Multi-photon microscopic imaging probe based on adaptive optics and imaging equipment

Abstract

The invention discloses a multi-photon microscopic imaging probe based on adaptive optics and imaging equipment. The probe comprises a photoelectric composite cable, a handle-shaped shell, an imaging module and a self-adaptive optical module. Wherein the imaging module is used for transmitting and controlling exciting light and receiving a fluorescence signal, and comprises a scanning galvanometer for controlling the exciting light. The adaptive optical module is arranged between the photoelectric composite cable and the imaging module. The adaptive optical module comprises a wavefront regulator and two lens groups, the wavefront regulator and the scanning galvanometer form 4f conjugation through the two lens groups, and the wavefront regulator can change the wavefront of a light beam entering the wavefront regulator based on wavefront control parameters. Based on the imaging probe provided by the specification, the self-adaptive optical module is integrated in the optical system of the micro multi-photon microscopy through 4f conjugation, and interference formed at each focusing depth due to the internal structure of an imaging object can be pre-compensated, so that the imaging quality at each focusing depth is improved.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging, and in particular to a multi-photon microscopic imaging probe and imaging device based on adaptive optics. Background Art

[0002] Multiphoton microscopy is a highly sensitive imaging technology based on the multiphoton phenomenon, in which the multiphoton phenomenon refers to the photoluminescence effect produced by fluorescent substances when they are excited by at least two photons at the same time, releasing fluorescent signals. Considering the complexity of the optical structure, multiphoton microscopy generally uses two photons, namely two-photon microscopy.

[0003] Compared with traditional fluorescence imaging technology, the excitation light of multiphoton microscopy is composed of multiple photons, and the attenuation at the depth level is weak, which can perform microscopic imaging of the internal structure of the imaging object within a certain depth range. However, in actual imaging, the internal structure of the imaging object will interfere with the excitation light, thereby affecting the imaging quality. Therefore, how to improve the quality of multiphoton microscopy is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] In view of this, the embodiments of this specification provide a multiphoton microscopic imaging probe and imaging device based on adaptive optics. The present application pre-adjusts the excitation light through adaptive optics technology, thereby reducing the influence of the internal structure of the imaging object on the multiphoton microscopic imaging, thereby overcoming the shortcomings of the prior art.

[0005] In a first aspect, an embodiment of the present application provides a multi-photon microscopic imaging probe based on adaptive optics, the imaging probe comprising: an optoelectronic composite cable for connecting to an imaging host; wherein the optoelectronic composite cable is used to transmit excitation light and transmit fluorescence signals; a handle-shaped shell, wherein the optoelectronic composite cable is connected to the first end of the handle-shaped shell; an imaging module based on micro-laser scanning microscopy technology, wherein the imaging module is arranged in the handle-shaped shell and connected to the optoelectronic composite cable, and is used to transmit and control the excitation light and receive the fluorescence signal, and the imaging module comprises a scanning galvanometer for controlling the excitation light; and an adaptive optical module, wherein the adaptive optical module is arranged between the optoelectronic composite cable and the imaging module; wherein the adaptive optical module comprises a wavefront adjuster and two lens groups, wherein the wavefront adjuster and the scanning galvanometer form a 4f conjugate through the two lens groups, and the wavefront adjuster can change the wavefront of the light beam incident on the wavefront adjuster based on the wavefront control parameters.

[0006] In a second aspect, an embodiment of the present application provides a multi-photon microscopic imaging device based on adaptive optics, the device comprising: an imaging host, used to generate excitation light and generate a fluorescence image based on a fluorescence signal; and the multi-photon microscopic imaging probe described in the first aspect, wherein the multi-photon microscopic imaging probe is connected to the imaging host via an optoelectronic composite cable, and is used to release the activation light to the imaging object and collect the fluorescence signal generated by the imaging object based on the photoluminescence effect.

[0007] The embodiments of the present specification provide a multi-photon microscopy imaging probe and imaging device based on adaptive optics. Based on the imaging probe provided in the present specification, an adaptive optics module is integrated into the optical system of a micro-multiphoton microscope through 4f conjugation, wherein the adaptive optics module can adjust the wavefront of the excitation light, thereby overcoming the wavefront distortion at different focusing depths, so that the excitation light has a well-shaped wave plate at the focal plane, thereby improving the penetration rate and excitation rate of the imaging object, thereby improving the imaging quality at each focusing depth. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0009] Figure 1 It is an application scenario diagram of a multi-photon microscopic imaging device provided by an exemplary embodiment of this specification.

[0010] Figure 2 It is a schematic diagram of the internal structure of a multiphoton microscopic imaging probe provided by an exemplary embodiment of this specification.

[0011] Figure 3 This is a schematic diagram of the first excitation light path of an imaging probe provided by an exemplary embodiment of this specification.

[0012] Figure 4 This is a schematic diagram of a second excitation light path of an imaging probe provided by an exemplary embodiment of this specification.

[0013] Figure 5 This is a schematic diagram of the third excitation light path of an imaging probe provided by an exemplary embodiment of this specification.

[0014] Figure 6 This is a first overall optical path schematic diagram of an imaging probe provided by an exemplary embodiment of this specification.

[0015] Figure 7This is a second overall optical path schematic diagram of an imaging probe provided by an exemplary embodiment of this specification.

[0016] Figure 8 It is an exemplary flow chart of a multiphoton microscopic imaging method provided by an exemplary embodiment of this specification.

[0017] Fig. 9 It is an exemplary flow chart of determining a first wavefront control parameter group based on an iterative algorithm provided by an exemplary embodiment of this specification.

[0018] Fig.10 It is an exemplary structural diagram of a multiphoton microscopic imaging device provided by an exemplary embodiment of this specification. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] Application Overview:

[0021] Wavefront refers to a beam of light or the front wave surface during propagation. In optics, wavefront refers to a series of equal phase points in the direction of light propagation, forming a curved surface. Wavefront can be used to describe the propagation direction, propagation speed and wave shape of light.

[0022] When light passes from one medium into a medium with a higher refractive index, the light bends toward the normal and the wavefront becomes concave; and when light passes from one medium into a medium with a lower refractive index, the light bends away from the normal and the wavefront becomes convex.

[0023] Considering multi-photon imaging, the excitation light will penetrate the imaging object and form a focal plane inside the imaging object. At this time, the complex structure inside the imaging object (such as uneven internal refractive index) will cause great interference to the wavefront of the excitation light, thereby causing the wavefront of the excitation light to be distorted.

[0024] The wavefront distortion of the excitation light mainly affects the penetration efficiency of the excitation light and the excitation efficiency at the focal plane. When the wavefront of the excitation light is distorted, it will affect the intensity of the light focused at the focal plane, thereby affecting the excitation efficiency of the imaging object (multi-photon fluorescent material) and whether the excitation light can reach the focal plane.

[0025] In some embodiments of the related art, the wavefront distortion inside the imaging object can be compensated by detecting it with a wavefront sensor. That is, the specific wavefront distortion can be detected by a wavefront sensor (such as an interferometer, a Hartmann sensor, etc.), and then the excitation light is compensated by a spatial light modulator, and the compensation value is continuously changed so that the wavefront sensor does not have wavefront distortion.

[0026] The following technical problems may arise in the actual application of relevant technologies:

[0027] First, the dry wavefront sensor is placed at the focal plane to be detected to collect the wavefront distortion at the focal plane position, that is, the imaging object must be possible to detect (such as an in vitro sample), which limits the imaging object. For living imaging objects, especially when multiphoton microscopy is used for non-invasive examination of the human body, it is impossible to detect the wavefront distortion at the focal plane inside the imaging object, and thus it is impossible to compensate.

