting stage can drive the high-magnification objective lens to move along the incident light path direction to achieve clear imaging of different focal planes.

[0034] (3) In the deep-sea micro Raman imaging system provided by this invention, the focal length of the high-magnification objective is less than 20 mm, and the focal length ratio between the imaging relay mirror and the high-magnification objective is greater than 20. Given a fixed aperture of the high-magnification objective, a smaller focal length results in a larger aperture angle, a larger numerical aperture, and thus a higher resolution. The fact that the focal length ratio between the imaging relay mirror and the high-magnification objective is greater than 20 ensures a higher imaging magnification. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of a first embodiment of a deep-sea micro Raman imaging system of the present invention (the XY linear translation stage and the piezoelectric ceramic lifting stage are not shown in the figure);

[0036] Figure 2 This is a schematic diagram of a second embodiment of a deep-sea micro Raman imaging system of the present invention (the XY linear translation stage and the piezoelectric ceramic lifting stage are not shown in the figure);

[0037] Figure 3 This is a schematic diagram of a third embodiment of the deep-sea micro Raman imaging system of the present invention (the XY linear translation stage and the piezoelectric ceramic lifting stage are not shown in the figure).

[0038] The annotations in the attached figures are explained as follows:

[0039] 1-Pressure-resistant window; 2-High-power objective lens; 21-First cemented lens group; 22-First lens; 23-Second cemented lens group; 24-Third cemented lens group; 25-First aperture stop; 3-Low-power objective lens; 31-Fourth cemented lens group; 32-Fifth cemented lens group; 33-Second lens; 34-Third lens; 35-Fourth lens; 36-Second aperture stop; 301-Fourteenth lens; 302-Fifteenth lens; 303-Sixteenth lens; 304-Seventeenth lens; 305-Third aperture stop; 306-Eighteenth lens; 307-Nineteenth lens; 308-Second aperture stop 10-Lens 309-Twenty-first Lens; 4-Dichroic Mirror; 5-Imaging Relay Mirror; 51-Fifth Lens; 52-Sixth Lens; 53-Seventh Lens; 54-Sixth Cemented Lens Group; 6-First Camera; 7-Beam Expander; 71-Eighth Lens; 72-Ninth Lens; 73-Tenth Lens; 8-Scanning Galvanometer; 9-Beam Splitter; 10-Raman Coupler; 101-Eleventh Lens; 102-Twelfth Lens; 103-Thirteenth Lens; 104-Seventh Cemented Lens Group; 105-Focusing Surface; 11-Filter; 12-Laser Source; 13-Second Camera. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and exemplary embodiments.

[0041] Reference Figures 1-3 An embodiment of the deep-sea micro Raman imaging system of the present invention is as follows: Figure 1As shown, a pressure-resistant window 1 made of sapphire glass is installed at the observation position. This material is scratch-resistant and has high hardness, making it suitable for use in environments with high deep-sea pressure. The pressure-resistant window 1 isolates the seawater from the entire system, and its inner surface is coated with a high-transmittance film to increase light transmission. A low-magnification objective lens 3 and a high-magnification objective lens 2 are installed inside the pressure-resistant window 1. Both objectives are mounted on an XY linear translation stage (model E24-6020) manufactured by Shanghai Huafu Information Technology Co., Ltd. The XY linear translation stage allows movement perpendicular to the optical path, enabling switching between the high-magnification objective lens 2 and the low-magnification objective lens 3 within the optical path. The high-magnification objective lens 2 is mounted on the XY linear translation stage via a piezoelectric ceramic lifting stage (model P115ZS) manufactured by Liaoning Yansheng Technology Co., Ltd. The piezoelectric ceramic lifting stage moves the high-magnification objective lens 2 along the optical path to achieve clear imaging of different focal planes.

