Imaging device based on multi-photon depth imaging and imaging probe thereof

Abstract

An imaging device (10) based on multi-photon depth imaging and an imaging probe (100) thereof. By using a split structure, a miniaturized imaging probe (100) is achieved. During imaging, the imaging probe (100) can be fixedly connected to an imaging object (20), and a multi-photon effect is excited inside the imaging object (20) by means of excitation light to acquire optical signals inside the imaging object (20), so as to detect internal conditions inside the imaging object (20).

Description

An imaging device and its imaging probe based on multiphoton depth imaging Technical Field

[0001] This application relates to the field of multiphoton imaging technology, specifically to an imaging device and its imaging probe based on multiphoton depth imaging. Background Technology

[0002] Multiphoton depth imaging is a technique for depth imaging based on the nonlinear optical effects of multiple photons. Specifically, multiphoton imaging utilizes various nonlinear phenomena and effects generated when multiple photons interact with matter simultaneously to produce corresponding optical signals, thereby performing depth scanning imaging. For example, the nonlinear optical effects of multiphoton depth imaging include second harmonic generation (SHG), third harmonic generation (THG), two-photon excited fluorescence (TPEF), three-photon excited fluorescence (3PEF), and coherent anti-Stokes Raman scattering (CARS).

[0003] In practical applications, because the internal optical path of multiphoton imaging equipment is complex, it often relies on large-scale equipment.

[0004] Summary of the Invention

[0005] Therefore, how to realize multiphoton imaging technology based on micro-devices and present good imaging results is a technical problem that urgently needs to be solved by those skilled in the art.

[0006] In view of this, the present application provides an imaging device that achieves probe miniaturization through a separate design of the probe and the host, while the input optical fiber used to transmit the excitation light has a target bandwidth that meets the requirements of multiphoton imaging and can carry composite lasers containing different wavelength components, thereby improving the imaging effect of the probe and solving the above-mentioned technical problems.

[0007] In a first aspect, this application provides an imaging probe that is applied to an imaging device based on multiphoton depth imaging. The imaging probe is connected to the imaging host of the imaging device via an optoelectronic composite cable. The imaging probe includes a housing, at least two input optical fibers, a coupling module, and an imaging device based on multiphoton depth imaging.

[0008] One end of the housing is provided with a first port for fixed connection with the organism to be tested, and the other end is provided with a second port, which is connected to the first port; wherein, at least two input optical fibers and a coupling module are sequentially arranged in the second port, and the imaging device is arranged at the first port.

[0009] The at least two input fibers, the coupling module and the imaging device form an excitation light path, wherein the at least two input fibers are used to provide at least two lasers of different wavelengths, the coupling module is used to combine the at least two lasers into a composite laser, and the imaging device is used to convert the composite laser into excitation light; the excitation light is focused on an internal focus position of the imaging object to trigger a multi-photon effect at the internal focus position; an optical signal generated by the multi-photon effect is collected; and the internal focus position is changed to determine the optical signal at each position in a focal plane, wherein the optical signal at each position in the focal plane is used to generate an optical image of the imaging object at the focal plane.

[0010] In a second aspect, the application provides an imaging device based on multi-photon depth imaging and an imaging probe thereof, wherein the imaging device comprises an imaging host, at least two lasers, a beam splitting module, at least two optical detectors and the imaging probe of the first aspect.

[0011] The imaging host contains at least two lasers, and the lasers are used to provide lasers meeting the imaging requirements and transmit the lasers to the imaging probe through corresponding output fibers in the imaging probe, wherein the wavelengths of the lasers emitted by each of the at least two lasers are different.

[0012] The imaging probe is internally provided with a coupling module and an imaging device based on multi-photon depth imaging, the coupling module is used to convert multiple lasers into a composite laser, and the imaging device is used to convert the composite laser into excitation light; the excitation light is focused on an internal focus position of the imaging object to trigger a multi-photon effect at the internal focus position; an optical signal generated by the multi-photon effect is collected; and the internal focus position is changed to determine the optical signal at each position in a focal plane, wherein the optical signal at each position in the focal plane is used to generate an optical image of the imaging object at the focal plane.

[0013] The beam splitting module is used to split the optical signal into at least two optical signal branches, and the optical detector is used to receive a corresponding optical signal branch and detect the signal intensity of the corresponding optical signal branch, wherein the optical signal branch reflects the signal component of the optical signal generated by the multi-photon effect at each wavelength, the optical signal branch corresponds to the optical detector one by one, and the signal intensity of the optical signal reflects the pixel value of a corresponding pixel in the optical image.

[0014] The application provides an imaging device based on multi-photon depth imaging and an imaging probe thereof. By adopting a split structure, a miniaturized imaging probe is realized. During imaging, the imaging probe can be fixedly connected with an imaging object, multi-photon effect is excited in the imaging object by excitation light, and optical signals in the imaging object are collected, so as to detect the internal condition of the imaging object. In particular, a plurality of input optical fibers are arranged in the imaging probe, and a plurality of wavelengths of laser light are simultaneously provided in the multi-photon imaging process, so that the optical signals can simultaneously include optical signal components responding to excitation light of different wavelengths, and different internal structures are simultaneously excited and detected. In addition, in the probe, the laser light of each wavelength is subjected to beam combination processing by a coupling module to form composite laser light, and the subsequent processing of the composite laser light is similar to that of the excitation light, and the internal structure of the imaging probe is further simplified. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Fig. 1 is an application scenario diagram of the imaging device provided by some embodiments of the present application.

[0017] Fig. 2 is a transmission schematic diagram of photoelectric signals in the imaging device provided by some embodiments of the present application.

[0018] Fig. 3 is a system block diagram of the imaging device provided by some embodiments of the present application.

[0019] Fig. 4 is another system block diagram of the imaging device provided by some embodiments of the present application.

[0020] Fig. 5 is a structural schematic diagram of the imaging probe provided by some embodiments of the present application.

[0021] Fig. 6 is another structural schematic diagram of the imaging probe provided by some embodiments of the present application.

[0022] Figs. 7A-7D are optical path schematic diagrams of the coupling module provided by some embodiments of the present application.

[0023] Fig. 8 is an optical path schematic diagram of parallel excitation light in the imaging probe provided by some embodiments of the present application.

[0024] Figs. 9A-9C are a plurality of structural schematic diagrams when the scanning galvanometer and the excitation light form an acute angle provided by some embodiments of the present application.

[0025] FIG. 10 is a structural schematic diagram of a zoom imaging probe according to some embodiments of the present application.

[0026] FIGS. 11A-11C are structural schematic diagrams of an optogenetic stimulation imaging probe according to some embodiments of the present application.

