Multiphoton microscopic imaging probe, imaging device, and imaging method

Abstract

The present application discloses a multiphoton microscopic imaging probe, an imaging device, and an imaging method. The imaging probe comprises: a medium assembly, wherein the medium assembly comprises a solid elastic medium and an imaging window plate that carries the solid elastic medium and is used for being in contact with an imaging object; a handle-shaped housing, wherein the medium assembly is detachably arranged at a first end of the handle-shaped housing; and an imaging module based on miniature laser scanning microscopy technology, wherein the imaging module is arranged in the handle-shaped housing, connected to a photoelectric composite cable and used for transmitting and controlling exciting light and receiving a fluorescence signal; and the imaging module comprises an objective lens in contact-fit with the solid elastic medium. Therefore, the objective lens provided by the present application comes into contact with the imaging object on the basis of the solid medium, eliminating the limitations of conventional immersion media on microscopic imaging apparatuses, thereby allowing the imaging probe to come into contact with the imaging object at any angle.

Description

A multiphoton microscopy probe, imaging device and imaging method

[0001] This application claims priority to Chinese Patent Application No. 202510032727.6, filed with the State Intellectual Property Office of China on January 8, 2025, entitled "A Multiphoton Microscopic Imaging Probe, Imaging Device and Imaging Method", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of microscopic imaging, specifically to a multiphoton microscopic imaging probe, imaging device, and imaging method. Background Technology

[0003] Multiphoton microscopy is a nonlinear optical imaging technique based on multiphoton absorption / excitation phenomena. Multiphotons typically include one or more imaging modes such as two-photon, three-photon, second-harmonic, and third-harmonic. Multiphoton absorption / excitation phenomena refer to nonlinear optical phenomena involving the interaction of multiple photons with matter during optical processes.

[0004] In label-free in vivo imaging applications, the extremely weak autofluorescence of biological tissues necessitates imaging systems with exceptionally high imaging performance, stable imaging characteristics, and user-friendly interface. Compared to traditional fluorescence imaging techniques, multiphoton microscopy utilizes excitation light in the near-infrared band, enabling microscopic imaging of the internal structure of the object at a certain depth. However, in practical imaging, in vivo microscopy suffers from focal plane instability issues, ambient light interference, and internal scattering problems compared to traditional stage-based microscopy, all of which severely impact image quality. Therefore, improving the quality of in vivo multiphoton microscopy is a crucial technical challenge that urgently needs to be addressed by those skilled in the art. Summary of the Invention

[0005] In view of this, embodiments of this application provide a multiphoton microscopy imaging probe, imaging device, and imaging method. The objective lens contacts the imaging object through a solid elastic medium, reducing aberrations between the medium and the imaging object, thereby overcoming the shortcomings of the prior art.

[0006] In a first aspect, embodiments of this application provide a multiphoton microscopy imaging probe, the imaging probe comprising: a medium component, wherein the medium component includes a solid elastic medium and an imaging window that carries the solid elastic medium and is used to contact the imaging object; a handle-shaped housing, wherein the medium component is detachably disposed at a first end of the handle-shaped housing; and an imaging module based on micro-laser scanning microscopy technology, wherein the imaging module is disposed within the handle-shaped housing and is used to transmit and control excitation light and receive fluorescence signals, the imaging module including an objective lens that contacts and engages with the solid elastic medium.

[0007] In some embodiments of this application, the imaging module further includes a displacement stage, which is used to control the overall movement of the imaging module within the handle-shaped housing to adjust the focal plane position of the imaging module.

[0008] In some embodiments of this application, the medium assembly further includes a first fixing structure, and a second fixing structure matching the first fixing structure is provided at the first end of the handle-shaped housing. The medium assembly is detachably disposed at the first end of the handle-shaped housing through the first fixing structure and the second fixing structure.

[0009] In some embodiments of this application, a first fixing structure ring is disposed around the imaging window and extends along the placement direction of the solid elastic medium.

[0010] In some embodiments of this application, the first fixing structure is made of a light-shielding material. When the first fixing structure and the second fixing structure cooperate and the imaging window contacts the imaging object, a low-light environment is formed between the imaging object and the objective lens.

[0011] In some embodiments of this application, the refractive index of the solid elastic medium is matched to that of the imaging object.

[0012] In some embodiments of this application, the elastic deformation range of the solid elastic medium is matched with the imaging depth range.

[0013] In some embodiments of this application, the imaging probe further includes an optoelectronic composite cable for connection to the imaging host. The optoelectronic composite cable is connected to the second end of the handle-shaped housing and to the imaging module. The optoelectronic composite cable includes a first optical fiber for transmitting excitation light, a second optical fiber for transmitting fluorescence signals, and a control bus for transmitting control signals.

[0014] Secondly, embodiments of this application provide a multiphoton microscopy imaging device, which includes: an imaging host for generating excitation light and generating a fluorescence image based on a fluorescence signal; and the multiphoton microscopy imaging probe shown in the first aspect, wherein the multiphoton microscopy imaging probe is connected to the imaging host via an optoelectronic composite cable and is used to release excitation light to the imaging object and collect the fluorescence signal generated by the imaging object based on the photoluminescence effect.

[0015] Thirdly, embodiments of this application provide a multiphoton microscopy imaging method, applied to the multiphoton microscopy imaging probe shown in the first aspect or the multiphoton microscopy imaging device shown in the second aspect. The imaging method includes: controlling the position of the imaging module in the imaging direction so that the focal plane of the imaging module is at the target focusing depth, wherein when the focal plane is at the target focusing depth, the objective lens is in contact with a solid elastic medium. Multiphoton microscopy imaging is performed on the imaging object based on the target focusing depth.

[0016] This application provides a multiphoton microscopy imaging probe, imaging device, and imaging method. By employing a solid elastic medium as the medium for indirect contact between the objective lens and the imaging object, it overcomes the limitations of traditional immersion media on microscopy imaging devices. This allows the imaging probe to contact the imaging object at any angle and to perform reciprocating imaging at depth levels. Furthermore, in this application, the solid elastic medium is solidified into a medium assembly and detachably connected to the probe body (handle-shaped housing). During use, the objective lens and solid elastic medium can be assembled directly by disassembling the medium assembly, eliminating the frequent and complex assembly process required by traditional immersion media. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 is a schematic diagram of an imaging situation based on the contact between the immersion medium and the imaging object provided in an exemplary embodiment of this application.