[0042] The high-magnification objective 2 in Embodiment 1 includes a first cemented lens group 21, a first lens 22, a second cemented lens group 23, a third cemented lens group 24, and a first aperture 25 arranged sequentially along the optical path. All lenses are spherical lenses, and their thicknesses are all between 1.2mm and 15mm. The distance from the outer surface of the first cemented lens group 21 to the outer surface of the first aperture 25, i.e., the total length of the high-magnification objective 2, is less than 65mm. This compact structure facilitates the manufacturing of the entire high-magnification objective 2. The working distance of the high-magnification objective 2 is 7mm, the numerical aperture is greater than 0.34, and the focal length is less than 20mm. With a fixed aperture of the high-magnification objective 2, the smaller the focal length, the larger the aperture angle, the larger the numerical aperture, and the higher the resolution.

[0043] The low-magnification objective 3 comprises a fourth cemented lens group 31, a fifth cemented lens group 32, a second lens 33, a third lens 34, a fourth lens 35, and a second aperture 36 arranged sequentially along the optical path. All lenses are spherical mirrors with a thickness between 1.2 mm and 15 mm. The total length of the low-magnification objective 3, i.e., the distance from the outer surface of the fourth cemented lens group 31 to the outer surface of the second aperture 36, is less than 110 mm. The fourth cemented lens group 31 and the fifth cemented lens group 32 are used to correct for higher-order aperture aberrations, on-axis chromatic aberration, and magnification chromatic aberration in the low-magnification microscopy imaging optical path. The working distance of the entire low-magnification objective 3 is 136 mm, and the numerical aperture is greater than 0.04.

[0044] Along the optical path, a dichroic mirror 4, an imaging relay mirror 5, and a first camera 6 are sequentially arranged. The imaging relay mirror 5 and the first camera 6 are located on the transmission optical path of the dichroic mirror 4. The first camera 6 uses a Medvision MV-XG6500GC / MT color / black and white camera with a chip pixel count greater than 36 million and a pixel size of 3-5 micrometers, which can increase the overall imaging size and improve the system's imaging resolution. The microscopic imaging signal light is transmitted light, and the Raman signal light is reflected light. The dichroic mirror 4 is mainly used to separate the microscopic imaging signal light and the Raman signal light to ensure that they do not interfere with each other.

[0045] The imaging relay mirror 5 includes a fifth lens 51, a sixth lens 52, a seventh lens 53, and a sixth cemented lens group 54 arranged sequentially along the optical path. The imaging relay mirror 5 is used to converge the microscopic imaging signal light transmitted from the dichroic mirror 4 and finally image it onto the first camera 6. All the lenses of the imaging relay mirror 5 are spherical, and the thickness of all the lenses is between 1.5mm and 15mm. The distance from the outer surface of the fifth lens 51 to the outer surface of the sixth cemented lens group 54 is less than 53mm, which ensures a compact structure and facilitates the manufacturing of the lens group. The focal length ratio between the imaging relay mirror 5 and the high-magnification objective lens 2 is greater than 20, which ensures the imaging magnification.

[0046] The observable sample diagonal field of view of the high-power objective lens 2 is 1.41 mm. The overall imaging magnification of the combination of high-power objective lens 2, dichroic mirror 4, and imaging relay mirror 5 is 21.4. When the microscopic imaging signal light generated by the target particles passes through the pressure-resistant window 1, high-power objective lens 2, dichroic mirror 4, and imaging relay mirror 5 and is finally imaged on the first camera 6, the imaging spot size is less than 29 micrometers, the MTF is greater than 0.3 at 161 p / mm, and the corresponding object-space resolution is less than 1.5 micrometers. The aberration correction is good, which effectively ensures the imaging resolution of the system. The observable sample diagonal field of view of the low-power objective lens 3 is 28 mm. The overall imaging magnification of the combination of low-power objective lens 3, dichroic mirror 4, and imaging relay mirror 5 is 1.1. When the microscopic imaging signal light generated by the target particles passes through the pressure-resistant window 1, low-power objective lens 3, dichroic mirror 4, and imaging relay mirror 5 and is finally imaged on the first camera 6, the imaging blur spot is less than 11 micrometers, the MTF is greater than 0.27 at 401p / mm, and the aberration correction is good. While ensuring a large field of view observation, the imaging quality is effectively improved.