[0027] Reference signs: 10, imaging device; 20, imaging object; 100, imaging probe; 200, imaging host; 300, photoelectric composite cable; 210, laser group; 220, computing device; 410, beam splitting module; 420, optical detector group; 340, coupling module; 310, input fiber group; 211, laser; 311, input fiber; 320, control cable; 330, output fiber; 341, beam combining mirror; 342, beam combining element; 412, decoupling dichroic mirror; 413, decoupling mirror; 411, environmental filter; 414, beam splitting filter; 421, optical detector; 430, collection lens; 110, collimation module; 120, scanning module; 130, lens module; 140, objective module; 121, scanning galvanometer; 122, scanning mirror; 150, motorized zoom module; 160, optogenetic module; 161, optogenetic light source; 162, optogenetic beam combining element; 163, optogenetic beam merging element; 350, optogenetic fiber; 1611, optogenetic laser; 1612, beam combining element; 3421, polarization beam splitter; 3422, half-wave plate. DETAILED DESCRIPTION

[0028] As described above, in multi-photon imaging, the excitation light is often relied on to trigger multi-photon linear, thereby obtaining the corresponding optical signal. For example, second harmonic generation (SHG) is a second-order multi-photon phenomenon, in which two photons of the same frequency interact with a nonlinear material to combine into a new photon of twice the frequency. For another example, three-photon excitation fluorescence (3PEF) requires three photons to be excited simultaneously to generate a fluorescence signal.

[0029] In practical applications, based on the imaging characteristics of multi-photon, it is often applied to depth imaging, i.e., generally requires the excitation light containing multiple photons to be focused at a certain depth to trigger the corresponding multi-photon effect to generate the corresponding optical signal. Thus, in multi-photon, the frequency and power of the excitation light have certain requirements. In addition, further considering that multi-photon often relies on excitation light of a specific frequency band. Thus, in practical applications, multi-photon imaging can often be realized based on excitation light of a single frequency band. Especially in the process of miniaturizing the imaging device, considering the complexity of the internal space, it cannot be propagated.

[0030] To further illustrate the practical application of the miniaturized equipment of the multi-photon imaging technology, the application provides an application scenario diagram of an imaging equipment (Figure 1). The application scenario shown in Figure 1 can reflect the process of the imaging equipment performing optical brain imaging on a mouse.

[0031] As shown in Figure 1, the application scenario of the imaging equipment can include an imaging equipment 10 and an imaging object 20.

[0032] The imaging equipment 10 is the execution subject of multi-photon imaging (for example, the imaging equipment 10 can be configured as a multi-photon microscope), which can release excitation light to trigger a multi-photon effect (such as a multi-photon fluorescence effect) inside the imaging object 20, thereby collecting the optical signal of the imaging object 20 after being excited by the excitation light, and generating a fluorescence image of the imaging object 20.

[0033] Exemplarily, the imaging equipment 10 can focus multiple photons of excitation light on a point at a target focal depth inside the imaging object 20 based on the principle of multi-photon fluorescence imaging, to obtain the fluorescence signal of the point. Then, by changing the focusing position at the target focal depth through scanning technology to perform plane scanning, the fluorescence signals at various positions at the target focal depth inside the imaging object 20 are obtained, so as to determine the fluorescence image.

[0034] The imaging object 20 can refer to an object that needs to be subjected to multi-photon imaging. For example, the imaging object 20 can be an ex vivo sample, a living sample, etc. Exemplarily, the imaging object 20 in the application can be a mouse, a rabbit, a bird, a non-human primate macaque, a marmoset, etc. The imaging object 20 can generally be used to image the internal structure (brain nerves, spinal nerves, etc.) of the imaging object.

[0035] As shown in Figure 1, the imaging object 20 in Figure 1 can be a mouse, and the brain of the mouse can be subjected to multi-photon imaging. The imaging equipment 10 can be fixedly connected with the brain of the imaging object 20, and the imaging object 20 can be freely active. The imaging equipment 10 can periodically scan the imaging object 20 during its free activity, to obtain the optical signal of each position in a specific focal plane of the brain of the imaging object 20, thereby determining the time sequence optical image of the position. The optical image collected generally can include the neural synapses of the position.

[0036] It should be noted that before the imaging object 20 is fixedly connected with the imaging equipment 10, the imaging object 20 can be subjected to adaptive processing. For example, the fixed connection part of the imaging object 20 and the imaging equipment 10 can be subjected to skin preparation. For another example, for the mouse shown in Figure 1, a skull window can be arranged on the brain of the mouse during the fixed connection, to improve the imaging effect. For another example, a fluorescent probe can be injected into the imaging object 20 in advance, to perform multi-photon fluorescence imaging based on the fluorescent probe.

[0037] To miniaturize the pre-imaging device, the imaging device 10 provided in the specification can be in a split configuration, thereby further comprising an imaging probe 100 and an imaging host 200, wherein the imaging probe 100 and the imaging host 200 can be communicatively connected based on an opto-electric composite cable 300.

[0038] The imaging probe 100 can be a device that releases excitation light and detects optical signals. Among them, the imaging probe 100 can convert the laser generated by the laser source into excitation light focused to the focal plane and having scanning capability through the optical assembly. In some embodiments, the imaging probe 100 can be a contact imaging probe.

[0039] The imaging host 200 can be a set of devices that cannot be integrated into the imaging probe 100, and can be specifically used to provide laser and image based on optical signals. Among them, the imaging host 200 can generally include a laser, a fluorescence collection device, and a scanning acquisition controller.

[0040] The opto-electric composite cable 300 can be a communication cable between the imaging probe 100 and the imaging host 200, used to realize data transmission of optical signals and electrical signals between the imaging host 200 and the imaging probe 100. Among them, the opto-electric 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 optical signals collected by the imaging probe 100 to the imaging host 200, to form an optical image.

[0041] Based on the foregoing imaging device 10, multi-photon imaging (such as multi-photon fluorescence imaging) inside the imaging object 20 can be realized. However, considering that the spectral bandwidth of the optical fiber is limited at present, and the imaging device 10 often needs highly stable and high-power laser, the optical fiber can usually only transmit laser of a single wavelength. Therefore, the optical fiber transmitting excitation light in the imaging device 10 generally only transmits one excitation light of a specific wavelength, and the imaging device 10 often performs multi-photon imaging based on only one excitation light of a specific wavelength.

[0042] However, in actual application, there is a need for simultaneous imaging of multiple substances. For example, when performing brain multi-photon imaging on a mouse, calcium ions, plaques and synapses in the brain are all objects that need to be observed, which often need excitation light of different wavelengths to be excited.

[0043] To realize excitation light of multiple wavelengths, in related technologies, multi-photon imaging of excitation light of multiple wavelengths can be realized by switching the laser source. That is, when performing multi-photon scanning, the laser source connected with the optical fiber at the host can be switched, thereby providing different wavelengths at different times, to realize multi-photon imaging of multiple wavelengths.

[0044] However, when time-division multiplexing is used for imaging, the optical signals corresponding to different wavelengths are not synchronized in time, and this method cannot meet the actual needs when analyzing specific parameters (such as calcium transient changes). At the same time, considering the bandwidth of the optical fiber, when the foregoing method is executed, the excitation efficiency of the photons can be affected, so that some fluorescent substances are not excited under optimal excitation conditions, affecting the imaging quality.

[0045] In addition, in traditional fluorescence imaging, there is also a technology of transmitting different fluorescent light sources in one cable at the same time. Specifically, different optical signals can be transmitted by using a composite optical fiber to form a composite light field. However, the principle of conventional fluorescence imaging is completely different from that of multi-photon depth imaging, and the transmission of the foregoing composite optical fiber can only provide the field light source required by conventional fluorescence imaging and cannot meet the requirements of multi-photon imaging.