[0019] Figure 2 is an application scenario diagram of the multiphoton microscopy imaging device provided in an exemplary embodiment of this application.

[0020] Figure 3 is a schematic diagram of the structure of a multiphoton microscopy probe provided in an exemplary embodiment of this application.

[0021] Figure 4 is a schematic diagram of a detachable arrangement of a media component and a handle-shaped housing provided in an exemplary embodiment of this application.

[0022] Figure 5 is a schematic diagram of the contact fit between the objective lens and the solid elastic medium provided in an exemplary embodiment of this application.

[0023] Figure 6 is an exemplary flowchart of a multiphoton microscopy imaging apparatus provided in an exemplary embodiment of this application.

[0024] Figure 7 is an exemplary flowchart of a multiphoton microscopy imaging method provided in an exemplary embodiment of this application.

[0025] Figure 8 is an exemplary flowchart of a method for determining a solid elastic medium provided in an exemplary embodiment of this application.

[0026] Among them, 110 is an optical element; 120 is an imaging sample; 130 is an immersion environment; 140 is an immersion medium; 10 is an imaging device; 20 is an imaging object; 200 is an imaging probe; 300 is an imaging host; 400 is an optoelectronic composite cable; 221 is a solid elastic medium; 222 is an imaging window; 231 is an objective lens; 230 is an imaging module; 220 is a medium assembly; 210 is a handle-shaped housing; 232 is a displacement stage; 223 is a first fixing structure; 211 is a second fixing structure; 310 is a power supply; 320 is a laser source; 330 is a collection module; and 340 is a signal control and processing module. Detailed Implementation

[0027] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0028] Application Overview:

[0029] In multiphoton microscopy, the influence of the imaging object on the excitation light is mainly manifested as aberrations. Aberrations refer to the deviations in image quality caused by refraction and reflection of light at different points in the optical system during imaging, resulting in image distortion or blurring. Different refractive indices cause light to refract when entering and passing through different media, thus affecting the direction and speed of light propagation and leading to aberrations.

[0030] For example, the refractive index of human skin is generally between 1.3 and 1.6, while the refractive index of the conventional air medium is 1. This means that when excitation light enters human skin from air, a significant refraction phenomenon occurs, resulting in aberrations.

[0031] To reduce aberrations, some existing multiphoton microscopy devices use an immersion medium instead of air, thereby reducing aberrations between the medium and the imaging object. For example, water or a similar translucent liquid mixture can be used as the immersion medium. For instance, water has a refractive index of 1.33, and this refractive index can be altered by mixing it with specific reagents or using a specific liquid medium, making it close to the refractive index of human skin. Thus, when excitation light enters human skin from the liquid immersion medium, no significant refraction occurs, resulting in smaller aberrations.

[0032] Based on the immersion medium, multiphoton microscopy generally includes two implementation methods: complete immersion and partial immersion.

[0033] Figure 1 is a schematic diagram of an imaging situation based on the contact between the immersion medium and the imaging object provided in an exemplary embodiment of this application.

[0034] The imaging schematic diagram shown in Figure 1 can specifically include a fully immersed mating relationship 100a based on the immersion environment and a partially immersed mating relationship 100b based on the immersed glass slide. In each mating relationship schematic, the description is based on optical element 110 and imaging sample 120. Here, optical element 110 only represents a portion of the optical elements (such as the objective lens) that emit excitation light in the existing embodiment, and can also be understood as a collection of optical devices in a multiphoton microscopy imaging device. Imaging sample 120 can be an imaging object that satisfies the corresponding constraints in various mating relationships. For example, an ex vivo sample.

[0035] In the fully immersed mating relationship 100a, the immersion medium can form an immersion environment 130, thereby completely immersing the optical element 110 and the imaging sample 120 in the immersion environment 130.

[0036] As shown in Figure 1, during imaging, both the optical element 110 and the imaging sample 120 are placed within the immersion environment 130, with the imaging direction of the optical element 110 aligned with that of the imaging sample 120. Based on this, the excitation light emitted by the optical element 110 enters the interior of the imaging sample 120 via the immersion environment 130. When the refractive index of the immersion environment 130 is close to that of the imaging sample 120, no significant refraction occurs when the excitation light enters the imaging object, resulting in smaller aberrations.

[0037] Based on the above technical solutions, an immersion environment must be formed when performing multiphoton microscopy, which limits the imaging object (generally an off-site object) and the imaging scene (which must be immersed in the immersion environment).

[0038] In the partial immersion mating diagram 100b, the immersion medium 140 can be constructed by using a coverslip to create an immersion slide, so that the objective lens is immersed in the immersion environment on the immersion slide.

[0039] As shown in Figure 1, during imaging, the coverslip can support the liquid immersion medium 140, which forms a small, spherical immersion environment due to its own surface tension. The coverslip contacts the imaging sample 120, and the optical element 110 is immersed in the immersion environment 130 formed by the immersion medium 140.

[0040] Therefore, the excitation light emitted by the optical element 110 always enters the imaging sample 120 through the immersion medium 140 and the coverslip to form a focal plane. During depth imaging, the position of the focal plane within the imaging sample 120 can be adjusted by adjusting the position of the objective lens. Specifically, when the refractive index of the immersion medium 140 matches that of the imaging sample 120, the displacement of the optical element 110 is consistent with the displacement of the focal plane. Throughout the imaging process, the refractive index along the path of the excitation light forming the focal plane remains essentially unchanged, thereby significantly reducing aberrations.