[0028] Second, according to the nature of the wavefront, the adjustment of the wavefront generally needs to be made at the place where the excitation light is released in the two-photon microscopy imaging device (such as the imaging probe). In the miniature multiphoton imaging device, the imaging probe is small and it is difficult to integrate the spatial light modulator. Among them, considering that the excitation light in the multiphoton imaging device needs to be transmitted through the optical fiber, optical modules such as collimation are generally set at the interface of the optical fiber for processing so as to transmit the plane wavefront in the optical fiber. Therefore, it is impossible to pre-compensate the excitation light at the place where the excitation light occurs (imaging host). It should be noted that if the optical fiber does not have a collimation module at the interface, the movement of the optical fiber and its own properties will produce wavefront distortion that is more difficult to eliminate.

[0029] Based on the above technical problems, the embodiments of this specification provide a multi-photon microscopic imaging probe and imaging device based on adaptive optics. Based on the imaging probe provided in this specification, an adaptive optics module is integrated into the optical system of a micro-multiphoton microscope through 4f conjugation, wherein the adaptive optics module can adjust the wavefront of the excitation light, thereby overcoming the wavefront distortion at different focal depths, so that the excitation light has a well-shaped wave plate at the focal plane, thereby improving the penetration rate and excitation rate of the imaging object, thereby improving the imaging quality at each focal depth. Various non-limiting embodiments of the present application will be specifically described below with reference to the accompanying drawings.

[0030] It should be noted that, in this specification, adaptive optics (Adaptive Optics) only refers to a technology for real-time correction of wavefront distortion in light transmission, which means that the multiphoton microscopy imaging probe provided in this specification can perform real-time pre-compensation for the wavefront distortion of the excitation light, and does not limit the technical solution adopted in this specification to include all optical structures of the adaptive optical system. For example, the imaging probe provided in this specification does not require a wavefront sensor during compensation.

[0031] Example application scenarios:

[0032] Figure 1 It is an application scenario diagram of a multi-photon microscopic imaging device provided by an exemplary embodiment of this specification.

[0033] like Figure 1 As shown, the application scenario diagram of the multiphoton microscopic imaging device may include a multiphoton microscopic imaging device 10 and an imaging object 20 .

[0034] The imaging device 10 is the executor of multiphoton microscopic imaging, which can release excitation light and collect fluorescence signals of the imaging object 20 after being excited by the excitation light, so as to generate a fluorescence image of the imaging object 20. In some embodiments, the imaging device 10 can focus multiple photons of the excitation light on a point at the target focus depth inside the imaging object 20 based on the principle of multiphoton imaging to obtain the fluorescence signal at the point. Then, the focus position at the target focus depth is changed by scanning technology to perform plane scanning, so as to obtain the fluorescence signals at various locations inside the imaging object 20 at the target focus depth to determine the fluorescence image.

[0035] The imaging object 20 is the object of multiphoton microscopic imaging, that is, the imaging object 20 can be an object for multiphoton imaging as needed. For example, the imaging object 20 can be an in vitro sample, a living sample, etc. In some embodiments, the imaging device 10 provided in this specification can be used as a non-invasive medical detection device, and the imaging object 20 can be a part of a patient to be detected, such as skin.

[0036] In order to achieve multi-angle detection of living samples, the imaging device 10 provided in this specification can adopt a split setting, thereby further including a multi-photon microscopic imaging probe 100 and an imaging host 200, wherein the multi-photon microscopic imaging probe 100 and the imaging host 200 can be communicatively connected based on an optoelectronic composite cable 300.

[0037] The imaging probe 100 may be a device that releases excitation light and detects fluorescent signals. The imaging probe 100 may convert the laser light generated by the laser source into excitation light that is focused on the focal plane and has scanning capability through an optical component. In some embodiments, the imaging probe 100 may be a contact imaging probe, that is, the imaging probe 100 may be in direct contact with the imaging object 20 from any angle. In some embodiments, the position where the imaging probe 100 contacts the imaging object 20 may be provided with a medium component to fill an imaging medium (such as a gel medium, a solid elastic medium, etc.) whose refractive index matches that of the imaging object 20, so as to reduce the aberration of the excitation light of the imaging probe 100. For more information about the imaging probe 100, please refer to Figure 2 .

[0038] The imaging host 200 may be a collection of devices that cannot be integrated into the imaging probe 100, and may be specifically used to provide excitation light and perform imaging based on fluorescent signals. For more information about the imaging host 200, see Fig.10 .

[0039] The optoelectronic composite cable 300 can be a communication cable between the imaging probe 100 and the imaging host 200, and is used to realize the data transmission of optical signals and electrical signals between the imaging host 200 and the imaging probe 100. The optoelectronic composite cable 300 can transmit the excitation light generated by the imaging host 200 to the inside of the imaging probe 100, and transmit the fluorescence signal collected by the imaging probe 100 to the imaging host 200 to form a fluorescence image.

[0040] Exemplary Imaging Probes:

[0041] Based on the aforementioned application scenarios, this specification provides a multi-photon microscopic imaging probe based on a solid elastic medium. Figure 2 It is a schematic diagram of the internal structure of a multiphoton microscopic imaging probe provided by an exemplary embodiment of this specification.

[0042] like Figure 2 As shown, the multiphoton microscopic imaging probe 100 may include an optoelectronic composite cable 300 , a handle-shaped housing 110 , an imaging module 120 , and an adaptive optical module 130 .

[0043] The optoelectronic composite cable 300 may refer to a collection of optical cables and electrical cables for data transmission between the imaging probe 100 and the imaging host 200. For example, the optoelectronic composite cable 300 may include a first optical fiber for transmitting excitation light, a second optical fiber for transmitting a fluorescent signal, and a control bus for transmitting a control signal. The control bus may be an integrated control cable for each device in the imaging probe 100.

[0044] It should be noted that, considering the complexity of the connection between the optoelectronic composite cable 300 and the imaging probe 100, the optoelectronic composite cable 300 is generally regarded as a part of the imaging probe 100. However, the imaging probe 100 described in this specification includes the optoelectronic composite cable 300, which can also be understood as including a data interface for connecting to the optoelectronic composite cable 300, and does not forcibly limit the imaging probe 100 to include a physical optoelectronic composite cable 300. Considering that the imaging probe 100 must be connected to the imaging host 200 through an optical cable when in use, the imaging probe 100 that does not directly include the optoelectronic composite cable 300 but can be used to connect to the optoelectronic composite cable 300 (such as being provided with an interface for the optoelectronic composite cable 300) can also be regarded as the imaging probe 100 provided in the embodiment of this specification.

[0045] In addition, to ensure the stability of data transmission, the optoelectronic composite cable 300 in this specification can be constructed in the form of an optical cable and an electrical cable. In practice, electrical signal transmission can also be carried out in the form of wireless communication. In this case, the optoelectronic composite cable 300 only needs to transmit optical signals without integrating electrical cables.

[0046] The handle-shaped housing 110 may be a housing of the multiphoton microscopic imaging probe 100, and the components of the multiphoton microscopic imaging probe 100 are integrated and arranged inside the handle-shaped housing 110. The relevant staff may hold the handle-shaped housing 110 in a holding method similar to holding a handle.