[0046] Therefore, in order to realize excitation light of multiple wavelengths, the present application provides an imaging device based on micro multi-photon. Specifically, the present application is provided with multiple input optical fibers in the imaging probe, and multiple wavelengths of laser are provided at the same time in the multi-photon imaging process, so that the optical signal can simultaneously include optical signal components responding to excitation light of different wavelengths, and different internal structures are simultaneously excited and detected. In addition, inside the probe, the laser of each wavelength is subjected to beam combining processing by the coupling module to form a composite laser, and thus the subsequent processing of the composite laser is similar to that of the excitation light, further simplifying the internal structure of the imaging probe.

[0047] The various non-limiting embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0048] Exemplary imaging device

[0049] In order to further illustrate the imaging device provided by the present application which can realize simultaneous excitation of multiple wavelengths, the present application also provides a schematic block diagram of the imaging device (Figure 2) to illustrate the transmission of the photoelectric signal (especially the laser of each wavelength) in the imaging device.

[0050] As shown in Figure 2, the actual functional modules in the imaging device can include a processing module, a signal collection module, a scanning control module, a laser group, an optical detector group, a coupling module, a beam splitting module, and an imaging device.

[0051] It should be noted that, considering that the imaging host and the imaging probe in the foregoing imaging device are actually a containing structure containing components / modules, the imaging host and the imaging probe are not shown in the structure shown in Figure 2.

[0052] As shown in FIG. 2, during imaging, the processing module can generate a control signal and apply it to the laser group, and each laser of the laser group can release laser of corresponding wavelength in response to the control signal and send it to the coupling module. The coupling module can perform spatial coupling processing on each laser to make it beam-combined into a composite laser and transmit it into the imaging device. The imaging device can convert the composite laser into excitation light capable of triggering multiphoton effect and apply it to a certain depth of the imaging object, thereby triggering multiphoton effect at the depth to collect corresponding optical signal.

[0053] Considering that the multiphoton effect triggered by laser of different wavelengths generally presents as optical signal of different wavelengths, the aforementioned excitation light containing different components generally produces optical signal containing different wavelengths when triggering multiphoton effect. Therefore, when the imaging device collects the optical signal, the optical signal can be transmitted to the beam splitting module, thereby decoupling the components of different wavelengths in the optical signal, thereby determining a plurality of light signal beams. The light signal beams can be transmitted to the corresponding optical detectors, thereby sensing the intensity of the corresponding signals. The signal collection module can receive the timing light signal intensity sensed by each optical detector in the timing of the optical detector group and forward it to the processing module. The processing module can determine the pixel value of the corresponding pixel in the optical image according to the received light signal.

[0054] Considering that multiphoton depth imaging generally can only excite a single point, i.e., during imaging, the imaging device often needs to focus the excitation light at the focal position inside the imaging object. In order to realize optical imaging of a specific region, in the present application, the focal position can be changed by the electrically controlled equipment in the imaging device. Specifically, the scanning process of multiphoton depth imaging is generally horizontal scanning, i.e., the imaging device often focuses the excitation light at the focal position of the focal plane inside the imaging object, thereby realizing scanning of each point on the focal plane by changing the focal position.

[0055] Therefore, as shown in FIG. 2, the processing module can generate a scanning control signal during imaging and convert it into a galvanometer control signal through the scanning control module and apply it to the imaging device to change the position of the excitation light on the focal plane. In the processor, the timing light signal intensity can be determined according to the scanning control signal and aligned with each position to render the optical image.

[0056] In the present application, the focal plane can also be changed by the imaging device, at this time, the processing module can also generate a depth control signal to generate a zoom control signal of the imaging device through the scanning control module to control the imaging depth of the imaging device. In some embodiments, the optical images of different depths can be reconstructed in three dimensions to determine a three-dimensional image.

[0057] In the present application, the processing module, the signal collection module, the scanning control module and the laser group in FIG. 2 can be integrated in the imaging host, and the imaging device and the coupling module can be integrated in the imaging probe. Considering the split design of the imaging probe and the imaging host, a plurality of output optical fibers can be arranged between the imaging host and the imaging probe to realize the transmission of each laser.

[0058] As an example, in the practical application of two-photon fluorescence microscopic imaging in the field of brain imaging of mice, the aforementioned laser group includes at least one of a first laser for emitting 780 nm laser (i.e., the center wavelength is 780±20 nm), a second laser for emitting 920 nm laser (i.e., the center wavelength is 920±20 nm), and a third laser for emitting 1030 nm (or 1060 nm) laser (i.e., the center wavelength is 1030±20 nm and / or 1060±20 nm). Then, the input optical fiber group for connecting the imaging host and the imaging probe can include three corresponding optical fibers. Each optical fiber in the input optical fiber group can have a high transmission effect (e.g., low spherical aberration and high power transmission efficiency) at the corresponding wavelength. For example, each optical fiber in the input optical fiber group can be configured as a photonic bandgap hollow photonic crystal fiber or an anti-resonant hollow photonic crystal fiber, and is designed to have a good transmission effect at the corresponding wavelength.

[0059] The 780 nm excitation light is used to excite the fluorescence information of amyloid plaques in the imaging object. The fluorescence probe of the 780 nm excitation light can be Methoxy-X04, and the wavelength of the optical signal of the probe is generally 460±25 nm. The 920 nm excitation light is used to excite the fluorescence information of mitochondria and calcium ion channels in the imaging object. The fluorescence probe of the 920 nm excitation light can be AAV-mito-GCaMP6f+CaMKII-Cre, and the wavelength of the optical signal of the probe is generally 520±35 nm. The 1030 nm excitation light (or 1060 nm excitation light) is used to excite the fluorescence information of neurons in the imaging object. The fluorescence probe of the 1030 nm excitation light can be AAV-hSyn-jRGECO1a, and the wavelength of the optical signal of the probe is generally 625±45 nm. For specific content of each fluorescence probe and its mechanism, please refer to the related technology which is not described herein.

[0060] In some embodiments, the aforementioned beam splitting module and the optical detector group can be arranged in the imaging probe or the imaging host according to actual needs. Considering the difference caused by the two arrangement modes, the present application also provides two system block diagrams of the imaging equipment (FIGS. 3 and 4). FIG. 3 can reflect the imaging equipment when the beam splitting module and the optical detector group are integrated in the imaging host. FIG. 4 can reflect the imaging equipment when the beam splitting module and the optical detector group are integrated in the imaging probe.

[0061] As shown in FIG. 3, the imaging device 10 can specifically include an imaging probe 100, an imaging host 200, and an opto-electric composite cable 300. The imaging host 200 can at least include a laser group 210 and a computing device 220, a beam splitting module 410, and an optical detector group 420. The opto-electric composite cable 300 can at least include an input optical fiber group 310 for connecting the laser group 210 and a coupling module 340 in the imaging probe 100. In consideration of the fact that the coupling module 340 is integrated in the imaging probe 100, the input optical fiber group 310 is generally fixedly connected with the imaging probe 100 to ensure the stability of the position of the coupling module 340, and can also be understood as a part of the imaging probe 100.

[0062] The laser group 210 can reflect a set of at least two lasers 211. Each laser 211 can emit laser light of different wavelengths to meet the requirements of multi-photon imaging. The specific wavelength of the laser can be determined according to the actual selected multi-photon effect and the imaging purpose, which is not limited here. In some embodiments, the laser 211 in the present application can be a femtosecond laser. For example, the laser 211 can be a fiber femtosecond laser, a titanium sapphire femtosecond laser, etc.