[0041] Based on the above technical solutions, at least the following technical problems exist in practical applications:

[0042] First, the immersion medium 140 itself is affected by gravity, thus limiting the application scenarios of the imaging device. Specifically, the immersion medium 140 is a fluid or semi-fluid. When the coverslip is placed in a non-horizontal orientation, the immersion medium 140 will shift under the influence of gravity, making it impossible to stably form the aforementioned small immersion environment, thus preventing imaging. For example, when the immersion medium 140 is applied to the skin, it will quickly slide down. Furthermore, when the immersion medium 140 is in a weightless environment, it cannot achieve stable contact with the objective lens and imaging window, thus preventing imaging.

[0043] Second, the immersion medium 140 is a disposable device by its very nature. After completing an imaging task, the shape of the immersion medium 140 cannot be restored, and it cannot be reused, often requiring reassembly of the immersion slide. For example, when the optical element 110 moves within the immersion medium 140, the lateral area of ​​the immersion medium 140 significantly increases due to the volume of the optical element 110 immersed in the immersion medium 140. When the optical element 110 leaves the immersion medium 140, its shape differs significantly from its original shape, making it impossible to repeat subsequent imaging tasks and requiring reassembly.

[0044] In related technologies, immersion media can also include colloidal media. Colloidal media can overcome some of the aforementioned technical problems by adsorbing onto the imaging window and objective lens. However, in practice, colloidal media still cannot completely solve these problems. For example, with colloidal media, when the objective lens moves towards the imaging window, the colloidal media can achieve an adsorbing fit with both the imaging window and the objective lens. However, when the objective lens moves away from the imaging window, the colloidal media may adsorb onto both the imaging window and the objective lens separately, thus splitting the colloidal media into two parts, resulting in the introduction of gaseous media and causing aberrations. Based on this, it can be seen that colloidal media is still a disposable device, requiring reassembly each time it is used. For imaging at specific angles, it is still affected by gravity, and there is a possibility that imaging may fail.

[0045] To address the aforementioned technical problems, this application provides an imaging probe and imaging device based on a solid-state elastic medium. By employing a solid-state elastic medium as the medium for indirect contact between the objective lens and the imaging object, the limitations imposed by traditional immersion media on microscopic imaging devices are eliminated. This allows the imaging device to contact the imaging object at any angle and perform reciprocating imaging at depth levels, functioning as an imaging probe. Furthermore, in this specification, the solid-state elastic medium is solidified into a medium assembly and detachably connected to the probe body (handle-shaped housing). During use, the objective lens and solid-state elastic medium can be assembled directly by disassembling the medium assembly, eliminating the frequent and complex assembly process required by traditional immersion media. Various non-limiting embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0046] Exemplary application scenarios:

[0047] Figure 2 is an application scenario diagram of the multiphoton microscopy imaging device provided in an exemplary embodiment of this application.

[0048] As shown in Figure 2, the application scenario diagram of the multiphoton microscopy imaging device can include a multiphoton microscopy imaging device 10 and an imaging object 20. The multiphoton microscopy imaging device 10 can be designed as a split unit based on a solid elastic medium, further including a multiphoton microscopy imaging probe 200 and an imaging host 300. The multiphoton microscopy imaging probe 200 can be used in conjunction with the imaging object 20 based on a solid elastic medium, and the multiphoton microscopy imaging probe 200 and the imaging host 300 can be connected via a photoelectric composite cable 400. For more information about the multiphoton microscopy imaging device 10, please refer to Figure 6 and its related description.

[0049] As shown in the partial schematic diagram in Figure 2, the area in contact between the multiphoton microscopy imaging probe 200 and the imaging object 20 may include a solid elastic medium 221, an imaging window 222 supporting the solid elastic medium, and an objective lens 231. Further details regarding the multiphoton microscopy imaging probe can be found in Figure 3 and its related description.

[0050] When performing multiphoton microscopy imaging, the objective lens 231 is in contact with the solid elastic medium 221, and one side of the imaging window 222 supports the solid elastic medium 221, while the other side is in contact with the imaging object 20. At this time, the excitation light generated by the objective lens 231 enters the imaging object 20 through the solid elastic medium 221 and the imaging window 222. When the objective lens 231 (imaging module 230) is moved, the solid elastic medium 221 compresses or expands due to its elastic properties, thus ensuring that the objective lens 231 remains in contact with the solid elastic medium 221. Because the solid elastic medium 221 is in a contact-fitting state (compressed state) during imaging, the elastic force generated by its compression can overcome the influence of gravity, allowing the imaging probe 200 to contact the imaging object 20 at any angle. Furthermore, in a weightless environment, the solid elastic medium 221 can also be fixed by its own elastic force, thus enabling multiphoton microscopy imaging even in a weightless environment. For details regarding the contact fit between the objective lens 231 and the solid elastic medium 221, please refer to Figure 4 and its related description.

[0051] Exemplary imaging probe:

[0052] Based on the aforementioned application scenarios, this application provides a multiphoton microscopy imaging probe based on a solid elastic medium. Figure 3 is a schematic diagram of the structure of a multiphoton microscopy imaging probe provided in an exemplary embodiment of this application.

[0053] As shown in Figure 3, the multiphoton microscopy imaging probe 200 may include an optoelectronic composite cable 400, a handle-shaped housing 210, a dielectric component 220, and an imaging module 230.

[0054] The optoelectronic composite cable 400 can be used to connect to the imaging host 300 and realize data transmission between the imaging host 300 and the imaging probe 200. Specifically, the optoelectronic composite cable 400 can transmit the excitation light generated by the imaging host 300 to the inside of the imaging probe 200, and transmit the fluorescence signal collected by the imaging probe 200 to the imaging host 300 to form a fluorescence image.

[0055] In some embodiments, the various devices within the imaging probe 200 can be connected to the imaging host 300 via an optoelectronic composite cable 400, thereby being controlled by the imaging host 300 to perform multiphoton microscopy imaging. In some embodiments, the optoelectronic composite cable 400 may include a first optical fiber for transmitting excitation light, a second optical fiber for transmitting fluorescence signals, and a control bus for transmitting control signals. The control bus may be an integrated control cable for the various devices within the imaging probe 200.