[0047] A beam expander 7, a scanning galvanometer 8, a beam splitter 9, and a Raman coupler 10 are sequentially arranged on the reflected light path of the dichroic mirror 4. The Raman coupler 10 is used to couple the Raman signal light to the focusing surface 105 of the Raman spectrometer to achieve Raman spectral imaging. A laser source 12 is arranged on the reflected light path of the beam splitter 9. A filter 11 for filtering out clutter is arranged between the beam splitter 9 and the laser source 12.

[0048] The beam expander 7 expands the incident laser beam to fill the entrance pupil of the high-power objective lens 2, thereby increasing the cone angle of the laser beam on the target particle and improving the imaging resolution. On the other hand, when the Raman signal light excited by the laser beam after irradiating the target particle passes through the high-power objective lens 2 and the dichroic mirror 4 and exits as parallel light to the beam expander 7, the beam expander 7 can compress the diameter of the laser beam to fit the size of the scanning galvanometer 8. The beam expander 7 includes an eighth lens 71, a ninth lens 72, and a tenth lens 73 arranged sequentially along the optical path. All lenses are spherical mirrors, which facilitates manufacturing. Furthermore, the ratio of the focal length of the eighth lens 71 to the combined focal length of the ninth and tenth lenses 73 is greater than -1.9, satisfying the imaging requirements of the high-power objective lens 2 after the laser beam is expanded.

[0049] The scanning galvanometer 8 is a model A5L2.2 manufactured by Mirrocle Technologies Inc. The scanning galvanometer 8 scans the laser beam and the Raman signal light to excite the Raman signal of the target at different positions on the object surface.

[0050] The beam splitter 9 is located between the scanning galvanometer 8 and the Raman coupler 10. It is used to reflect the laser beam generated by the laser source 12 and transmit the Raman signal light. The Raman signal light enters the Raman coupler 10 after being transmitted through the beam splitter 9.

[0051] The Raman coupler 10 includes an eleventh lens 101, a twelfth lens 102, a thirteenth lens 103, and a seventh cemented lens group 104 arranged sequentially along the optical path. All lenses are spherical mirrors with a focal length of less than 25 mm. The distance from the outer surface of the eleventh lens 101 to the outer surface of the seventh cemented lens group 104 is less than 60 mm, meaning the total length of the Raman coupler 10 is less than 60 mm. This makes the overall structure compact and easy to manufacture. The Raman signal light generated by the laser beam excited by the target particles passes sequentially through the pressure-resistant window 1, the high-power objective lens 2, the dichroic mirror 4, the beam expander 7, the scanning galvanometer 8, and the beam splitter 9 before entering the Raman coupler 10 and finally coupling to the focusing surface 105 of the Raman spectrometer. Under the diagonal field of view of the sample observable by the high-power objective lens 2, the RMS radius of the emitted light spot is less than 9 micrometers, resulting in good light focusing effect, which is beneficial for the reception of Raman spectral signal light and the improvement of Raman imaging resolution.

[0052] The specific parameters of each lens in the pressure-resistant window 1 and the high-magnification objective lens 2 in Example 1 are shown in the table below:

[0053]

[0054] The specific parameters of each lens in the pressure-resistant window 1 and the low-magnification objective lens 3 in Example 1 are shown in the table below:

[0055]

[0056]

[0057] The parameters of each lens in the imaging transfer mirror 5 in Example 1 are shown in the table below:

[0058]

[0059] The parameters of each lens in the beam expander 7 of Example 1 are shown in the table below:

[0060]

[0061] The parameters of each lens in the Raman coupling lens 10 of Example 1 are shown in the table below:

[0062]

[0063] Example 1 also provides a deep-sea micro Raman imaging method, including the following steps:

[0064] S1: The low-power objective lens 3 is switched into the optical path through the XY linear translation stage. The microscopic imaging signal light of the target particle passes through the pressure-resistant window 1, the low-power objective lens 3, the dichroic mirror 4, and the imaging transfer mirror 5 in sequence and is then focused on the first camera 6 to achieve low-power imaging of the target particle and obtain the abundance and basic position of the target particle.