[0047] like Figure 3 As shown, the handle-shaped housing 110 can be presented as a cylindrical structure, and is provided with a first end and a second end, wherein the cylindrical structure between the first end and the second end forms a gripping portion. The first end is used to access the optoelectronic composite cable 300 so that the optoelectronic composite cable 300 is connected to each component in the imaging probe 100. The second end is used to contact the imaging object 20 (for example, through a dielectric component). The gripping portion is used to be held by a relevant staff member / mechanical arm, so as to contact the imaging object 20 at a preset angle.

[0048] In some embodiments, the second end may be provided with a medium component to accommodate an imaging medium that matches the imaging object 20. For example, the imaging medium may be a colloidal immersion medium or a solid elastic medium. The medium component may realize multi-angle in vivo imaging. For more information, please refer to the relevant technology, which will not be described in detail here.

[0049] The imaging module 120 can be understood as a collection of optical elements in the imaging probe 100. The imaging module 120 can construct an internal optical path of the imaging probe 100 based on micro laser scanning microscopy technology, thereby achieving control of the excitation light.

[0050] The imaging module 120 may include an excitation light path and a fluorescence light path, wherein the excitation light path may control the excitation light to scan the imaging object 20 point by point, and the fluorescence light path may be used to use the fluorescence signal generated after the imaging object 20 is excited. The excitation light path may be connected to the first optical fiber in the optoelectronic composite cable 300, and the fluorescence signal light path may be connected to the second optical fiber in the optoelectronic composite cable 300.

[0051] In some embodiments, the internal optical path of the imaging module 120 can be constructed based on micro laser scanning microscopy technology. The imaging module 120 at least includes a scanning galvanometer 121 for controlling the excitation light to scan the imaging object 20 point by point.

[0052] The scanning galvanometer 121 may refer to an optical element that can change the direction of a light beam by vibration or rotation. Among them, the scanning galvanometer 121 generally includes at least one reflective surface, and the light beam can be reflected in different directions by controlling the angle of the reflective surface. In the imaging module 120, the scanning galvanometer 121 can change the angle of the excitation light so that multiple photons of the excitation light converge to a point on the focal plane, thereby realizing multi-photon activation at the position, so as to collect the fluorescence signal generated by the imaging object 20 at the position based on the multi-photon phenomenon. And by changing the angle of the excitation light, the convergence point of multiple photons traverses the focal plane, thereby realizing multi-photon microscopic imaging of each position on the focal plane.

[0053] In some embodiments, for the convenience of integration inside the imaging probe 100, the scanning galvanometer 121 can be an electrically controlled galvanometer based on a micro-electromechanical system. Its specific implementation structure can be determined according to the application scenario. For example, the scanning galvanometer 121 can include at least one of an electromagnetic micro-electromechanical system galvanometer, an electrostatic micro-electromechanical system galvanometer, and a thermoelectric micro-electromechanical system galvanometer.

[0054] In some embodiments, in order to ensure that the wavefront of the excitation light at the focal plane has a good imaging effect, the imaging probe 100 may further be provided with an adaptive optical module 130 between the optoelectronic composite cable 300 and the imaging module 120 .

[0055] The adaptive optical module 130 may be a set of optical devices that can realize pre-compensation of the excitation light. That is, after passing through the adaptive optical module 130, the wavefront of the excitation light may be in a preset shape, so that when the excitation light reaches the target focus position inside the imaging object 20, the influence of the imaging object 20 on the excitation light wavefront is offset by the preset shape, so that the wavefront of the excitation light in the focal plane of the target focus position is similar to a plane wavefront, so that the excitation light has better penetration and excitation intensity (excitation efficiency).

[0056] like Figure 2 As shown, the adaptive optical module 130 may include a wavefront adjuster 131 for adjusting the wavefront and two lens groups 132 for optical coupling with the scanning galvanometer.

[0057] The wavefront adjuster 131 may be an optical device that can change the wavefront of the light beam incident into the wavefront adjuster 131 based on the wavefront control parameter. Among them, the optical device that can change the wavefront of the light beam can generally be called a spatial light modulator.

[0058] In the present specification, the wavefront adjuster 131 may be a small spatial light modulator that can be integrated into the imaging probe 100. In some embodiments, the wavefront adjuster 131 includes one of a micro deformable mirror and a liquid crystal spatial light modulator.

[0059] Taking a micro deformable mirror as an example, when the light beam enters the wavefront adjuster 131, the deformable mirror array on the micro deformable mirror can deform in response to the wavefront control parameter, so that the light beam is emitted through the deformable mirror array to change the wavefront of the light beam.

[0060] In some embodiments, a collimation module may be provided at the connection between the optoelectronic composite cable 300 and the wavefront adjuster 131 to collimate the excitation light transmitted by the optoelectronic composite cable 300 so that it has a relatively flat wavefront. If the collimation module is not provided, the deformation of the optoelectronic composite cable 300 itself may produce a relatively complex wavefront, thereby increasing the difficulty of adjusting the adaptive optics.

[0061] In some embodiments, the wavefront adjuster 131 can determine the second wavefront control parameter (target wavefront control parameter) based on the first wavefront control parameter group (multiple candidate wavefront control parameters), thereby achieving wavefront adjustment with the focal plane located at the target focal depth. For more information on determining the second wavefront control parameter based on the first wavefront control parameter group, please refer to Fig. 9 , 10 Related description.

[0062] The two lens groups 132 can be used to optically couple the wavefront adjuster 131 and the scanning galvanometer 121 in a 4f-conjugated form. The two lens groups 132 can include two lens groups (such as a first lens group 1321 and a second lens group 1322).

[0063] Each lens group in the two lens groups 132 (such as the first lens group 1321 and the second lens group 1322) can refer to a combination of optical devices that can realize the function of a lens. For example, the first lens group 1321 and the second lens group 1322 can both be double-cemented lens groups. The optical properties of each lens group correspond to the lens, for example, they all have optical properties such as focus, focal length, front and rear focal planes, etc.

[0064] In 4f conjugation, f refers to the focal length of the lens group. In the 4f conjugation system formed by the two lens groups 132, the focal lengths of the first lens group 1321 and the second lens group 1322 are equal within the tolerance range. The distance between the first lens group 1321 and the second lens group 1322 is 2 times the focal length, the wavefront adjuster 131 is located at the front focal plane of the first lens group 1321, and the scanning galvanometer 121 is located at the back focal plane of the second lens group 1322.

[0065] In some embodiments, if the scanning galvanometer 121 and the wavefront adjuster 131 are directly optically coupled, the scanning galvanometer 121 and the wavefront adjuster 131 are generally set at 45 degrees. In this case, two lens groups 132 need to be tilted between the scanning galvanometer 121 and the wavefront adjuster 131. To ensure the stability of the adaptive optical module 130, the adaptive optical module 130 may also include multiple optical elements to adjust the internal optical path so that the optical path is arranged in the horizontal and vertical directions. For more information on the specific optical path and its optical elements, please refer to Figure 3-Figure 7 and its related description.

[0066] Based on the imaging probe provided in this specification, an adaptive optical module is integrated into the optical system of a micro-multiphoton microscope through 4f conjugation, wherein the adaptive optical module can adjust the wavefront of the excitation light, thereby overcoming the wavefront distortion at different focusing depths, so that the excitation light has a well-shaped wave plate at the focal plane, thereby improving the penetration rate and excitation rate of the imaging object, thereby improving the imaging quality at each focusing depth.

[0067] In some embodiments, Figure 2 As shown, the imaging module 120 may further include an objective lens 122 and a translation stage 123 .