[0056] The handle-shaped housing 210 can be the outer shell of the multiphoton microscopy imaging probe 200, with the components of the multiphoton microscopy imaging probe 200 integrated inside the handle-shaped housing 210. The handle-shaped housing 210 can be understood as a device that allows personnel to hold it in a manner similar to gripping a handle.

[0057] As shown in Figure 3, the handle-shaped housing 210 can be presented as a near-cylindrical structure, with a second end and a first end. The near-cylindrical structure between the second end and the first end forms a gripping part. The second end is used to connect to the optoelectronic composite cable 400, so that the optoelectronic composite cable 400 is correspondingly connected to the imaging module 230. The first end is used to detachably engage with the media assembly 220, and when the media assembly 220 is positioned at the first end, it contacts the imaging object 20 through the imaging window 222 in the media assembly 220. The gripping part is used to be held by relevant personnel / robotic arms, thereby contacting the imaging object 20 at a preset angle.

[0058] In some embodiments, the first end of the handle-shaped housing 210 may have an opening, and the objective lens 231 is disposed at the opening at the first end. When the media assembly 220 is assembled at the first end, the solid elastic medium 221 can contact and engage with the objective lens 231. Exemplarily, when the media assembly 220 is assembled at the first end, the axial direction of the solid elastic medium 221 coincides with the axial direction of the objective lens 231, thereby allowing the solid elastic medium 221 to contact and engage with the objective lens 231 by shifting the objective lens 231. More details regarding the engagement relationship between the solid elastic medium 221 and the objective lens 231 can be found in Figure 4 and its related description.

[0059] The medium assembly 220 can refer to the component that enables indirect contact between the objective lens 231 and the imaging object 20. The medium assembly 220 can be implemented based on the aforementioned solid elastic medium 221 and imaging window 222. That is, the medium assembly 220 may include the solid elastic medium 221 and the imaging window 222. One side of the imaging window 222 carries and is fixedly connected to the solid elastic medium 221, while the other side is used for contact-type engagement with the imaging object 20 during multiphoton microscopy imaging.

[0060] In some embodiments, the media assembly 220 is detachably disposed at the first end of the handle-shaped housing 210. Detachable disposal can refer to the media assembly 220 being detachably engaged with the second end via mechanical components (in both a fixed connection state and a separated state). Further details regarding detachable engagement can be found in Figure 4 and its related description.

[0061] The solid elastic medium 221 can be a transparent, solid, and elastic material. In some embodiments, when the solid elastic medium 221 is in contact with the objective lens 231, the solid elastic medium 221 itself is compressed by the pressure of the objective lens 231. When the objective lens 231 moves further, the solid elastic medium 221 can maintain contact through compression and expansion, thereby ensuring the contact between the objective lens 231 and the solid elastic medium 221. In some embodiments, the elastic modulus of the solid elastic medium 221 can be related to the imaging depth range, as shown in Figure 5 and related content.

[0062] In some embodiments, the refractive index of the solid elastic medium 221 can be matched with that of the imaging object 20. Matching can mean that the refractive index of the solid elastic medium 221 is within the allowable range of the refractive index of the imaging object 20 (e.g., ±5%, ±10%). The specific material of the solid elastic medium 221 can be selected based on the imaging object 20. For example, the refractive index of skin in type A populations is generally 1.45, so silica gel (whose refractive index can be made approximately 1.45 through doping) or butyl rubber (with a refractive index of approximately 1.46) can be selected as the solid elastic medium. As another example, the refractive index of skin in type B populations is generally above 1.3, so fluororubber (with a refractive index of 1.35) can be selected as the solid elastic medium.

[0063] It should be noted that in practical use, the refractive index of the solid elastic medium 221 does not necessarily have to be strictly consistent with that of the imaging object 20. It can also be used solely based on the elastic properties of the solid elastic medium 221 in resisting gravity, that is, the imaging probe 200 can be used at different imaging angles.

[0064] The imaging window 222 refers to the medium that directly contacts the imaging object 20. The imaging window 222 operates on the same principle as a coverslip in a microscope sample, facilitating cleaning of the side in contact with the imaging object 20. The imaging window 222 can be made of common coverslip materials, such as glass.

[0065] In some embodiments, the imaging window 222 may also be integrated into the solid elastic medium 221. For example, the side of the solid elastic medium 221 closest to the imaging object 20 may be cured to form the imaging window 222.

[0066] In some embodiments, to further reduce aberrations, the refractive index of the imaging window 222 can also be matched with that of the imaging object 20. The imaging window 222 can also be selected based on the refractive index of the imaging object 20. That is, the imaging window 222 can be selected as a solid, transparent medium with a refractive index matching that of the imaging object 20, capable of forming a smooth interface. For example, for the skin of type A individuals, the imaging window 222 can be made of ultra-transparent glass (refractive index of 1.45 to 1.55).

[0067] The imaging module 230 can be understood as a collection of optical elements within the imaging probe 200. Specifically, the imaging module 230 can construct the internal optical path of the imaging probe 200 based on micro laser scanning microscopy technology, thereby enabling control of the excitation light.

[0068] The imaging module 230 may include an excitation light path and a fluorescence signal path. The excitation light path controls the excitation light to scan the imaging object 20 point by point, and the fluorescence signal path is used to capture the fluorescence signal generated after the imaging object 20 is excited. The excitation light path can be connected to the first optical fiber in the optoelectronic composite cable 400, and the fluorescence signal path can be connected to the second optical fiber in the optoelectronic composite cable 400.

[0069] In some embodiments, the internal optical path of the imaging module 230 can be constructed based on micro-laser scanning microscopy. For example, refer to the internal structure schematic diagram shown in Figure 7 of CN109662696A. Furthermore, the internal optical path of the imaging module 230 can be adjusted according to actual needs. For example, a collimation module can be provided at the interface of the first optical fiber entering the imaging module 230 to collimate the excitation light.

[0070] Objective lens 231 can refer to the optical element that indirectly contacts the imaging module 230 and the imaging object 20. It can serve as a window for contact between the excitation light path and the fluorescence signal path and the imaging object 20, and is used to release excitation light and collect fluorescence signals.