[0065] S2: The high-power objective lens 2 is switched into the optical path through the XY linear translation stage. The microscopic imaging signal light of the target particle passes through the pressure-resistant window 1, the high-power objective lens 2, the dichroic mirror 4, and the imaging transfer mirror 5 in sequence and is then focused on the first camera 6 to achieve high-power imaging of the target particle and obtain the size, shape and precise position of the target particle.

[0066] S3: The laser light source 12 emits a laser beam with a wavelength of 785nm. After being reflected by the beam splitter 9, scanned by the scanning galvanometer 8, expanded by the beam expander 7, reflected by the dichroic mirror 4, it enters the high-power objective lens 2. Finally, after passing through the pressure-resistant window 1, it converges to the target particle and excites Raman signal light at different positions of the target particle.

[0067] S4: Raman signal light excited at different positions of the target particle passes sequentially through the pressure-resistant window 1 and the high-power objective lens 2, and is reflected by the dichroic mirror 4. It is then compressed by the beam expander 7, scanned by the scanning galvanometer 8, and coupled to the focusing surface 105 of the Raman spectrometer by the beam splitter 9 and the Raman coupler 10, thus realizing Raman spectral imaging. The high-power objective lens 2 is moved along the optical path by the piezoelectric ceramic lifting platform to achieve clear Raman spectral imaging of different focal planes, and the tomography function of the target particle is realized based on the Raman signal light.

[0068] Embodiment 2 of the deep-sea micro Raman imaging system of the present invention is as follows: Figure 2As shown, the difference from Embodiment 1 is that the scanning galvanometer 8 is removed, which further shortens the distance between the beam expander 7 and the Raman coupler 10, thereby improving the compactness of the entire system. In order to adapt to the structural change, the lens composition of the beam expander 7 and the Raman coupler 10 remains unchanged in this embodiment, but the specific parameters are changed. The beam expander 7 still includes the eighth lens 71, the ninth lens 72 and the tenth lens 73 arranged sequentially along the optical path, and the Raman coupler 10 still includes the eleventh lens 101, the twelfth lens 102, the thirteenth lens 103 and the seventh cemented lens group 104 arranged sequentially along the optical path.

[0069] The laser beam emitted from the laser source 12 passes sequentially through the beam splitter 9, beam expander 7, dichroic mirror 4, high-magnification objective lens 2, and pressure-resistant window 1 before converging onto the target particle. Within a 1.41mm image-square field of view, the RMS radius of the light spot emitted onto the target particle from different fields of view is less than 6 micrometers, effectively improving the target resolution capability of Raman imaging.

[0070] The Raman signal light passes sequentially through the pressure-resistant window 1, high-magnification objective lens 2, dichroic mirror 4, beam expander 7, beam splitter 9, and Raman coupler 10 before converging at the focal plane 105 and entering the Raman spectrometer. Within a 1.41 mm object-side field of view, the RMS radius of the emitted light spot in different fields of view is less than 9 micrometers, resulting in good light focusing, which is beneficial for receiving Raman spectral signals and improving Raman imaging resolution.

[0071] The parameters of each lens in the beam expander 7 of Example 2 are shown in the table below:

[0072]

[0073]

[0074] The parameters of each lens in the Raman coupling lens 10 in Example 2 are shown in the table below.

[0075]

[0076] Example 2 also provides a deep-sea micro Raman imaging method, including the following steps:

[0077] S1: The low-power objective lens 3 is switched into the optical path through the XY linear translation stage. The microscopic imaging signal light of the target particle passes through the pressure-resistant window 1, the low-power objective lens 3, the dichroic mirror 4, and the imaging transfer mirror 5 in sequence and is then focused on the first camera 6 to achieve low-power imaging of the target particle and obtain the abundance and basic position of the target particle.

[0078] S2: The high-power objective lens 2 is switched into the optical path through the XY linear translation stage. The microscopic imaging signal light of the target particle passes through the pressure-resistant window 1, the high-power objective lens 2, the dichroic mirror 4, and the imaging transfer mirror 5 in sequence and is then focused on the first camera 6 to achieve high-power imaging of the target particle and obtain the size, shape and precise position of the target particle.