[0068] In some embodiments, the excitation light can be focused on the focal plane by the objective lens 122. When the excitation light transmitted in the optoelectronic composite cable 300 is controlled by the scanning galvanometer and emitted from the objective lens 122, the processed excitation light forms a stable focal plane. Each emitted excitation light includes a plurality of photons, and the plurality of photons are focused on a focal position of the focal plane, thereby activating the imaging object 20 corresponding to the focal position.

[0069] In some embodiments, the objective lens 122 may be in contact with the imaging object 20 through a dielectric component. The focal plane may be at a fixed preset distance from the objective lens 122.

[0070] Considering that the relative positions of the focal plane and the objective lens 122 are fixed, the focal plane position within the imaging object 20 can be adjusted by adjusting the position of the objective lens 122 , thereby imaging the internal structure of the imaging object 20 .

[0071] In some embodiments, considering that the imaging module 120 itself is based on micro laser scanning microscopy technology, when the position of the objective lens 122 is adjusted alone, other internal optical elements are difficult to cooperate with the adjustment. Therefore, the imaging module 120 can be moved as a whole in the handle-shaped housing 110 to adjust the focal plane position. That is, the imaging module 120 also includes a displacement stage 123. The displacement stage 123 is used to control the imaging module 120 to move as a whole in the handle-shaped housing 110 to adjust the focal plane position of the imaging module 120.

[0072] In some embodiments, to prevent the movement of the imaging module 120 from affecting the coupling relationship between the wavefront adjuster 131 and the scanning galvanometer 121 , the adaptive optics module 120 may be fixedly connected to the imaging module 120 and move together with the imaging module 120 .

[0073] Exemplary excitation light path:

[0074] Figure 3 - Figure 5 Schematic diagram of three excitation light paths of an imaging probe provided by an exemplary embodiment of this specification. Figure 3 Mainly, the excitation light path for releasing excitation light inside the imaging probe is explained.

[0075] exist Figure 3 The optical path structure shown may be a vertical cross-sectional view of the imaging probe 100. Figure 3 The first direction and the second direction may be included in Figure 3 In the embodiment, the second direction may be the direction in which the optoelectronic composite cable 300 releases the excitation light, or may be used as Figure 3 The first direction may be a direction in the plane that is perpendicular to the second direction, or may be a Figure 3 Horizontal direction in .

[0076] It should be noted that in Figure 3-Figure 5 In the embodiment, the optoelectronic composite cable 300 releases the excitation light, which is processed by the wavefront adjuster 131 and the scanning galvanometer 121, and then enters the imaging object 20 through the objective lens 122 to form an excitation light path. The light path of the objective lens 122 part in the excitation light path can be adjusted according to the fluorescence light path. For details, see Figure 6 , Figure 7 For the related description of , only the separate excitation light path is explained here.

[0077] In some embodiments, the internal light path can be adjusted through optical elements such as a polarization splitter and a quarter-wave plate, so that the scanning galvanometer 121 and the wavefront adjuster 131 are set along the first direction or the second direction, so that the excitation light is incident on the scanning galvanometer 121 and the wavefront adjuster 131 in a nearly vertical manner, thereby facilitating the adjustment of relevant parameters of the scanning galvanometer 121 and the wavefront adjuster 131.

[0078] A polarizing beam splitter (PBS) is an optical device used to separate light of different polarization states from an incident light beam. A light beam in a first polarization state can pass through the polarizing beam splitter, and a light beam in a second polarization state is reflected by the polarizing beam splitter. The first polarization state is generally orthogonal to the second polarization state.

[0079] A quarter-wave plate (QWP) is a birefringent single-crystal wave plate of a certain thickness. When light passes through the wave plate from a normal incidence direction, the quarter-wave plate can change the polarization state of the light beam.

[0080] In some embodiments, the adaptive optical module 130 may further include an optical element for arranging the wavefront adjuster 131 along the first direction, wherein the optical element for arranging the wavefront adjuster 131 along the first direction may include a first polarization beam splitter 133 and a first quarter wave plate 134 .

[0081] like Figure 3 As shown, the first polarization splitter 133 can be vertically arranged below the optoelectronic composite cable 300 along the second direction in the optoelectronic composite cable 300, the wavefront adjuster 131 is arranged along the first direction at the same height as the first polarization splitter 133, and the first quarter wave plate 134 is arranged between the wavefront adjuster 131 and the first polarization splitter 133.

[0082] The polarization state of the excitation light transmitted by the optoelectronic composite cable 300 is the second polarization state of the first polarization beam splitter 133 , that is, after the excitation light reaches the first polarization beam splitter 133 , it is reflected by the first polarization beam splitter 133 and propagates toward the wavefront adjuster 131 along the first direction.

[0083] The excitation light reflected by the first polarization beam splitter 133 enters the wavefront adjuster 131 through the first quarter wave plate 134, and is adjusted by the wavefront adjuster 131 to be emitted along the incident direction, and then passes through the first quarter wave plate 134 to reach the first polarization beam splitter 133. At this time, the polarization state of the excitation light changes from the second polarization state to the first polarization state through the first quarter wave plate 134, and can be directly projected into the first polarization beam splitter 133 and enter the two lens groups 132.

[0084] Among them, when the excitation light transmitted by the optoelectronic composite cable 300 is linearly polarized light, when it passes through the first surface of the first quarter wave plate 134, the angle between the polarization direction of the excitation light and the fast axis and the slow axis of the first quarter wave plate 134 can be 45°, and the excitation light will become circularly polarized light after passing through the first quarter wave plate 134. The circularly polarized light is adjusted by the wavefront adjuster 131 and passes through the second surface of the first quarter wave plate 134. At this time, the excitation light will become linearly polarized light again. The current polarization direction of the excitation light (the polarization direction after passing through the second surface of the first quarter wave plate 134) is orthogonal to the initial linear polarization direction (the polarization direction before passing through the first surface of the first quarter wave plate 134).

[0085] Based on the adjustment of the optical path by the first polarization beam splitter 133 and the first quarter wave plate 134, the wavefront adjuster 131 is located at the first focal plane of the two lens groups 132 to achieve 4f conjugation. The first focal plane of the two lens groups 132 may refer to the front focal plane of the first lens group 1321. The adjustment of the optical path by the first polarization beam splitter 133 and the first quarter wave plate 134 may be understood as being affected by the first polarization beam splitter 133 and the first quarter wave plate 134, and the wavefront adjuster 131 is equivalently located at the front focal plane of the first lens group 1321. For example, an optical path simulation may be performed based on the first polarization beam splitter 133 and the first quarter wave plate 134, and the theoretical position of the front focal plane of the first lens group 1321 may be determined in the simulated optical path, so that the wavefront adjuster 131 is placed at the theoretical position of the front focal plane of the first lens group 1321, thereby achieving 4f conjugation.

[0086] Based on the above settings, the excitation light can be incident vertically on the wavefront adjuster 131, which facilitates the adjustment of the excitation light by the wavefront adjuster 131 and also facilitates the quantification of the adjustment of the excitation light by the wavefront adjuster 131 to generate appropriate wavefront control parameters.

[0087] In some embodiments, the scanning galvanometer 121 may be arranged along the second direction based on an optical structure similar to the above. In this case, the adaptive optical module 130 may further include a second polarization beam splitter 135 and a second quarter wave plate 136 .

[0088] like Figure 3 As shown, the second polarization beam splitter 135 can be set along the first direction on the other side of the two lens groups 132, the scanning galvanometer 121 is set along the second direction and at the same horizontal position as the second polarization beam splitter 135, and the second quarter wave plate 136 is set between the scanning galvanometer 121 and the second polarization beam splitter 135.