[0071] When the excitation light transmitted in the optoelectronic composite cable 400 is controlled by the imaging module 230 and emitted from the objective lens 231, the processed excitation light forms a stable focal plane. Each emitted excitation light contains multiple photons, which are focused at a focal point on the focal plane, thereby activating the imaging object 20 corresponding to that focal point. The imaging module 230 can control the focusing position of the excitation light using internal optical components (such as a scanning galvanometer) to scan the imaging object 20 at the focal plane.

[0072] Since the relative position of the focal plane and the objective lens 231 is fixed, the position of the focal plane within the imaging object 20 can be adjusted by adjusting the position of the objective lens 231, thereby achieving imaging of the internal structure of the imaging object 20.

[0073] In some embodiments, considering that the imaging module 230 itself is based on micro-laser scanning microscopy, it is difficult to coordinate the adjustment of other internal optical components when adjusting the position of the objective lens 231 alone. Therefore, the imaging module 230 can be moved as a whole within the handle-shaped housing 210 to adjust the focal plane position. That is, the imaging module 230 also includes a displacement stage 232. The displacement stage 232 is used to control the overall movement of the imaging module 230 within the handle-shaped housing 210 to adjust the focal plane position of the imaging module 230.

[0074] In some embodiments, the various components within the imaging module 230 can be controlled via a control bus in the optoelectronic composite cable 400. Specifically, the control bus may include a position control cable for controlling the displacement stage, a scanning control cable for controlling the scanning galvanometer, a communication cable for communicating with the imaging probe 200 body (such as the internal integrated circuit or control board of the imaging probe 200), and a ground wire.

[0075] Based on some embodiments of the multiphoton microscopy imaging probe provided in this application, by using a solid elastic medium as the medium for indirect contact between the objective lens and the imaging object, the limitations of traditional immersion media on the use of microscopy imaging devices are eliminated. This allows the imaging probe to contact the imaging object at any angle and to perform reciprocating imaging at depth levels. Furthermore, in this application, the solid elastic medium is solidified into a medium assembly and connected to the probe body (handle-shaped housing) in a detachable manner. During use, the objective lens and the solid elastic medium can be assembled directly by disassembling the medium assembly, eliminating the need for the frequent and complex assembly process of traditional immersion media.

[0076] Exemplary detachable fit:

[0077] Figure 4 is a schematic diagram of a detachable arrangement of a media component and a handle-shaped housing provided in an exemplary embodiment of this application.

[0078] As shown in Figure 4, the detachable connection can include a separated state 400a and a fixed connection state 400b. When the media assembly 220 and the handle-shaped housing 210 are in the separated state 400a, they are separated. When the media assembly 220 and the handle-shaped housing 210 are in the fixed connection state 400b, the axis of the solid elastic medium 221 coincides with the axis of the objective lens 231, and the objective lens 231 can be moved to make the objective lens 231 contact the solid elastic medium 221.

[0079] As shown in Figure 4, the media assembly 220 may further include a first fixing structure 223 for achieving a detachable connection. A second fixing structure 211 matching the first fixing structure 223 is then provided at the first end of the handle-shaped housing 210. The media assembly 220 can be detachably mounted on the first end of the handle-shaped housing 210 via the first fixing structure 223 and the second fixing structure 211.

[0080] In some embodiments, the first fixing structure 223 and its corresponding second fixing structure 211 can be common detachable components. For example, the first fixing structure 223 can be a slot or groove, and the second fixing structure 211 can be a pin for holding in the slot or groove. As another example, both the first fixing structure 223 and the second fixing structure 211 can be threaded, wherein the thread ridges and thread valleys of the first fixing structure 223 and the second fixing structure 211 correspond to each other.

[0081] In some embodiments, to avoid affecting the contact between the imaging window 222 and the imaging object 20, the first fixing structure 223 may be arranged around the imaging window 222 and extend along the placement direction of the solid elastic medium 221. That is, the medium assembly 220 may form a concave structure, wherein the imaging window 222 is disposed on the bottom surface of the concave structure, the solid elastic medium 221 is disposed on the inner wall of the bottom surface, and the first fixing structure 223 is disposed on the side wall of the concave structure.

[0082] Corresponding to the first fixing structure 223, the first end of the handle-shaped housing 210 can form a boss structure corresponding to the concave structure. The second fixing structure 211 can be provided on the side wall of the boss structure so that the concave structure and the boss structure cooperate to detachably set the medium assembly 220 at the first end.

[0083] Considering that the imaging probe 200 provided in this application can directly contact the imaging object 20, imaging can be performed based on a low-light environment. Low-light environment imaging can refer to blocking external light sources during the imaging process, using excitation light and fluorescence signals as the primary light sources for imaging.

[0084] In some embodiments, a low-light environment can be achieved through the first fixing structure 223. That is, the first fixing structure 223 can be made of a light-shielding material. When the first fixing structure 223 cooperates with the second fixing structure 211 (i.e., fixed connection state 400b) and the imaging window 222 is in contact with the imaging object 20 (i.e., the imaging window 222 does not introduce an external light source), a low-light environment is formed between the imaging object 20 and the objective lens 231.

[0085] Based on the first fixing structure 223 on the medium component 220 provided in some embodiments of this application, it can not only achieve detachable engagement with the second fixing structure 211, but also block external light sources, thereby forming a weak light environment to improve the recognition effect of fluorescence signals.

[0086] Exemplary contact mating:

[0087] Figure 5 is a schematic diagram of the contact fit between the objective lens and the solid elastic medium provided in an exemplary embodiment of this application.

[0088] In some embodiments, the mating process between the objective lens and the solid elastic medium may include a non-contact state and a contact mating state. As shown in Figure 5, an example of a contact mating may include a separation state 500a, a reference state 500b, an intermediate state 500c, and a limiting state 500d. Separation state 500a is a type of non-contact state, while reference state 500b, intermediate state 500c, and limiting state 500d are types of contact mating states.

[0089] The non-contact state can refer to the state in which the objective lens 231 and the solid elastic medium 221 have not made contact. Specifically, it can include the separation state 500a.