[0063] In some embodiments, after each laser in the laser group 210 emits laser light, the laser light can enter the corresponding input optical fiber group 310 through a plurality of optical elements. For example, the optical elements can be position sensors, power sensors, etc., so as to detect the stability of the laser light.

[0064] The computing device 220 can be a set of devices with computing capability in the imaging host, for example, the computing device 220 can include various processors and various memories in the imaging device. The computing device 220 can generally include a processing module, a signal collection module, and a scanning control module. In consideration of the fact that the computing device 220 is composed of computing devices, the wires related to the computing device are generally realized by electrical cables.

[0065] The input optical fiber group 310 can specifically include a plurality of input optical fibers (hereinafter referred to as input optical fiber 311). The number of input optical fibers in the input optical fiber group 310 corresponds to the aforementioned lasers, and is used to transmit the corresponding laser light. In application, each input optical fiber can be constructed based on the femtosecond value and the wavelength of the laser light it transmits, so as to be configured as a pulse optical fiber within the corresponding wavelength bandwidth, so as to have good transmission effect (high transmission power and low spherical aberration) for the laser light of the wavelength.

[0066] The coupling module 340 can be a set of optical elements for coupling multiple lasers into a composite laser. The multiple lasers input into the imaging probe 100 are offset in space, and in order to simultaneously excite different multi-photon effects corresponding to different wavelengths, the different lasers need to be coupled into one beam. In some embodiments, the coupling module 340 can be constructed by using conventional beam combining elements (such as dichroic mirrors) and related optical elements to form a coupling light path so that multiple lasers enter the coupling light path to form a composite laser.

[0067] The imaging probe 100 can be internally provided with an imaging device based on multi-photon effects, which is used to convert the composite laser into excitation light when fixedly connected to the imaging object and focus on the focal position in the internal focal plane of the imaging object to trigger the multi-photon effect at the focal position. Considering that the composite laser contains multiple wavelengths of laser, the excitation light can simultaneously excite multi-photon effects of different wavelengths.

[0068] It should be noted that the specific structure and composition of the imaging device can be adaptively modified based on the multi-photon effect adopted. Considering that the nonlinear optical effect based on multi-photons is generally excited based on multiple photons focusing on the same position at the same time, the aforementioned excitation light can be a parallel light beam that meets the requirements of multi-photon imaging, which can trigger the multi-photon effect to generate an optical signal when the parallel light beam converges at a point.

[0069] For example, the aforementioned imaging device can be constructed based on the second harmonic generation or high harmonic generation effect, and the aforementioned excitation light can be a parallel light beam of a specific frequency that meets the requirements of multi-photon imaging, which can trigger harmonic generation to generate new photons of double / multiple frequency when the parallel light beam converges at a point. In addition, the aforementioned imaging device can also be constructed based on multi-photon excited fluorescence, coherent anti-Stokes Raman scattering (CARS), and other multi-photon effects, and the specific structure can be adjusted according to the corresponding multi-photon effect. In the following, the imaging device based on the two-photon excited fluorescence effect will be described, and other multi-photon imaging effects can be adaptively adjusted.

[0070] The control cable 320 can be used to transmit electrical signals between the imaging probe 100 and the imaging host 200. The control cable 320 can transmit control signals of the scanning galvanometer to the imaging probe 100, so as to control the focal position to scan each position in the focal plane. Specifically, considering that the multi-photon effect often needs to converge the excitation light to the focal position when depth imaging, in order to obtain an optical image of a specific region, the imaging probe 100 can control the focal position of the excitation light in the internal focal plane of the imaging object during multi-photon imaging, and by scanning the focal plane, the optical signals of each position in the focal plane can be collected. In addition, the control signals of other controllable devices (such as an electric zoom module) of the imaging probe 100 can also be transmitted by the control cable.

[0071] The output optical fiber 330 is used to transmit the optical signal generated by the multi-photon effect between the imaging probe 100 and the imaging host 200. Different from the composite laser, considering that the optical signal generated by the multi-photon effect often judges the actual situation of the corresponding position (such as whether there is fluorescence or high-frequency signal), the output optical fiber 330 can generally transmit the optical signal of the corresponding frequency band, and there is no excessive requirement for its energy loss and other performances. Among them, further considering that the optical signal includes the signals corresponding to the lasers of various wavelengths, the output optical fiber 330 can transmit the optical signal. Preferably, in order to reduce the influence of the output optical fiber 330 on the free activity of the imaging object, the output optical fiber 330 can be configured as a flexible optical fiber bundle (SFB).

[0072] The beam splitting module 410 can be an optical element-based light beam splitting module, which can be used to split the optical signal based on the nonlinear optical effect triggered by the multi-photon effect to determine a plurality of light signal splits of different wavelengths. Among them, considering that the aforementioned composite laser includes at least two lasers of different wavelengths, each wavelength of laser can trigger a nonlinear optical effect of a corresponding wavelength / frequency band during the aforementioned multi-photon imaging, thereby containing optical signals responding to different wavelengths. Therefore, the responses of each wavelength laser in the optical signal can be separated by the beam splitting module 410, thereby obtaining a plurality of light signal splits.

[0073] The optical detector group 420 can be a collection of various optical detectors in the imaging host 200. Among them, the optical detector can correspond to the light signal split and be arranged towards the corresponding light signal split for detecting the signal intensity of the corresponding light signal split. In the actual detection process, considering that the scanning process changes quickly, each optical detector in the optical detector group 420 can be configured as an analog signal-based sensor (such as a photomultiplier tube (PMT)), thereby outputting the time sequence intensity of each light signal split (denoted as time sequence light signal intensity).

[0074] The processing module in the computing device 220 can obtain the aforementioned time sequence light signal intensity through the signal collection module, and process the aforementioned time sequence light signal intensity according to the scanning control signal issued by the scanning control module through the processing module, thereby determining the light signal image. Among them, different wavelengths of light signal splits can be rendered by different colors in the light signal image, and the pixel value of each light signal split in the image can be determined based on the signal intensity at the corresponding time. That is, the time sequence light signal intensity on the focal plane is converted into the intensity distribution at the focal plane based on the scanning time sequence in the scanning control signal, and then the intensity is converted into gray / color value to determine the image under the light signal split, and the images of each split are superimposed to obtain the light signal image.

[0075] In some embodiments, the aforementioned computing device 220 or its processing module can further process the optical signal images. For example, the optical signal images can be continuously acquired during actual detection, and subsequent analysis can be performed based on the time-sequenced optical signal images. Illustratively, the aforementioned intracranial imaging of mice can be used to determine the transient variation curve of calcium ions in the mice based on multiple lasers. For another example, the aforementioned processing module can further perform three-dimensional spatial reconstruction based on the depth control signal and the optical signals collected by the imaging probe corresponding to the signals to determine the corresponding three-dimensional model.

[0076] As shown in FIG. 3, the laser group 210 can include 4 lasers 211, wherein each laser 211 emits laser light of different wavelength, and the specific wavelength can be determined according to the imaging requirements of the specific nonlinear optical effect. In addition, the number of lasers 211 can be adjusted according to actual needs. For example, the aforementioned content in the mouse brain imaging can be 3 lasers, the laser 211 in FIG. 4 can be 4 lasers, and the laser 211 in FIG. 5 can be 2 lasers.