[0089] The propagation of the excitation light by the above optical structure is similar to that of the aforementioned first polarization beam splitter 133 and the first quarter wave plate 134. The excitation light transmitted from the two lens groups 132 can be in the second polarization state of the second polarization beam splitter 135, so that it is reflected by the second polarization beam splitter 135 and propagates toward the scanning galvanometer 121 in the opposite direction of the second direction, and propagates to the surface of the scanning galvanometer 121 through the first surface of the second quarter wave plate 136, and is adjusted by the scanning galvanometer 121, and propagates to the second polarization beam splitter 135 through the second surface of the second quarter wave plate 136. At this time, the excitation light is in the first polarization state of the second polarization beam splitter 135 and can propagate outward through the second polarization beam splitter 135.

[0090] Based on the adjustment of the optical path by the second polarization beam splitter 135 and the second quarter wave plate 136, the scanning galvanometer 121 is located at the second focal plane of the two lens groups 132 to achieve 4f conjugation. The second focal plane of the two lens groups 132 may refer to the back focal plane of the second lens group 1322, that is, the scanning galvanometer 121 is located at the theoretical position of the back focal plane of the second lens group 1322, and the theoretical position may be a simulation result based on the adjustment of the optical path by the second polarization beam splitter 135 and the second quarter wave plate 136.

[0091] In some embodiments, considering that the excitation light transmitted by the first polarization beam splitter 133 is orthogonal to the polarization direction of the original state, when the second polarization beam splitter 135 is made of the same material and configured in the same manner as the first polarization beam splitter 133, the excitation light entering the second polarization beam splitter 135 is in the first polarization state of the second polarization beam splitter 135, i.e., it is directly projected through the second polarization beam splitter 135.

[0092] In some embodiments, the aforementioned adjustment structure can be adjusted based on this situation, so that the scanning galvanometer 121 is arranged along the first direction and at the same height as the second polarization beam splitter 135, so that the excitation light passing through the second surface of the second quarter-wave plate 136 is in the second polarization state of the second polarization beam splitter 135 and is reflected by the second polarization beam splitter 135 into the objective lens 122. The specific optical path is as follows Figure 4 shown.

[0093] In some embodiments, to ensure Figure 3 The optical path structure shown can also adjust the second polarization beam splitter 135 or related optical elements so that the excitation light entering the second polarization beam splitter 135 is in the second polarization state of the second polarization beam splitter 135 .

[0094] In some embodiments, the second polarization beam splitter 135 made of a material different from that of the first polarization beam splitter 133 may be directly selected, wherein the first polarization state of the first polarization beam splitter 133 and the second polarization beam splitter 135 is opposite to the second polarization state.

[0095] In some embodiments, the adaptive optical module 130 may further include a half glass plate 137 disposed between the two lens groups 132 and the second polarization beam splitter 135. The half glass plate 137 may be used to change the polarization state of the excitation light. The excitation light passing through the two lens groups 132 is changed from the first polarization state of the second polarization beam splitter 135 to the second polarization state. At this time, the second polarization beam splitter 135 is made of the same material and the same configuration as the first polarization beam splitter 133. The specific optical path is as follows: Figure 5 shown.

[0096] In some embodiments, the assembly method of the second polarization beam splitter 135 can also be changed. For example, the second polarization beam splitter 135 can be rotated with the first direction as the axis so that the excitation light passing through the two lens groups 132 is in the second polarization state of the second polarization beam splitter 135. For example, the second polarization beam splitter 135 can be rotated 90° (perpendicular to the page), and the excitation light of the two lens groups 132 is in the second polarization state of the rotated second polarization beam splitter 135.

[0097] Example imaging probe internal optical path:

[0098] Figure 6 , Figure 7 These are two schematic diagrams of overall optical paths of an imaging probe provided by an exemplary embodiment of this specification.

[0099] exist Figure 6 and Figure 7 In the embodiment, the imaging module 120 may include a beam splitter 124, wherein the beam splitter 124 is arranged along the axial direction of the objective lens 122 to separate the fluorescence light path and the activation light path. In the optoelectronic composite cable 300, the first optical fiber 310 releases the excitation light to form the excitation light path, and the second optical fiber 320 receives the fluorescence signal to form the fluorescence light path.

[0100] The fluorescence signal of the fluorescence light path and the excitation light of the activation light path are in the first polarization state and the second polarization state of the beam splitter 124 respectively, so that the fluorescence signal and the activation light path form different light paths, and the fluorescence light path and the activation light path can share the objective lens 122.

[0101] In some embodiments, the fluorescence signal is generally in the first polarization state of the beam splitter 124, and the excitation light is generally in the second polarization state of the beam splitter 124. That is, the fluorescence signal can directly pass through the beam splitter 124 to form a fluorescence light path in the axial direction of the objective lens 122, and the excitation light will be reflected by the beam splitter 124 and then enter the objective lens 122, so that the fluorescence light path and the excitation light path outside the objective lens 122 do not overlap.

[0102] exist Figure 6 In the above, the direction in which the excitation light enters the imaging probe 100 is parallel to the direction in which the fluorescent signal leaves the imaging probe 100. Figure 6 The reference coordinate system in Figure 3-Figure 5 The specific excitation light path setting relationship can be referred to Figure 3-Figure 5 The relevant description is not repeated here.

[0103] In some embodiments, the imaging module 120 may further include a reflector 125 arranged according to the positions of the scanning galvanometer 121 and the beam splitter 124. The reflector 125 is generally arranged at 45°. The reflector 125 is aligned with the beam splitter 124 in a first direction and aligned with the scanning galvanometer 121 in a second direction. Thus, the excitation light emitted from the scanning galvanometer 121 can pass through the reflector 125 and then enter the beam splitter 124 and the objective lens 122.

[0104] Based on the above-mentioned reflector 125, the direction and position of the excitation light passing through the scanning galvanometer 121 entering the objective lens 122 can be changed, so that the excitation light path and the fluorescence light path are staggered, thereby ensuring that the excitation light path and the fluorescence light path do not interfere with each other.

[0105] like Figure 6 As shown, the horizontal position of the reflector 125 is consistent with the scanning galvanometer 121, and the vertical position is consistent with the beam splitter 124. The reflector 125 can be used to reflect the excitation light passing through the scanning galvanometer 121 to the beam splitter 124, thereby offsetting the excitation light path and the fluorescence light path in the horizontal direction.

[0106] exist Figure 7 In the embodiment, the direction in which the excitation light enters the imaging probe 100 is perpendicular to the direction in which the fluorescent signal leaves the imaging probe 100. Figure 7 The second direction may be the direction in which the optoelectronic composite cable 300 releases the excitation light, or may be used as Figure 7 The first direction may be a direction perpendicular to the second direction in the plane, and may be used as Figure 7 The vertical direction in .

[0107] exist Figure 7 In the figure, when the excitation light enters the imaging probe 100 in the horizontal direction, the propagation direction of the excitation light is changed to the vertical direction through the first polarization beam splitter 133 and enters the wavefront adjuster 131 through the first quarter wave plate 134. After being adjusted by the wavefront adjuster 131, the excitation light propagates in the vertical direction to the second polarization beam splitter 135. After the excitation light passes through the second polarization beam splitter 135, the propagation direction is changed to the horizontal direction again, and the excitation light passes through the second quarter wave plate 136 to scan the galvanometer 121. The excitation light enters the beam splitter 124 and the objective lens 122 in the horizontal direction through the scanning galvanometer 121, thereby staggering the excitation light path and the fluorescence light path in the horizontal direction.