[0090] When in the separated state 500a, the solid elastic medium 221 is separated from the objective lens 231. At this time, the solid elastic medium 221 is not compressed, and its height relative to the imaging window 222 can be denoted as h0. The objective lens 231 can form a focal plane. The focal plane can be seen in the trapezoidal area marked with a dashed line in the figure; its base can be considered as the focal plane, and the trapezoidal body can be regarded as the collection of excitation light that forms the focal plane. When the excitation light is emitted from the objective lens 231, it will converge at the focal plane as shown in the figure. When in the non-contact state, the focal plane position generally does not reach the contact interface between the imaging window 222 and the imaging object 20.

[0091] When the objective lens 231, which is in a separated state, moves toward the solid elastic medium 221, it can make the objective lens 231 and the solid elastic medium 221 meet the matching conditions, and the objective lens 231 and the solid elastic medium 221 form a contact fit.

[0092] The conditions for cooperation may include the following restrictions:

[0093] First, the objective lens 231 is in contact with the solid elastic medium 221, and the solid elastic medium 221 is deformed under force.

[0094] Second, the light rays that form the focal plane do not pass through any medium other than the solid elastic medium. That is, the excitation light that forms the focal plane only passes through the solid elastic medium and the imaging window before entering the imaging object 20.

[0095] Third, the focal plane is located inside the imaging object 20.

[0096] In some embodiments, the contact area between the solid elastic medium 221 and the imaging window 222 is larger than the focal plane area. In this case, to ensure that the aforementioned first and second conditions are met, it is only necessary to ensure that the contact interface formed between the objective lens 231 and the solid elastic medium 221 is horizontal with the focal plane and can completely cover the area in the objective lens 231 where the excitation light is emitted.

[0097] Based on the above-mentioned mating conditions, during the contact mating process in which the objective lens 231 moves toward the solid elastic medium 221, the reference state 500b, the intermediate state 500c, and the extreme state 500d can be formed sequentially.

[0098] The reference state 500b can refer to the engagement state when the focal plane is located at the contact interface between the imaging window 222 and the imaging object 20.

[0099] At this time, the solid elastic medium 221 is compressed, and its height relative to the imaging window 222 can be denoted as h1. The relative depth between the focal plane of the objective lens 231 and the imaging object 20 is 0.

[0100] The intermediate state 500c can refer to the state in which the focal plane is located inside the imaging object 20 and the objective lens 231 can reciprocate. Here, the reciprocating motion of the objective lens 231 can be understood as the objective lens 231 being able to move towards the imaging object 20 or away from the imaging object 20 within a certain range in the direction of movement of the objective lens 231.

[0101] At this time, the solid elastic medium 221 is compressed, and its height relative to the imaging window 222 can be denoted as h2. The relative depth between the focal plane of the objective lens 231 and the imaging object 20 is d1.

[0102] The extreme state 500d can refer to the engagement state when the objective lens 231 can no longer move toward the imaging object 20 (i.e., the extreme compression state of the solid elastic medium 221).

[0103] At this point, the solid elastic medium 221 is compressed to its limit, and its height relative to the imaging window 222 can be denoted as h3. The relative depth between the focal plane of the objective lens 231 and the imaging object 20 is d2.

[0104] In actual imaging, contact engagement includes at least an intermediate state. Whether other states are included can be determined based on the specific imaging task. For example, when the imaging task includes detecting a relative depth from 0 to a target depth, the depth 0 can be determined first in a reference state, thereby adjusting the objective lens depth to an intermediate state for imaging, thus ensuring the relative depth between the focal plane and the imaging object 20 reaches the target depth. As another example, when the imaging task includes a first target depth to a second target depth, imaging can be performed directly in an intermediate state, thus making d1 represent each target depth from the first target depth to the second target depth.

[0105] It should be noted that, considering the refractive index of the solid elastic medium matches the imaging object, the displacement of the objective lens (or the deformation of the solid elastic medium) is basically consistent with the displacement of the focal plane. For example, the height difference between h1 and h3 (i.e., the objective lens displacement distance from the reference state to the limiting state) is the same as d2.

[0106] Based on the aforementioned fit, it can be seen that the elastic deformation range of the solid elastic medium can match the imaging depth range. Here, the imaging depth range refers to the imaging depth range required by the imaging task. As shown in the previous description, the elastic deformation range of the solid elastic medium can include the elastic range required for contact fit (h0 to h1) and the elastic range after contact (h1 to h3), while the imaging depth range can be any range from 0 to d2. Therefore, it can be concluded that the elastic range after contact (such as the height difference between h1 and h3) within the elastic deformation range of the solid elastic medium is greater than or equal to the imaging depth range (generally any range from 0 to d2).

[0107] It should be noted that, considering different media components may be used for different imaging tasks using the same probe, the contact fit between the media component and the objective lens only needs to meet the requirements of the imaging task. For example, when the focal plane position coincides with the contact interface between the imaging window and the imaging object, the objective lens and the solid elastic medium can be in a non-contact state. When implementing contact fit later, imaging of the object within the imaging depth range is directly based on the aforementioned intermediate state. The focal plane position can be directly determined based on the displacement parameters of the objective lens itself.

[0108] Exemplary imaging device:

[0109] Figure 6 is an exemplary flowchart of a multiphoton microscopy imaging apparatus provided in an exemplary embodiment of this application.

[0110] As shown in Figure 6, the multiphoton microscopy imaging device 10 may include an imaging host 300 and a multiphoton microscopy imaging probe 200, which are connected by an optoelectronic composite cable 400. The specific internal structure of the multiphoton microscopy imaging probe 200 can be found in the description in Figure 3 above, and will not be repeated here.

[0111] The imaging host 300 can be a collection of components that cannot or are inconvenient to integrate into the imaging probe 200 in the imaging device 10. For example, the imaging host 300 can be used to generate excitation light and generate fluorescence images based on fluorescence signals. The imaging host 300 can be connected to the multiphoton microscopy imaging probe 200 via an optoelectronic composite cable 400 to transmit excitation light and control signals and receive fluorescence signals.