[0077] In some embodiments, in order to realize the coupling of the laser light emitted by different lasers 211, the aforementioned coupling module 340 can include multiple coupling elements. Wherein the multiple coupling elements are arranged along the corresponding laser direction to form a coupling light path for outputting the composite laser light. When the laser light of each wavelength enters the coupling light path, it will overlap in space to form a composite laser light.

[0078] In actual application, the coupling element can generally include a reflecting element (such as a mirror) for changing the light propagation path and a beam combining element (such as a dichroic mirror, PBS, etc.) for beam combining. Specifically, for 4 lasers 211, the coupling module 340 can include 1 beam combining mirror 341 and three beam combining elements 342. Wherein the beam combining mirror 341 is arranged towards the laser 211 at the edge, and the beam combining element 342 of the middle laser 211 can reflect the corresponding laser light and project the upstream laser light, so that the laser light converges downstream, so that the laser light of each laser 211 converges at the same position, thereby forming a composite laser light.

[0079] It should be noted that the aforementioned is only an exemplary structure of the coupling light path, and the specific structure can be adjusted according to actual needs, and the coupling elements that function can also be selected as needed. For example, PBS and lenses can also be used for beam coupling. For specific details, please refer to the related description of FIGS. 7A-7D.

[0080] Similar to the coupling module 340, the aforementioned beam splitting module 410 (also referred to as a decoupling module) can also be constructed based on similar optical elements to decouple different wavelength components in the optical signal, considering the reversibility of light propagation. That is, the beam splitting module 410 can include multiple optical elements to achieve decoupling (i.e., light splitting based on wavelength or other optical characteristics), similar to the coupling module 340. For example, as shown in FIG. 4, the beam splitting module 410 can include a decoupling dichroic mirror 412 and a decoupling mirror 413. The optical path in the beam splitting module 410 is similar to that in the coupling module 340, and will not be described here.

[0081] In some embodiments, to ensure the purity of light signal splitting, a filter for filtering other light can be included in the aforementioned beam splitting module 410. Specifically, an ambient filter 411 for filtering ambient light and a beam splitting filter 414 for filtering light other than the light signal splitting can be included.

[0082] Similar to the aforementioned laser group 210, multiple optical detectors 421 can also be provided in the aforementioned optical detector group 420. It should be noted that the wavelengths collected by the optical detectors 421 and the number of settings can be adjusted according to the actual dependent nonlinear optical effect and imaging requirements, and are not strictly consistent with the number of lasers 211.

[0083] In some embodiments, considering the heat accumulation caused by long-term operation of the optical detectors 421, a temperature control device (such as a semiconductor heat sink) is also provided in the aforementioned imaging host 200, wherein the temperature control device is at least used to reduce the operating temperature of the optical detector group 420.

[0084] In some embodiments, the aforementioned temperature control device can be provided in the space where the optical detectors 421 are located, so as to achieve heat dissipation of the optical detectors 421 by controlling the temperature of the space where the optical detectors 421 are located. In some embodiments, the temperature control device can also be in direct contact with each optical detector 421, so as to directly dissipate heat from the optical detectors 421. For example, the temperature control device can be integrally provided inside the optical detectors 421.

[0085] In some embodiments, considering that the optical signal collected in the imaging probe 100 can be divergent with the propagation in the optical path, and thus cannot be transmitted through the aforementioned output optical fiber 330 (or cannot enter the beam splitting module 410 in FIG. 4), a collecting device (see the collecting lens 430 in the subsequent FIG. 5 and the like) can also be provided in the imaging probe 100. The collecting device can be constructed based on a lens, so as to perform converging processing on the optical signal transmitted through the collecting device.

[0086] Specifically, the lens constituting the aforementioned collection device can be configured to adopt an aspheric lens to reduce the focal point of the optical signal on the surface of the output optical fiber 330 to 1 mm, thereby allowing the use of a thinner output optical fiber 330 diameter to minimize the obstruction to the movement of the imaging object.

[0087] As mentioned above, in some embodiments, the beam splitting module 410 and the optical detector group 420 can also be arranged inside the imaging probe 100, as shown in FIGS. 4 and 6.

[0088] As shown in FIG. 6, the beam splitting module 410 and the optical detector group 420 can be directly integrated inside the imaging probe 100, in which case the imaging probe 100 does not need to be provided with the aforementioned output optical fiber 330, but transmits the optical signal to the beam splitting module 410 through an internal light path. Only the timing optical signal intensity of the optical detector group 420 needs to be transmitted to the signal collection module through a cable (denoted as a transmission cable).

[0089] Considering that the multi-photon imaging device in the imaging probe 100 in the present application also needs to be connected to the aforementioned computing device 220 through a cable, the cable can be multiplexed so that the transmission cable can transmit both the control signal of the multi-photon imaging device as the control cable 320 and the sensor signal of the optical detector group 420.

[0090] In some embodiments, considering that the imaging probe 100 itself has a small volume, each optical detector in the aforementioned optical detector group 420 integrated in the imaging probe 100 can be configured as a micro detector capable of collecting the intensity of the optical signal to reduce the volume of the imaging probe 100. The micro detector can include but is not limited to a silicon photomultiplier (SiPM), a micro photomultiplier, a photodiode (PD), an avalanche photodiode (APD), a single-photon avalanche diode (SPAD), etc.

[0091] Thus, the imaging equipment based on multi-photon depth imaging shown in FIGS. 1-6 adopts a split structure to realize a miniaturized imaging probe. During imaging, the imaging probe can be fixedly connected to the imaging object, and the multi-photon effect is excited in the imaging object by the excitation light to collect the optical signal inside the imaging object to detect the internal condition of the imaging object. In particular, the present application provides a plurality of input optical fibers in the imaging probe, which simultaneously provide laser beams of multiple wavelengths during multi-photon imaging, so that the optical signal can simultaneously include optical signal components in response to excitation light of different wavelengths, and different internal structures are simultaneously excited and detected. In addition, inside the probe, the laser beams of different wavelengths are combined by the coupling module to form a composite laser beam, so that the subsequent processing of the composite laser beam is similar to that of the excitation light, further simplifying the internal structure of the imaging probe.

[0092] Exemplary imaging probe

[0093] To further illustrate the light path propagation in the process of multi-photon imaging, the present application also provides a structural diagram of an imaging probe (Fig. 5, Fig. 6). Wherein, Fig. 5 and Fig. 6 can present a cross-sectional view along the center line of the imaging probe 100. Fig. 5 can reflect the imaging probe when the aforementioned beam splitting module and optical detector group are arranged in the imaging host, and Fig. 6 can reflect the imaging probe when the beam splitting module and optical detector group are arranged in the imaging probe.

[0094] As shown in Fig. 5, there is an excitation light path in the body of the imaging probe 100, which connects the first end and the second end of the imaging probe 100. The first end is used for fixed connection with the imaging object, and the input optical fiber is communicated with the imaging probe at the second end for releasing the composite laser at the second end.

[0095] Based on the aforementioned excitation light path, the imaging device of the imaging probe 100 can include a plurality of input optical fibers 311, a coupling module 340, a collimating module 110, a scanning module 120 and a lens module 130 arranged in sequence along the propagation direction of the composite laser at the second end, an objective lens module 140 arranged at the first end, and a collection lens 430. In order to accommodate the above-mentioned devices, a first opening is provided at the first end, and a second opening is provided at the second end. That is, the plurality of input optical fibers 311, the coupling module 340, the collimating module 110, the scanning module 120 and the lens module 130 are arranged in sequence in the second opening, and the objective lens module 140 is arranged in the first opening.