[0108] It should be noted that Figure 6 and Figure 7 The optical path can be adjusted according to actual needs, as long as the excitation light path and the fluorescence light path are staggered. Figure 7 The positions of the middle wavefront adjuster 131 and the scanning galvanometer 121 in the vertical direction can be exchanged and the corresponding optical devices can be adjusted accordingly. Figure 6 and Figure 7 The vertical and horizontal directions in are not limited to the positive directions. For example, Figure 7 When the excitation light enters the imaging probe 100 along the horizontal direction, the propagation direction of the excitation light is changed to the vertical direction by the first polarization beam splitter 133. It can be understood that the propagation direction of the excitation light is changed to the positive direction or the reverse direction of the vertical direction. The specific propagation direction only needs to ensure that the excitation light path and the fluorescence light path are staggered.

[0109] In addition, it should be noted that based on Figure 7 As shown in the fluorescent light path, the internal light path of the imaging probe 100 matches the probe shape, which can further improve the integration of the imaging probe 100.

[0110] Exemplary Imaging Methods:

[0111] Figure 8 is an exemplary flow chart of a multiphoton microscopic imaging method provided by an exemplary embodiment of this specification. In some embodiments, Figure 8 The process P800 shown is generally performed by a computing device (such as a signal control and processing module in the imaging host 200). In some embodiments, based on the split design of the aforementioned imaging device 10, the signal control and processing module in the imaging host 200 can control the imaging probe 100 through the optoelectronic composite cable 300, thereby implementing P800.

[0112] For the convenience of description, the optoelectronic composite cable 300 is used as the execution subject to describe P800.

[0113] like Figure 8 As shown, P800 includes the following steps:

[0114] S810, sending a displacement signal to the translation stage, so that the translation stage controls the position of the focal plane to a target focusing depth based on the displacement signal.

[0115] The displacement signal may refer to a control signal for controlling the position of the displacement stage. In some embodiments, the signal control and processing module may drive the imaging module to move by controlling the position of the displacement stage. Based on the fixed relative distance between the imaging module (objective lens) and the focal plane, when the channel controls the displacement stage to move, the focal plane may be driven to move synchronously to change the position of the focal plane.

[0116] In some embodiments, the imaging task of the multiphoton imaging device is generally embodied as imaging at each target focus depth within the imaging depth range to obtain a sequence of fluorescence images of the imaging object, thereby realizing analysis of the imaging object. The signal control and processing module can generally gradually adjust the position of the displacement stage along the direction of the excitation light release, so that the focal plane position is at each target focus depth.

[0117] When the focal plane position is at a target focal depth, the signal control and processing module can control the imaging probe to perform multiphoton imaging at the target focal depth. After completing the imaging at the target focal depth, the signal control and processing module can continue to control the translation stage along the release direction of the excitation light, so that the focal plane position is at the next target focal depth.

[0118] The target focus depth may refer to the depth within the imaging depth range of the imaging task where multiphoton imaging is required. In some embodiments, the target focus depth may be determined based on the actual structure of the imaging object. For example, when imaging human facial skin, the target focus depth may refer to the depth within the range that has imaging significance. Exemplarily, the human skin structure generally includes epidermis, dermis, and subcutaneous tissue. The target focus depth may at least include the depth that can reflect the structure of each layer.

[0119] In some embodiments, the target focus depth referred to in P800 can also be understood as the focus depth at which the excitation light wavefront needs to be adjusted. For example, when the image indicators (such as fluorescence signal intensity, image clarity, etc.) of the fluorescence image collected at the current focus depth do not meet the preset indicators, the current focus depth can be used as the target focus depth to execute P800, so that the wavefront adjuster in the imaging probe can change the beam wavefront of the excitation light based on the wavefront control parameters corresponding to the imaging effect at the target focus depth, thereby optimizing the imaging effect of the fluorescence image at the target focus depth.

[0120] In some embodiments, the image index of the fluorescence image may be the excitation efficiency. For example, the fluorescence intensity may be determined by the fluorescence signal corresponding to the fluorescence image, thereby estimating the excitation efficiency of the excitation light at this time. When the excitation efficiency is lower than a threshold, the current focus depth is used as the target focus depth.

[0121] S820, sending a scanning signal to the scanning galvanometer, so that the scanning galvanometer controls the excitation light to perform multi-photon microscopic imaging on the imaging object.

[0122] The scanning signal may refer to a control signal for controlling the reflection angle of the scanning galvanometer. In some embodiments, the signal control and processing module may control the focus position of the excitation light in the focal plane by controlling the reflection angle of the scanning galvanometer. When the signal control and processing module controls the scanning galvanometer to present various reflection angles, the excitation light traverses the focal plane to complete two-photon imaging at the current focal depth.

[0123] In some embodiments, the aforementioned scanning signal can be coupled with the aforementioned displacement signal, and after adjusting the focal plane position, the scanning galvanometer is controlled to scan, that is, the displacement signal and the scanning signal can be generated based on the response time of the translation stage to the displacement signal.

[0124] S830, sending a first wavefront control parameter group to a wavefront adjuster, so that the wavefront adjuster adjusts the wavefront of the excitation light based on the first wavefront control parameter group to generate a corresponding first fluorescence signal group.

[0125] The first wavefront control parameter group may be a set of first wavefront control parameters. That is, the first wavefront control parameter group may include multiple first wavefront control parameters. The first wavefront control parameters may be used to test the influence of different wavefront shapes of the excitation light on the imaging effect of the target focus position.

[0126] In some embodiments, the first wavefront control parameter group can be determined based on an optimization method. Before determining the first wavefront control parameter group, a mapping relationship between the wavefront control parameter and the fluorescence signal can be constructed based on historical data. Then the current fluorescence signal is input into the mapping relationship to determine multiple possible wavefront control parameters as the first wavefront control parameter group. The mapping relationship can be implemented based on a conventional modeling algorithm. For example, the modeling algorithm can be a data comparison table, a regression algorithm, a neural network algorithm, etc. It will not be described in detail here.

[0127] In some embodiments, the first wavefront control parameter group can also be determined based on a model-free optimization method based on perturbation analysis (such as a model-free optimization based on Zernike). The model-free optimization method based on perturbation analysis may refer to directly applying perturbations to the first wavefront control parameters without building a model, analyzing the changes in the corresponding first fluorescence signal, and then iterating. For more information about determining the first wavefront control parameter group based on an iterative algorithm, please refer to Fig. 9 and its related description.

[0128] In some embodiments, for each wavefront control parameter in the first wavefront control parameter group, the wavefront adjuster may first respond to the current wavefront control parameter, and after the wavefront adjuster completes the response, the scanning galvanometer is controlled to execute the scanning signal to determine the corresponding first fluorescent signal. After the first scanning signal is executed, the wavefront adjuster is made to respond to the next wavefront control parameter until the response to the first wavefront control parameter group is completed to determine the corresponding first fluorescent signal group.

[0129] The first fluorescence signal may refer to a set of fluorescence signals excited by the excitation light at various positions on the focal plane after adjustment of the first wavefront control parameter. The first fluorescence signal group may refer to a set of first fluorescence signals determined based on various first wavefront control parameters. The first fluorescence signals correspond one to one with the first wavefront control parameters.

[0130] In some embodiments, the first fluorescent signal may reflect the imaging effect of the target focus position. For example, the excitation efficiency of the excitation light at the target focus position based on the corresponding first fluorescent signal may be determined by the signal intensity of the first fluorescent signal.