[0079] S3: The laser light source 12 emits a laser beam with a wavelength of 785nm. After being reflected by the beam splitter 9, expanded by the beam expander 7, and reflected by the dichroic mirror 4, it enters the high-power objective lens 2. Finally, after passing through the pressure-resistant window 1, it converges to the target particle and excites Raman signal light at different positions of the target particle.

[0080] S4: Raman signal light excited at different positions of the target particle passes sequentially through the pressure-resistant window 1 and the high-power objective lens 2, and is reflected by the dichroic mirror 4. After being compressed by the beam expander 7, the beam splitter 9, and the Raman coupler 10, the light is coupled to the focusing surface 105 of the Raman spectrometer to achieve Raman spectral imaging. The high-power objective lens 2 is moved along the optical path by the piezoelectric ceramic lifting platform to achieve clear Raman spectral imaging of different focal planes, and the tomography function of the target particle is realized based on the Raman signal light.

[0081] Embodiment 3 of the deep-sea micro Raman imaging system of the present invention is as follows: Figure 3 As shown, the difference from Embodiment 1 lies in the low-magnification objective 3. The microscopic imaging signal light emitted by the sample under test passes through the pressure-resistant window 1 and the low-magnification objective 3 before directly entering the second camera 13, achieving low-magnification imaging. The high-magnification objective 2 is switched into the optical path via an XY linear translation stage. The microscopic imaging signal light emitted by the target particles under test passes through the pressure-resistant window 1, the high-magnification objective 2, the dichroic mirror 4, and the imaging transfer mirror 5 before entering the first camera 6, achieving high-magnification imaging.

[0082] In this embodiment, the low-magnification objective 3 includes a fourteenth lens 301, a fifteenth lens 302, a sixteenth lens 303, a seventeenth lens 304, a third aperture 305, an eighteenth lens 306, a nineteenth lens 307, a twentieth lens 308, and a twenty-first lens 309 arranged sequentially along the optical path. All lenses are spherical mirrors. The second camera 13 is set on the XY linear translation stage and located on the optical path between the low-magnification objective 3 and the dichroic mirror 4. The low-magnification objective 3 becomes an independent imaging system and does not share the imaging transfer mirror 5 with the high-magnification objective 2. Therefore, the distance between the high-magnification objective 2 and the dichroic mirror 4 can be significantly shortened, reducing the system size and thus improving the system's compactness. The remaining lens groups are the same as in Embodiment 1.

[0083] The microscopic imaging signal light of the target particle passes sequentially through the pressure-resistant window 1, the high-power objective lens 2, the dichroic mirror 4, and the imaging transfer mirror 5. Within a field of view of 1.41 mm, the imaging blur spot is less than 28 micrometers, the MTF is greater than 0.28 at 161 p / mm, and the corresponding object-side resolvable size is less than 1.5 micrometers. The aberration correction is good, effectively ensuring the imaging resolution of the system.

[0084] The microscopic imaging light of the target particle passes sequentially through the pressure-resistant window 1 and the low-power objective lens 3. Within a field of view of 28.18 mm, the imaging spot size is less than 4 micrometers, the MTF is greater than 0.3 at 801p / mm, and the corresponding object-side resolvable size is less than 4 micrometers. The aberration correction is good, which effectively improves the imaging quality of the system while ensuring large field of view observation.

[0085] The laser beam emitted by the laser source 12 is reflected by the beam splitter 9, scanned by the scanning galvanometer 8, expanded by the beam expander 7, reflected by the dichroic mirror 4, and then passed through the high-magnification objective lens 2 and the pressure-resistant window 1 before converging onto the target particle. Within the 0.71mm image-square field of view, the RMS radius of the light spot emitted onto the target particle in different fields of view is less than 6 micrometers, which effectively improves the target resolution capability of Raman imaging.

[0086] The Raman signal light of the target particle passes sequentially through the pressure-resistant window 1, high-power objective lens 2, dichroic mirror 4, beam expander 7, scanning galvanometer 8, beam splitter 9, and Raman coupler 10 before converging to the focusing surface 105 of the Raman spectrometer. Within an object-side field of view of 0.71 mm, the RMS radius of the emitted light spot in different fields of view is less than 9 micrometers, resulting in good light focusing effect, which is beneficial to the reception of Raman signal light and the improvement of Raman imaging resolution.