[0096] In the excitation light path, the plurality of input optical fibers 311 can provide lasers of different wavelengths and couple into a composite laser through the coupling module 340. The collimating module 110 can be used to convert the composite laser into excitation light. Wherein, the excitation light can release multiple photons at the same time, which can be understood as parallel light beams with the same wavelength component. The scanning module 120 can be used to control the exit angle of the excitation light, the lens module 130 can be used to transmit the excitation light to the objective lens module 140, and the objective lens module 140 can be used to focus the excitation light on the focus position corresponding to the exit angle in the focal plane inside the imaging object.

[0097] Specifically, considering the specific principle of multi-photon depth imaging, that is, multiple photons can be focused to the same position during imaging, thereby exciting the nonlinear optical effect (such as fluorescence effect) of the position. Then in the aforementioned multi-photon imaging device, the collimating module 110 can convert the composite laser into excitation light containing parallel light beams, wherein each parallel light beam in the excitation light contains all wavelengths of the composite laser, and is not split based on its wavelength. It should be noted that the parallel light beams are only used to illustrate that the excitation light provides multiple photons at the same time, and in actual application, the specific beam splitting of the excitation light can be configured with different refractive powers, thereby causing it to be non-parallel.

[0098] As shown in FIG. 5, when the beam splitting module and the optical detector group are arranged in the imaging main machine, the imaging probe 100 only needs to collect the corresponding optical signals and transmit them to the output optical fiber 330. That is, the collection optical path connecting the first end and the second end can also be included in the aforementioned imaging probe 100, and the output optical fiber 330 is arranged at the opening (marked as the third opening) of the collection optical path at the second end, so that the optical signals collected by the aforementioned objective lens module 140 are directly transmitted to the output optical fiber 330 through the collection optical path. Among them, the aforementioned collection lens 430 can be arranged in the collection optical path to focus the optical signals to the surface of the output optical fiber 330.

[0099] Specifically, the aforementioned output optical fiber 330 can be arranged at the third opening, wherein the third opening is in communication with the first opening to form a collection optical path, thereby transmitting the optical signals to the output optical fiber 330.

[0100] In the aforementioned optical path, the collimation module 110, the scanning module 120, the lens module 130, and the objective lens module 140 can all be a collection of optical elements that can achieve corresponding functions.

[0101] The collimation module 110 (also referred to as a collimator) can be a collection of optical elements that collimate the excitation light. That is, the main function of the collimation module 110 is to adjust the divergent laser beam into a parallel light beam. Generally, this is achieved by using collimating lenses (or a combination of lenses), which can convert the composite laser light that is approximately a point source into a beam of approximately parallel collimated light (i.e., the excitation light of the aforementioned parallel light beam).

[0102] The scanning module 120 can be a collection of scanning mirrors and related optical elements. The scanning module 120 can be used to control the propagation direction of the light (i.e., the excitation light of the parallel light beam) by mechanical movement to achieve scanning of a specific area. Specifically, the parallel light beam changes its propagation direction and exit angle after passing through the scanning module 120, so that the excitation light is focused on different positions of the imaging object based on different exit angles through the subsequent module. Among them, the scanning mirror in the scanning module 120 is generally a MEMS (Micro Electro Mechanical System) scanning mirror or other types of optical components (such as rotating mirrors, galvanometer mirrors, prisms, etc.). Preferably, a scanning galvanometer will be used as the main functional device in the aforementioned scanning module 120.

[0103] The lens module 130 can be a collection of optical elements for transmitting excitation light between the scanning module 120 and the objective lens module 140. Among them, the lens module 130 is generally a lens combination (such as a 4f conjugate lens group) to ensure the transmission of parallel excitation light.

[0104] In some embodiments, considering that the parallel light beams with different exit angles in the scanning module 120 have different focal positions on the focal plane, the aforementioned lens module 130 can cause the parallel light beams with different exit angles to enter the objective module 140 at different positions based on the combination of internal lenses, so that the objective module 140 only needs to focus the light rays at different positions, thereby directly ensuring that the light rays with different exit angles are focused on the same focal plane.

[0105] The objective module 140 can be a collection of optical elements (such as lenses) for focusing parallel light beams and collecting optical signals. Among them, the parallel light beams entering the objective module 140 are converged by each lens in the objective module 140, thereby focusing on a point on a specific plane (denoted as a focal plane). In addition, after the multi-photon effect of the focal position is excited, it can enter the imaging probe 100 along the objective module 140.

[0106] Based on the foregoing description and optical path, considering that there can be differences in different wavelengths when passing through optical elements, at least one set of doublet lenses can be included in the aforementioned collimating module 110, lens module 130 and objective module 140. Among them, the doublet lens is an optical lens composed of two lenses, which is used to correct spherical aberration, coma and chromatic aberration. That is, the propagation difference of excitation light of different wavelengths in the composite laser can be eliminated by adjusting the parameters of the doublet lens.

[0107] Specifically, in order to realize multi-color excitation, several sets of achromatic doublet lenses are introduced into the collimating module 110, the lens module 130 and the objective module 140. By using various optical material combinations with different dispersions, the system realizes achromatism in the excitation light wavelength band (such as 760±20nm-1060±20nm).

[0108] In addition, considering that the excitation light path and the collection light path multiplex the aforementioned objective module 140, a dichroic mirror or other beam splitting element can be provided in the aforementioned objective module 140 to multiplex the optical path without cross talk. For more information about the coupling module 340, please refer to the related description of FIGS. 7A-7D.

[0109] In particular, considering the actual application, the imaging probe 100 provided by the present application needs to be fixedly installed on the imaging object and imaged during the movement of the imaging object. In order to avoid the imaging probe 100 itself forming a biasing force on the imaging object, each input optical fiber 311 in the aforementioned input optical fiber group 310 can be uniformly distributed at the second opening along the first direction (the horizontal direction in the figure), so as to ensure that the input optical fiber group 310 will not generate a biasing force due to the arrangement problem to affect the movement of the imaging object.

[0110] In FIG. 5, the optical signal collected by the objective module 140 can be converged by the collection lens 430 to the output optical fiber 330, so as to be transmitted to the imaging host. In some alternative embodiments, the electrical signal can also be directly transmitted to the imaging host without transmitting the optical signal. At this time, the beam splitting module 410 and the optical detector group 420 for sensing the optical signal are integrated in the imaging probe.

[0111] For further illustration, the beam splitting module 410 and the optical detector group 420 are integrated in the imaging probe. Please refer to FIG. 6. In FIG. 6, the beam splitting module 410 and the optical detector group 420 are integrated in the imaging probe 100.

[0112] It should be noted that the beam splitting module 410 can be set according to actual needs. In actual application scenarios, the composite laser can have two wavelengths of excitation light, and then two optical detectors can be configured. Correspondingly, the beam splitting module 410 can be directly constructed based on a dichroic mirror to split the optical signal into two beams. Among them, considering that the scanning module 120 communicates with the computing device through the control cable 320, the aforementioned optical detector group 420 can also carry the control cable 320 to transmit the sensing signal to the computing device.