[0131] S840, sending a second wavefront control parameter to the wavefront adjuster to generate a second fluorescence signal.

[0132] The second wavefront control parameter may refer to a wavefront control parameter determined by the signal control and processing module that can produce a better imaging effect, wherein the second wavefront control parameter is determined based on the first wavefront control parameter group and the first fluorescence signal group.

[0133] In some embodiments, the second wavefront control parameter can be determined based on some wavefront control parameters with better display effects in the first wavefront control parameter group. For example, the second wavefront control parameter can be the wavefront control parameter with the highest excitation efficiency in the first wavefront control parameter group.

[0134] In some embodiments, the second wavefront control parameter may also be a wavefront control parameter when the imaging effect (corresponding to the first fluorescence signal) satisfies a preset termination condition during the iteration of the first wavefront control parameter group. For example, when the imaging effect (such as excitation efficiency) corresponding to the current wavefront control parameter is greater than a preset termination threshold (such as an excitation efficiency threshold), the iteration may be stopped and the current wavefront control parameter may be used as the second wavefront control parameter.

[0135] The second fluorescence signal may refer to a fluorescence signal determined based on the second wavefront control parameter. In some embodiments, the second fluorescence signal may be used to generate a fluorescence image of the target focus position.

[0136] In some embodiments, in order to further fine-tune the wavefront of the excitation light, the imaging effect of the focal plane can be partitioned, and each imaging area can be adjusted separately. At this time, the second wavefront control parameter may include the wavefront control parameters of multiple imaging areas. Taking into account that the wavefront adjuster has a response time to the wavefront control parameter, when performing the scan, the scanning batches can be delineated according to the wavefront control parameters, and each batch only scans the areas with the same wavefront control parameters. To adapt to the scanning method, the scanning can be performed based on the second scanning signal, wherein the second scanning signal includes scanning signals corresponding to multiple imaging areas. That is, when executing the second scanning signal, it can be coordinated with the aforementioned wavefront adjustment, that is, after completing the wavefront adjustment of each area, a corresponding second scanning signal is generated to scan the corresponding partition.

[0137] Based on the above multiphoton microscopy imaging method, during the multiphoton microscopy imaging method, the adaptive optics module can generate a suitable wavefront control parameter group for each target focus position. This process does not require the detection of the actual wavefront situation, and on the basis of ensuring the imaging effect, it greatly expands the types of imaging objects. For example, for living samples, imaging can also be performed directly.

[0138] Exemplary first wavefront control parameter determination method:

[0139] Fig. 9 It is an exemplary flow chart of determining a first wavefront control parameter group based on an iterative algorithm provided by an exemplary embodiment of this specification.

[0140] In some embodiments, Fig. 9 The process P900 shown may be a process of processing a certain wavefront control parameter (referred to as a target wavefront control parameter) in the first wavefront control parameter group during the first wavefront control parameter iteration process.

[0141] In some embodiments, P900 can be constructed based on a model-free optimization method based on perturbation analysis. The model-free optimization method based on perturbation analysis can be an iterative method, in which perturbations can be applied to the input data (target wavefront control parameters) of the current round based on the changes in the output data (target fluorescence signal) in each iteration, thereby optimizing the output data until the output data converges.

[0142] It should be noted that in the multiphoton imaging process, there must be an optimal solution for the wavefront interference at the target focal depth, so that the wavefront of the excitation light is similar to a plane wavefront when it reaches a certain focus position at the target focal depth. Therefore, in the iterative process of P900, the output data (target fluorescence signal) must converge.

[0143] like Fig. 9 As shown, for the target wavefront control parameters of the first wavefront control parameter group, P900 includes the following steps:

[0144] S910, sending a target wavefront control parameter to a wavefront adjuster and sending a corresponding target scanning signal to a scanning galvanometer to determine a target fluorescence signal corresponding to the target wavefront control parameter.

[0145] The target wavefront control parameter may refer to the first wavefront control parameter of the current iteration cycle. For ease of description, the target wavefront control parameter may be denoted as ai. Then the target scanning signal may refer to the scanning signal corresponding to the target wavefront control parameter. Among them, the target scanning signal is generally consistent with the first scanning signal. The target fluorescence signal may refer to the fluorescence signal determined based on the target wavefront control parameter.

[0146] In some embodiments, in the first iteration cycle, the value of the target wavefront control parameter a1 may be the wavefront control parameter when not adjusted. In the imaging process, the influence of the imaging object on the wavefront increases with the depth of the focus position, and the wavefront control parameter when not adjusted at the target focus position may be understood as the second wavefront control parameter at the previous focus position.

[0147] In some embodiments, in order to reduce the number of iterations, the target wavefront control parameter a1 may also be determined based on historical data or an artificial intelligence algorithm.

[0148] In some embodiments, the target wavefront control parameter ai (i>5) can be determined according to the target wavefront control parameter ai-1 and the target fluorescence signal bi-1 of the previous round. For details, please refer to the relevant description of S940.

[0149] S920, determining whether the target fluorescent signal meets a preset image quality index.

[0150] The image quality index may refer to a threshold index that the target fluorescence signal should meet when the iteration is stopped. In some embodiments, the image quality index may be understood as the aforementioned preset termination condition.

[0151] In some embodiments, based on the above discussion on convergence, the image quality index can be characterized as a limit on the change amount of the target fluorescence signal, such as a change amount threshold. When the change amount of the target fluorescence signal is less than the change amount threshold, the target fluorescence signal is deemed to meet the preset image quality index. Otherwise, it is deemed not to meet the preset image quality index.

[0152] In some embodiments, when the target fluorescence signal has no convergence trend after multiple iterations, the image quality indicator may also be an iteration number threshold. When the iteration number is greater than the iteration number threshold, the iteration may be stopped, and the wavefront control parameter with the best imaging effect of the target fluorescence signal may be used as the second wavefront control parameter.

[0153] S930: If yes, the target wavefront control parameter is used as the second wavefront control parameter, and the target fluorescence signal is used as the second fluorescence signal.

[0154] The aforementioned S920 and S930 can be understood as that when the target fluorescence signal meets the preset image quality index, the iteration can be stopped, and the target wavefront control parameter is used as the second wavefront control parameter and the target fluorescence signal is used as the second fluorescence signal.

[0155] S940: If not, determine the target wavefront control parameters for the next iteration based on the target fluorescence signal and the target wavefront control parameters, and resend them to the wavefront adjuster.

[0156] The aforementioned S920 and S940 can be understood as that when the target fluorescence signal does not meet the preset image quality index, the target wavefront control parameter ai+1 of the next iteration can be determined and i=i+1 is used to continue the iteration.

[0157] In some embodiments, the target wavefront control parameter ai+1 can be determined based on the target fluorescence signal bi and the target wavefront control parameter ai. Exemplarily, for the target wavefront control parameter a1 of the first iteration, the target wavefront control parameter a2 of the second iteration can be randomly determined. For the target wavefront control parameter ai of the i-th iteration, the change direction of the target wavefront control parameter when the target fluorescence signal increases can be determined based on the relationship between the change value ai-ai-1 of the target wavefront control parameter and the change value bi-bi-1 of the target fluorescence signal, and the target wavefront control parameter ai+1 of the i+1 iteration can be determined based on the change direction.

[0158] Based on the first wavefront control parameter group determined above, there is no need to model the mapping relationship between the wavefront control parameters and the fluorescence signal. In practice, the overall iteration process is faster, the calculation is simpler, and the calculation load is less.