[0087] The parameters of each lens in the pressure-resistant window 1 and the low-magnification objective lens 3 in Example 3 are shown in the table below:

[0088]

[0089]

[0090] Example 3 also provides a deep-sea micro Raman imaging method, including the following steps:

[0091] S1: The low-magnification objective lens 3 is switched into the optical path via the XY linear translation stage. The microscopic imaging signal light of the target particle passes through the pressure-resistant window 1 and the low-magnification objective lens 3 in sequence and is then focused on the second camera 13 to achieve low-magnification imaging of the target particle and obtain the abundance and basic position of the target particle.

[0092] S2: The high-power objective lens 2 is switched into the optical path through the XY linear translation stage. The microscopic imaging signal light of the target particle passes through the pressure-resistant window 1, the high-power objective lens 2, the dichroic mirror 4, and the imaging transfer mirror 5 in sequence and is then focused on the first camera 6 to achieve high-power imaging of the target particle and obtain the size, shape and precise position of the target particle.

[0093] S3: The laser light source 12 emits a laser beam with a wavelength of 785nm. After being reflected by the beam splitter 9, it enters the scanning galvanometer 8 for scanning and the beam expander 7 for beam expansion. After being reflected by the dichroic mirror 4, it enters the high-power objective lens 2. Finally, after passing through the pressure-resistant window 1, it converges to the target particle and excites Raman signal light at different positions of the target particle.

[0094] S4: Raman signal light excited at different positions of the target particle passes sequentially through the pressure-resistant window 1 and the high-power objective lens 2, and is reflected by the dichroic mirror 4. It is then compressed by the beam expander 7, scanned by the scanning galvanometer 8, and coupled to the focusing surface 105 of the Raman spectrometer by the beam splitter 9 and the Raman coupler 10, thus realizing Raman spectral imaging. The high-power objective lens 2 is moved along the optical path by the piezoelectric ceramic lifting platform to achieve clear Raman spectral imaging of different focal planes, and the tomography function of the target particle is realized based on the Raman signal light.

[0095] The embodiments described above are merely illustrative of specific implementations of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A deep-sea micro Raman imaging system, characterized in that: It includes a microscopy module, a microscopic imaging module, a separation module, and a Raman imaging module; The microscopic module includes a pressure-resistant window (1) set at the observation position. A low-power objective (3) and a high-power objective (2) are set inside the pressure-resistant window (1). Both the low-power objective (3) and the high-power objective (2) are mounted on an XY linear translation stage. The movement direction of the XY linear translation stage is perpendicular to the optical path direction, which is used for switching between the high-power objective (2) and the low-power objective (3) in the optical path. The separation module includes a dichroic mirror (4), which is located in the optical path between the microscopic module and the microscopic imaging module. It is used to separate the microscopic imaging signal light and the Raman signal light. The microscopic imaging signal light is transmitted light, and the Raman signal light is reflected light. The imaging module includes an imaging relay mirror (5) and a first camera (6) arranged sequentially on the transmission light path of the dichroic mirror (4). The Raman imaging module includes a laser source (12), a beam expander (7), a beam splitter (9), and a Raman coupler (10). The beam expander (7) and beam splitter (9) are sequentially arranged on the reflected light path of the dichroic mirror (4); The Raman coupler (10) is set on the transmission light path of the beam splitter (9), and the laser source (12) is set on the reflection light path of the beam splitter (9); the Raman coupler (10) is used to couple the Raman signal light to the focusing surface (105) of the Raman spectrometer to realize Raman spectral imaging. The high-power objective lens (2) includes a first cemented lens group (21), a first lens (22), a second cemented lens group (23), a third cemented lens group (24), and a first aperture (25) arranged sequentially along the optical path, wherein all lenses are spherical lenses; The imaging relay mirror (5) includes a fifth lens (51), a sixth lens (52), a seventh lens (53), and a sixth cemented lens group (54) arranged sequentially along the optical path. The low-power objective (3) includes a fourth cemented lens group (31), a fifth cemented lens group (32), a second lens (33), a third lens (34), a fourth lens (35), and a second aperture (36) arranged sequentially along the optical path, all of which are spherical mirrors; or, the low-power objective (3) includes a fourteenth lens (301), a fifteenth lens (302), a sixteenth lens (303), a seventeenth lens (304), a third aperture (305), an eighteenth lens (306), a nineteenth lens (307), a twentieth lens (308), and a twenty-first lens (309) arranged sequentially along the optical path, all of which are spherical mirrors, and a second camera (13) is arranged in the optical path between the low-power objective (3) and the dichroic mirror (4), and the second camera (13) is also arranged on the XY linear translation stage; The beam expander (7) includes an eighth lens (71), a ninth lens (72) and a tenth lens (73) arranged sequentially along the optical path, and all lenses are spherical mirrors; The Raman coupling mirror (10) includes an eleventh lens (101), a twelfth lens (102), a thirteenth lens (103), and a seventh cemented lens group (104) arranged sequentially along the optical path. A filter (11) is provided between the laser light source (12) and the beam splitter (9); it also includes a piezoelectric ceramic lifting stage, the high-magnification objective lens (2) is set on the XY linear translation stage through the piezoelectric ceramic lifting stage, the piezoelectric ceramic lifting stage is used to drive the high-magnification objective lens (2) to move along the optical path direction, so as to achieve clear imaging of different focal planes, the XY linear translation stage model is E24-6020, and the piezoelectric ceramic lifting stage model is P115ZS.