[0113] In addition, considering that the transmission of the optical signal does not need to be realized through the output optical fiber, in FIG. 6, the collection light path can not be connected at the second end, but only connected inside the imaging probe 100, so that the optical signal collected by the objective module 140 is directly transmitted to the beam splitting module. Among them, in FIG. 6, each optical detector in the optical detector group 420 can be configured as the aforementioned SiPM or other micro detectors to reduce the volume of the imaging probe 100.

[0114] In some embodiments, considering that only two optical detectors are involved in FIG. 7, the beam splitting module in FIG. 7 can directly include a decoupling dichroic mirror 412 for splitting. In order to filter other wavelengths of light signals, the beam splitting module can also include an ambient filter 411 for filtering ambient light and a beam splitting filter 414 for filtering light other than the split light signal. Specifically, considering that the beam splitting filter 414 can allow the split light signal to pass, the aforementioned ambient filter 411 can also not be provided.

[0115] In some embodiments, in order to adjust the imaging effect, the aforementioned objective module 140 can include a plurality of candidate objective lenses, wherein each candidate objective lens in the plurality of candidate objective lenses has a different focal position for the same propagation angle of the excitation light, so as to present different display fields of view. In order to assemble each candidate objective lens, the first end of the aforementioned imaging probe 100 can be detachably connected with each candidate objective lens, wherein the currently assembled candidate objective lens at the first end can be denoted as a target objective lens.

[0116] To further illustrate the specific implementation of different field of view ranges, the application also provides a light path schematic diagram of excitation light in an imaging probe based on different candidate objectives (Figure 8).

[0117] As shown in Figure 8, the light path schematic diagram can include a scanning galvanometer 121, a lens module 130, and multiple candidate objectives (denoted as HR objective, U objective, and LF objective, respectively). As shown in Figure 8, the scanning galvanometer 121 can adjust the exit angle of the excitation light, i.e., the exit angles of the light rays of different colors in Figure 8 are different, and pass through the same lens module 130 to enter different candidate objectives.

[0118] Specifically, the light beams of different exit angles (specifically, lines of different gray levels / colors in the figure) in Figure 8 are emitted from the scanning galvanometer 121, can be converged by the lens module 130 to different positions of the candidate objectives, and then converged by the candidate objectives to a point on the focal plane.

[0119] As can be seen from Figure 8, in different candidate objectives, the convergence positions of the candidate objectives for excitation light of different exit angles are different. Specifically, in different candidate objectives, the larger the field of view range, the smaller the displacement of the angle on the focal plane. Among them, the distance of the convergence position of the light rays of the HR objective on the focal plane relative to the center point is less than the distance of the convergence position of the light rays of the U objective on the focal plane relative to the center point, which is less than the distance of the convergence position of the light rays of the LF objective on the focal plane relative to the center point, then the field of view range of the corresponding HR objective is greater than the field of view range of the U objective, which is greater than the field of view range of the LF objective.

[0120] In some embodiments, the parfocal distance of each candidate objective can be the same, thereby ensuring that the focal plane positions of each candidate objective for excitation light are the same. In some embodiments, the first end of the candidate objective or the imaging probe is provided with an objective length adapter ring and / or a length adjusting device, wherein the objective length adapter ring and / or the length adjusting device are used to match the parfocal distances of different objectives.

[0121] In some embodiments, to ensure the performance of the candidate objectives and reduce the parameter adjustment process, the candidate objectives include common structures and adjustment structures, wherein the common structures are shared by multiple candidate objectives, the adjustment elements are used to adjust the focusing position of the excitation light, and the common elements at least include a doublet lens. For example, the lens types included in each candidate objective in Figure 8 are consistent, and only the sizes thereof are adjusted.

[0122] Based on the aforementioned achromatic design of the doublet lens, the residual lateral chromatic aberration is less than 0.5 microns at half of the field of view of each candidate objective lens. For the HR objective lens and the U objective lens, the residual axial chromatic aberration is about 1 micron in the 780-920 nm and 920-1030 nm wavelength bands, while for the LF objective lens, it is about 1 micron in the 780-920 nm wavelength band and 3 microns in the 920-1030 nm wavelength band. In practical applications, the chromatic aberration is extremely small and can be considered as a nonlinear optical effect at the same position.

[0123] In some embodiments, the aforementioned various candidate objective lenses can also be designed based on the spherical aberration of the actual application. Specifically, in the actual deep brain imaging process, since the refractive index of the cortex (about 1.38) is higher than that of water (about 1.33), the spherical aberration also increases with the increase of the imaging depth in the brain tissue. Therefore, the optical path of the HR / U / LF candidate objective lens is optimized so that the spherical aberration introduced in the 0-800 micron cortex depth range is lower than or slightly higher than the diffraction limit. In addition, at the collection level, the optical path is designed separately so that the collection numerical aperture (NA) of the HR, U and LF objectives is expanded to 0.74, 0.67 and 0.4 respectively, so that it matches the collection lens 430 in the figure, so that the focal point of the optical signal on the surface of the output optical fiber 330 is reduced to 1 millimeter, thereby allowing the use of a thinner output optical fiber 330 diameter to minimize the obstruction to the movement of the imaging object.

[0124] In practical applications, considering that the excitation light entering the scanning galvanometer at a large angle in the scanning module 120 can cause scanning distortion, the propagation direction can be adjusted by a mirror (denoted as a scanning mirror) to reduce the angle of the excitation light entering the scanning galvanometer to an acute angle as much as possible.

[0125] To achieve the excitation light entering the scanning galvanometer at an acute angle, there can be two technical directions in the design inside the imaging probe. First, at least one mirror (denoted as a scanning mirror) can be arranged between the scanning galvanometer and the collimation module, thereby adjusting the propagation angle of the excitation light so that the excitation light enters the scanning galvanometer at an acute angle. Second, the collimation module and the input optical fiber can be directly inclined to make the excitation light itself have a certain inclination angle so that it enters the scanning galvanometer at an acute angle.

[0126] In practical applications, the aforementioned technical directions can be combined to form three implementation manners. To illustrate the three cases, Figures 9A-9C of the present application reflect the three implementation manners.

[0127] As shown in FIG. 9A, the aforementioned scanning module 120 can include a scanning galvanometer 121 and a scanning mirror 122. The scanning mirror 122 is arranged between the collimating module 110 and the scanning galvanometer 121, and is used to change the propagation direction of the excitation light so that the excitation light reaches the scanning galvanometer 121 at an acute angle. The arrangement angle of the scanning mirror 122 can be adjusted based on the position of the scanning galvanometer 121 and the aforementioned excitation light, so that the excitation light propagates in the direction shown in the figure.

[0128] As shown in FIG. 9B, the aforementioned scanning module 120 can only include a scanning galvanometer 121. The input fiber group 310 has a second included angle with the second direction (i.e. the vertical direction, the objective direction) at the second end, so that the composite laser enters the imaging probe along the second included angle. Correspondingly, the collimating module is arranged downstream of the communication of the input fiber at the second end along the second included angle, and is used to convert the composite laser along the second included angle into the excitation light along the second included angle. As shown in FIG. 9B, the second included angle can be directly arranged based on the arrangement direction of the scanning galvanometer 121, so that the excitation light enters the scanning galvanometer 121 at an acute angle.