[0159] It should be noted that the methods shown in P800 and P900 can be separated from the actual imaging process. For example, the method of P800 or P900 can be executed on a standard imaging object to determine the second wavefront adjustment parameter, and the second wavefront adjustment parameter can be directly called in the actual imaging. When the second wavefront adjustment parameter of the standard imaging object cannot adapt to the current imaging task, P800 or P900 is re-executed.

[0160] Exemplary Imaging Devices:

[0161] Fig.10 It is an exemplary structural diagram of a multiphoton microscopic imaging device provided by an exemplary embodiment of this specification.

[0162] like Fig.10 As shown, the multiphoton microscopic imaging device 10 may include an imaging host 200 and a multiphoton microscopic imaging probe 100, and the imaging host 200 and the multiphoton microscopic imaging probe 100 are connected via an optoelectronic composite cable 300. The internal structure of the multiphoton microscopic imaging probe 100 can be seen in the aforementioned Figure 3 The relevant description is not repeated here.

[0163] The imaging host 200 may be a collection of components in the imaging device 10 that cannot or is not conveniently integrated into the imaging probe 100. For example, the imaging host 200 may be used to generate excitation light and generate a fluorescence image based on a fluorescence signal.

[0164] like Fig.10As shown, the imaging host 200 may specifically include a power supply 210, a laser light source 220, a collection module 230, and a signal control and processing module 240. The power supply 210 may be used to provide an operating voltage for the imaging device 10. The laser light source 220 may be used to generate excitation light. The collection module 230 may be used to generate a fluorescent image based on a fluorescent signal. The signal control and processing module 240 is used to generate a control signal for controlling the imaging probe 100.

[0165] Those of ordinary skill in the art will appreciate that the units and algorithm steps of each example 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 performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0166] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0167] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms.

[0168] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0169] In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit.

[0170] If the function is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present application can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product is stored in a storage medium, including several instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present application. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk, and other media that can store program check codes.

[0171] It should be noted that, in the description of this application, the terms "first", "second", "third", etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance. In addition, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0172] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A multiphoton microscopic imaging probe based on adaptive optics, characterized in that: The imaging probe comprises: An optoelectronic composite cable for connecting to an imaging host; wherein the optoelectronic composite cable is used to transmit excitation light and transmit fluorescence signals; A handle-shaped housing, wherein the optoelectronic composite cable is connected to a first end of the handle-shaped housing; An imaging module based on micro laser scanning microscopy technology, wherein the imaging module is disposed in the handle-shaped housing and connected to the optoelectronic composite cable, and is used to transmit and control the excitation light and receive the fluorescence signal, and the imaging module includes a scanning galvanometer for controlling the excitation light; and An adaptive optical module, wherein the adaptive optical module is arranged between the optoelectronic composite cable and the imaging module; wherein the adaptive optical module includes a wavefront adjuster and two lens groups, wherein the wavefront adjuster and the scanning galvanometer form a 4f conjugate through the two lens groups, and the wavefront adjuster can change the wavefront of the light beam incident on the wavefront adjuster based on the wavefront control parameters.

2. The imaging probe according to claim 1, characterized in that: The adaptive optics module also includes: a first polarization beam splitter disposed between the optoelectronic composite cable and the wavefront adjuster, for converting the direction of the excitation light so that the excitation light propagates along a first direction to the wavefront adjuster, and after adjustment, propagates outward through the first polarization beam splitter in a direction opposite to the first direction; and A first quarter wave plate disposed between the wavefront adjuster and the first polarization beam splitter, for changing the polarization state of the excitation light; Wherein, based on the adjustment of the optical path by the first polarization beam splitter and the first quarter-wave plate, the wavefront adjuster is located at the first focal plane of the two lens groups to achieve 4f conjugation.

3. The imaging probe according to claim 2, characterized in that: The adaptive optics module also includes: a second polarization beam splitter disposed between the two lens groups and the scanning galvanometer, for converting the direction of the excitation light so that the excitation light propagates to the scanning galvanometer in the opposite direction of the second direction and propagates outward through the second polarization beam splitter in the second direction after adjustment; and A second quarter wave plate disposed between the scanning galvanometer and the second polarization beam splitter, for changing the polarization state of the excitation light; Wherein, based on the adjustment of the optical path by the second polarization beam splitter and the second quarter-wave plate, the scanning galvanometer is located at the second focal plane of the two lens groups to achieve 4f conjugation.

4. The imaging probe according to claim 3, characterized in that: The adaptive optical module also includes a half glass plate arranged between the two lens groups and the second polarization beam splitter, which is used to change the polarization state of the excitation light.

5. The imaging probe according to claim 2, characterized in that: The wavefront adjuster includes one of a micro deformable mirror and a liquid crystal spatial light modulator; the scanning galvanometer includes at least one of an electromagnetic micro-electromechanical system galvanometer, an electrostatic micro-electromechanical system galvanometer, and a thermoelectric micro-electromechanical system galvanometer.

6. The imaging probe according to claim 1, characterized in that: The imaging module further comprises a displacement stage, and the displacement stage is used to control the overall movement of the imaging module in the handle-shaped housing to adjust the focal plane position of the imaging module.

7. The imaging probe according to claim 6, characterized in that: The optoelectronic device further includes a control bus connected to the translation stage, the scanning galvanometer and the wavefront adjuster, and the control bus is used to: sending a displacement signal to the translation stage, so that the translation stage controls the position of the focal plane to a target focusing depth based on the displacement signal; Sending a scanning signal to the scanning galvanometer so that the scanning galvanometer controls the excitation light to perform multi-photon microscopic imaging on an imaging object; Sending a first wavefront control parameter group to the wavefront adjuster, so that the wavefront adjuster adjusts the wavefront of the excitation light based on the first wavefront control parameter group to generate a corresponding first fluorescence signal group, wherein the first wavefront control parameter group includes a plurality of first wavefront control parameters, the first fluorescence signal group includes a plurality of first fluorescence signals, and the first fluorescence signals correspond to the first wavefront control parameters one by one; A second wavefront control parameter is sent to the wavefront adjuster to generate a second fluorescence signal, wherein the second wavefront control parameter is determined based on the first wavefront control parameter group and the first fluorescence signal group.

8. The imaging probe according to claim 7, characterized in that: The control bus is also used for: For the target wavefront control parameters of the first wavefront control parameter group, sending the target wavefront control parameter group to the wavefront adjuster and sending a corresponding target scanning signal to the scanning galvanometer to determine a target fluorescence signal corresponding to the target wavefront control parameter; Wherein, when the target fluorescence signal meets the preset image quality index, the target wavefront control parameter is used as the second wavefront control parameter, and the target fluorescence signal is used as the second fluorescence signal; When the target fluorescence signal does not meet the preset image quality index, the target wavefront control parameter is iterated based on the target fluorescence signal, the iterated target wavefront control parameter is used as the target wavefront control parameter again, and is resent to the wavefront adjuster.

9. The imaging probe according to claim 7, characterized in that: The second wavefront control parameters include wavefront control parameters of a plurality of imaging regions.

10. A multiphoton microscopic imaging device based on adaptive optics, characterized in that: The device comprises: an imaging host for generating excitation light and generating a fluorescence image based on the fluorescence signal; and The multi-photon microscopic imaging probe according to any one of claims 1 to 9, wherein the multi-photon microscopic imaging probe is connected to the imaging host via an optoelectronic composite cable, and is used to release the activation light to the imaging object and collect the fluorescence signal generated by the imaging object based on the photoluminescence effect.

Images