2. The deep-sea micro Raman imaging system according to claim 1, characterized in that: It also includes a scanning galvanometer (8), which is disposed in the optical path between the beam expander (7) and the beam splitter (9).

3. The deep-sea micro Raman imaging system according to claim 2, characterized in that: The focal length of the high-power objective lens (2) is less than 20 mm, and the focal length ratio between the imaging mirror (5) and the high-power objective lens (2) is greater than 20.

4. The deep-sea micro Raman imaging system according to claim 3, characterized in that: The pressure-resistant window (1) is made of sapphire glass.

5. A deep-sea micro-Raman imaging method, employing the deep-sea micro-Raman imaging system according to any one of claims 1-4, characterized in that: Includes the following steps: S1: Switch the low-power objective lens (3) to the optical path through the XY linear translation stage to achieve low-power imaging of the target particles and obtain the abundance and basic position of the target particles; S2: The high-power objective lens (2) is switched into the optical path through the XY linear translation stage so that the microscopic imaging signal light of the target particle is finally focused on the first camera (6) to achieve high-power imaging of the target particle and obtain the size, shape and precise position of the target particle. S3: The laser light source (12) emits a laser beam, which is reflected by the beam splitter (9), expanded by the beam expander (7), reflected by the dichroic mirror (4), and then enters the high-power objective lens (2). Finally, it passes through the pressure-resistant window (1) and converges to the target particle, and excites Raman signal light at different positions of the target particle. S4: The Raman signal light excited at different positions of the target particles passes through the pressure-resistant window (1) and the high-power objective lens (2), and is reflected by the dichroic mirror (4). It is then compressed by the beam expander (7), coupled to the beam splitter (9) and the Raman coupler (10) onto the focusing surface (105) of the Raman spectrometer to achieve Raman spectral imaging.

6. The deep-sea micro Raman imaging method according to claim 5, characterized in that: Steps S3 and S4 also include scanning the laser beam and the Raman signal light by scanning the scanning galvanometer (8) to excite the Raman signal of the target at different positions on the object surface.

7. The deep-sea micro Raman imaging method according to claim 5 or 6, characterized in that: In step S1, the low-power objective lens (3) is switched to the optical path, and the microscopic imaging signal light of the target particles is focused on the first camera (6) or the second camera (13).

8. The deep-sea micro Raman imaging method according to claim 7, characterized in that: In step S4, the high-power objective lens (2) moves along the optical path direction as a whole through the piezoelectric ceramic lifting platform to achieve clear Raman spectral imaging of different focal planes, and realizes the tomography function of target particles based on Raman signal light.

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