[0112] As shown in Figure 6, the imaging host 300 may specifically include a power supply 310, a laser source 320, a collection module 330, and a signal control and processing module 340. The power supply 310 provides the operating voltage for the imaging device 10. The laser source 320 generates excitation light. The collection module 330 generates fluorescence images based on fluorescence signals. The signal control and processing module 340 generates control signals for controlling the imaging probe 200. A cable interface is used for connection to the optoelectronic composite cable 400.

[0113] Exemplary imaging method:

[0114] Figure 7 is an exemplary flowchart of a multiphoton microscopy imaging method provided in an exemplary embodiment of this application. In some embodiments, the process P700 shown in Figure 7 can be executed by the signal control and processing module 340. The signal control and processing module 340 executes P700 by generating a corresponding control signal to be transmitted to the imaging probe 200. Therefore, P700 can also be understood as being executed by the imaging probe 200 or the imaging device 10.

[0115] As shown in Figure 7, P700 includes the following steps:

[0116] S710 controls the position of the imaging module in the imaging direction so that the focal plane of the imaging module is at the target focusing depth. When the focal plane is at the target focusing depth, the objective lens is in contact with the solid elastic medium.

[0117] The target depth of focus can refer to any value within the imaging depth range of an imaging task. It can reflect the position of the focal plane during imaging. For example, in a typical imaging task, the imaging depth range can be from 0 to a preset depth. For instance, in imaging human facial skin, it is typically 0 to 0.3 mm to ensure that the imaging depth range encompasses all structures of the human epidermis.

[0118] In some embodiments, adjusting the focal plane to the target depth of focus is generally determined based on the amount of displacement relative to a reference state. For example, a reference scale for the objective lens displacement in the reference state can be determined through image features, and the target depth of focus can be reached by displacing the objective lens based on this reference scale. The reference state can also be determined based on the factory parameters of the objective lens and media assembly.

[0119] The S720 performs multiphoton microscopy on the object being imaged based on the target focusing depth.

[0120] Since the focal plane is already located at the target focal depth, a scanning operation can be directly performed to achieve multiphoton microscopy imaging at the target focal depth. As an example, the focusing position of the excitation light on the focal plane can be adjusted by using a scanning galvanometer, allowing the focusing position to traverse the entire focal plane. When the object being imaged is excited by the excitation light at the corresponding position, a fluorescence signal is generated, which is acquired by the imaging probe and transmitted to the collection module. The collection module and controller determine the fluorescence image at the target focal depth based on the scanning timing signal and the fluorescence signal, thus completing the multiphoton microscopy imaging.

[0121] Exemplary method for determining solid elastic media:

[0122] For the aforementioned imaging device based on solid-state elastic media, Figure 8 of this application also provides a method for determining a suitable solid-state elastic media for the imaging module based on the imaging task.

[0123] Figure 8 is an exemplary flowchart of a method for determining a solid elastic medium provided in some embodiments of this application.

[0124] As shown in Figure 8, P800 may include the following steps:

[0125] S810 identifies the imaging equipment, imaging mission, and candidate solid-state elastic media.

[0126] An imaging task can refer to at least one imaging operation that an imaging device needs to perform during the actual imaging process. An imaging task can include the imaging object and the imaging depth.

[0127] In S810, the imaging device can refer to a device used to perform actual imaging. In some embodiments, the imaging device can be determined before the imaging task is performed, meaning the imaging task can be completed by the imaging device. In some embodiments, the imaging device can also be determined based on the imaging task. For example, an imaging device with better fluorescence effect can be selected for imaging based on the imaging object. Another example is selecting an imaging device whose imaging module's movement range meets the imaging depth requirements based on the imaging depth.

[0128] Candidate solid-state elastic media refer to solid-state elastic media that can be selected for this imaging mission. That is, for an imaging mission, the imaging device needs to determine a suitable solid-state elastic medium from the candidate solid-state media to perform the imaging mission, and then perform the imaging mission by cooperating with that solid-state elastic medium. The candidate solid-state elastic medium can be directly determined from existing elastic media.

[0129] In some embodiments, considering the imaging principle of the imaging device, solid-state elastic media can be screened to determine candidate solid-state elastic media, thereby reducing the workload of testing. The candidate solid-state elastic media should at least satisfy the following requirements: it should have a cross-section larger than the imaging module (light exit port / objective lens), and the imaging window corresponding to the candidate solid-state elastic media should be larger than the focal plane of the imaging module. Based on this, the candidate solid-state elastic media determined can at least guarantee that the candidate solid-state elastic media and the imaging module can maintain the aforementioned initial fit state.

[0130] S820 determines the reference position parameters, mating position parameters, and extreme position parameters of the imaging module based on the candidate solid elastic medium.

[0131] The reference position parameters can refer to the position parameters (such as position scale) of the displacement stage when it is in the reference state 500b in Figure 5. The configuration position parameters can refer to the position parameters of the displacement stage when the objective lens is just in contact with the solid elastic medium. The limit position parameters can refer to the position parameters of the displacement stage when it is in the limit state 500d in Figure 5.

[0132] In some embodiments, the aforementioned mating position parameters can be determined based on the morphology of the solid elastic medium. When the cross-sectional area of ​​the solid elastic medium is larger than the light-emitting area of ​​the objective lens, it can be considered that when the solid elastic medium deforms to this point, the objective lens and the solid elastic medium are just in contact and mating.

[0133] In some embodiments, the aforementioned parameters may also include the protrusion height of the solid elastic medium relative to the imaging device. As shown in Figure 4, the solid elastic medium and the imaging window can be assembled on the imaging device. During assembly, the top end of the solid elastic medium may be at a certain distance from the end end of the imaging device, and this distance can be denoted as the protrusion height.

[0134] In some embodiments, the reference position parameter can be estimated based on the difference in protrusion height of different solid elastic media. For example, if the protrusion height of solid elastic medium a is h1 and the reference position parameter is s1, then for solid elastic medium b with a protrusion height of h2, its reference position parameter can be s1 + h1 - h2.

[0135] In some embodiments, the protrusion height of candidate solid elastic media can be marked after production and directly invoked during use. In some embodiments, the difference in protrusion height between different solid elastic media can also be determined based on testing, thereby determining the reference position parameters of each solid elastic media.

[0136] In some embodiments, the position parameters can be determined based on the morphology of the solid elastic medium. Specifically, when the cross-section of the solid elastic medium is larger than the light exit port of the imaging module (e.g., the area of ​​the objective lens), it can be considered that the solid elastic medium covers the imaging module.

[0137] Similar to the aforementioned reference position parameters, in some embodiments, the mating position parameters can be determined based on the compression of the solid elastic medium when it is larger than the light outlet of the imaging module. For example, if the compression of the solid elastic medium b in the reference mating state is y1 and the compression in the initial mating state is y2, the mating position parameter of the solid elastic medium b can be (s1+h1-h2)-y1+y2.

[0138] In some embodiments, the limiting position parameter can be determined based on the compression amount under the limiting compression state of the solid elastic medium. The specific determination process is similar to that of the mating position parameter and will not be elaborated here.

[0139] S830 determines the target solid-state elastic medium from candidate solid-state elastic media based on reference position parameters, mating position parameters, and limit position parameters.

[0140] The target solid-state elastic medium can refer to a solid-state elastic medium that meets the requirements of the imaging task and can be used to perform subsequent imaging tasks.

[0141] In some embodiments, the target solid-state elastic medium can be determined by comparing the imaging range and imaging depth corresponding to the solid-state elastic medium. When the reference position parameter of the solid-state elastic medium is within the interval [fitting position parameter, extreme position parameter], the corresponding imaging range is the interval [0, extreme position parameter - reference position parameter]. When the imaging depth of the imaging task is within this interval, it can be determined that the solid-state elastic medium meets the imaging task and can be used as the target solid-state elastic medium.

[0142] In some embodiments, when the imaging depth of the imaging task is large, a combination of multiple solid elastic media with different imaging ranges can be used as the target solid elastic media.

[0143] When combining solid elastic media, it is advisable to consider solid elastic media with a reference position parameter less than the mating position parameter. When the reference position parameter of the solid elastic media is less than the mating position parameter, the corresponding imaging range is the interval [matting position parameter - reference position parameter, extreme position parameter - reference position parameter]. Because its reference position parameter is smaller, its imaging range is larger. Although it does not include the zero point, it can be combined with solid elastic media that do include the zero point to cover the imaging depth.

[0144] Therefore, the imaging device provided in this application can select a suitable solid elastic medium based on the imaging task, thereby ensuring the smooth execution of the imaging task.

[0145] All of the above-mentioned optional technical solutions can be combined in any way to form optional embodiments of this application, and will not be described in detail here.

[0146] Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0147] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, devices, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0148] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection between apparatuses or units may be electrical, mechanical, or other forms.

[0149] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0150] In addition, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit.

[0151] If the aforementioned functions are implemented as software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, or a portion of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this application. The aforementioned storage medium includes various media capable of storing program verification codes, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0152] It should be noted that in the description of this application, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Furthermore, in the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0153] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications or equivalent substitutions made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A multiphoton microscopic imaging probe, characterized in that, include: A media assembly, wherein the media assembly includes a solid elastic medium and an imaging window that carries the solid elastic medium and is used to contact the imaging object; A handle-shaped housing, wherein the medium assembly is detachably disposed at a first end of the handle-shaped housing; and An imaging module based on micro laser scanning microscopy technology is provided, wherein the imaging module is disposed within the handle-shaped housing and is used to transmit and control excitation light and receive fluorescence signals, and the imaging module includes an objective lens that contacts and engages with the solid elastic medium.

2. The imaging probe according to claim 1, characterized in that, The imaging module also includes a displacement stage, which is used to control the overall movement of the imaging module within the handle-shaped housing to adjust the focal plane position of the imaging module.

3. The imaging probe according to claim 1, characterized in that, The medium assembly further includes a first fixing structure, and a second fixing structure matching the first fixing structure is provided at the first end of the handle-shaped housing. The medium assembly is detachably disposed at the first end of the handle-shaped housing through the first fixing structure and the second fixing structure.

4. The imaging probe according to claim 3, characterized in that, The first fixing structure ring is disposed around the imaging window and extends along the placement direction of the solid elastic medium.

5. The imaging probe according to claim 4, characterized in that, The first fixing structure is made of light-shielding material. When the first fixing structure cooperates with the second fixing structure and the imaging window contacts the imaging object, a low-light environment is formed between the imaging object and the objective lens.

6. The imaging probe according to claim 1, characterized in that, The refractive index of the solid elastic medium matches that of the imaging object.

7. The imaging probe according to claim 1, characterized in that, The elastic deformation range of the solid elastic medium is matched with the imaging depth range.

8. The imaging probe according to claim 1, characterized in that, The imaging probe also includes an optoelectronic composite cable for connecting to the imaging host, the optoelectronic composite cable being connected to the second end of the handle-shaped housing and connected to the imaging module; The optoelectronic composite cable includes a first optical fiber for transmitting excitation light, a second optical fiber for transmitting fluorescence signals, and a control bus for transmitting control signals.

9. A multiphoton microscopy imaging device, characterized in that, include: An imaging host is used to generate excitation light and generate fluorescence images based on fluorescence signals; as well as The multiphoton microscopy imaging probe according to any one of claims 1 to 8, wherein the multiphoton microscopy imaging probe is connected to the imaging host via an optoelectronic composite cable, and is used to release the excitation light to the imaging object and collect the fluorescence signal generated by the imaging object based on the photoluminescence effect.

10. A multiphoton microscopy imaging method, characterized in that, The method, applied to the multiphoton microscopy imaging probe according to any one of claims 1 to 8 or the multiphoton microscopy imaging device according to claim 9, comprises: The position of the imaging module in the imaging direction is controlled so that the focal plane of the imaging module is at the target focusing depth, wherein when the focal plane is at the target focusing depth, the objective lens is in contact with the solid elastic medium. Multiphoton microscopy is performed on the imaging object based on the target focusing depth.

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