[0129] In combination with the arrangements of FIG. 9A and FIG. 9B, in FIG. 9C, the aforementioned scanning mirror 122 is arranged, and the input fiber group 310 has a second included angle with the second direction at the second end, so that the excitation light reaches the scanning galvanometer 121 at an acute angle. In particular, considering the influence of the scanning mirror 122 on the propagation, the included angle direction of the second included angle in FIG. 9C is different from that in FIG. 9B.

[0130] In particular, the included angle of the scanning galvanometer 121 along the first direction can also cause scanning distortion. The included angle of the scanning galvanometer 121 along the first direction can be an acute angle (such as 15°-30°), and preferably, the included angle of the scanning galvanometer along the first direction is 20°, wherein the first direction is the extension direction of the lens module 130.

[0131] In some embodiments, in actual applications, the multi-photon depth imaging can also change the focal plane position to obtain optical images at different depths, thereby performing three-dimensional modeling. In actual applications, the change of the aforementioned focal plane position is often realized by a displacement table or the like, however, considering the size of the imaging probe itself, the displacement table cannot be used to change the relative position of the imaging probe and the imaging object when changing the focal plane position, so as to change the focal plane position.

[0132] In order to realize the zoom of the imaging probe, the present application creatively changes the internal light path of the excitation light in the imaging probe. As shown in FIG. 10.

[0133] As shown in FIG. 10, the imaging probe 100 provided by the present application can be provided with an electric zoom module 150 between the aforementioned collimating module 110 and the scanning module 120.

[0134] The electric zoom module 150 can be a set of optical devices for changing the diopter of the excitation light. For example, the electric zoom module 150 can be constructed by an electric zoom device. Illustratively, the electric zoom module 150 can be implemented by a liquid zoom lens, a piezoelectric zoom lens, or the like electric zoom device.

[0135] That is, considering the principle of multi-photon depth imaging, the aforementioned excitation light is often configured as a parallel light beam, and the aforementioned electric zoom module 150 can change the diopter of the parallel light beam. The diopter of the parallel light beam refers to the refractive power of an optical system to an incident parallel light beam, which is used to represent the degree of focusing or divergence of the light beam after passing through the optical element.

[0136] As shown in the aforementioned FIG. 8, when the excitation light is a parallel light beam, the objective module 140 will converge the parallel light to the corresponding position in the focal plane. However, when the aforementioned parallel light beam is diverged or converged in advance, based on the unchanged refractive power of the objective module 140 itself, the corresponding excitation light will be converged later or converged in advance, thereby changing the depth of the focal plane.

[0137] In some embodiments, the depth change of the focal plane caused by each diopter can be calibrated in advance. Thus, in actual control, the electric zoom module can be communicatively connected based on the aforementioned computing device, and the processing module in the computing device can issue a control signal based on the depth of the desired imaging and the aforementioned depth transformation corresponding relationship, thereby adjusting the depth of the focal plane.

[0138] In the actual application of mouse brain imaging, based on the aforementioned electric zoom module, the focal plane can be changed from 0 μm to 850 μm, and then after completing the scanning of a position in the aforementioned scanning process, the depth of the focal plane can be changed by the electric zoom module, thereby determining the fluorescence images of multiple depths. Thus, based on the fluorescence images of each depth, the region can be three-dimensionally modeled. For example, the present application can realize three-dimensional modeling of amyloid plaques, mitochondria, calcium ion channels, and neurons in mice within 0 μm to 850 μm.

[0139] In summary, based on the aforementioned designed imaging device, multi-color excitation, extended imaging depth, and switching between different magnification targets can be realized to achieve an expandable field of view.

[0140] In some embodiments, considering that there can be light-sensitive proteins (such as ChR2 and NpHR) in the imaging object, the light-sensitive proteins can be optogenetically stimulated during the imaging process of the aforementioned multi-photon depth imaging (especially multi-photon fluorescence excitation), thereby activating or inhibiting specific neurons. The optogenetic stimulation generally refers to stimulating the corresponding light-sensitive protein by using a light beam of a specific wavelength.

[0141] Therefore, considering that the application can detect the optical image of the brain nerve (such as the brain nerve of a mouse), the optogenetic light beam is applied at the same time during imaging to realize optogenetic stimulation.

[0142] For example, during the aforementioned two-photon brain imaging, the ChR2 inhibitory optogenetic protein can be stimulated using a 635 nm LED light source. At this time, two-photon fluorescence excitation can use a 920 nm light beam to image the GCaMP green fluorescent indicator. In addition, to avoid the influence of the aforementioned LED light source on the collection of the fluorescence signal, a filter (such as the aforementioned environmental filter 411) can be used at the beam splitting module to filter out the 635 nm LED light.

[0143] For another example, during the aforementioned two-photon brain imaging, the NpHR excitatory optogenetic protein can be stimulated using a 488 nm LED light source. At this time, two-photon uses a 1030 nm light beam to image the jRGECO red fluorescent indicator. In addition, to avoid the influence of the aforementioned LED light source on the collection of the fluorescence signal, a filter (such as the aforementioned environmental filter 411) can be used at the beam splitting module to filter out the 488 nm LED light.

[0144] To realize the aforementioned optogenetic stimulation, the aforementioned imaging probe 100 can also be provided with an optogenetic module. To illustrate the specific structure of the optogenetic module, the application also provides various structural diagrams (such as FIGS. 11A-11C).

[0145] Please refer to FIG. 11A, the aforementioned imaging probe 100 can also include an optogenetic module 160. Wherein, the optogenetic module 160 is used to release an optogenetic light beam and converge into the excitation light path at the objective module 140, and form an optogenetic stimulation area at the focal depth of the focal plane, the optogenetic stimulation area covers the focal plane. That is, the aforementioned optogenetic light beam can be irradiated on the focal plane area at the same time as the excitation light. Considering the realization principle of optogenetic stimulation, to ensure that each position in the focal plane is stimulated by optogenetic stimulation, the aforementioned optogenetic stimulation area can include the focal plane area formed by the excitation light.

[0146] In some embodiments, to realize the generation of the aforementioned optogenetic light beam, the aforementioned optogenetic module 160 can include an optogenetic light source 161, an optogenetic converging element (for example: a lens or a lens group with converging function) 162, and an optogenetic converging element 163. Wherein, the optogenetic light source 161 is used to release the optogenetic light beam, the optogenetic converging element 162 is used to converge the optogenetic light beam in the optogenetic optical fiber, and the optogenetic converging element 163 is based on the objective module 140 to input the optogenetic light beam into the objective module 140.

[0147] In consideration of the fact that the optogenetic light beam covers the focal region of the excitation light at the focal plane, the aforementioned optogenetic light source can generally be configured as a surface light source. Specifically, in the aforementioned FIG. 11A, the aforementioned optogenetic light source 161 can be an optogenetic laser capable of emitting an optogenetic light beam. For example, the optogenetic light source 161 can be an LED light source of a specific wavelength. Further considering the divergence of the aforementioned surface light source, the aforementioned optogenetic condensing element 162 can condense the optogenetic optical fiber to meet the optogenetic stimulation requirements.

[0148] The aforementioned optogenetic in-coupling element 163 can be constructed based on a conventional light beam in-coupling element. As shown in FIG. 11A, the aforementioned optogenetic in-coupling element 163 can be configured as a dichroic mirror. Considering that the optogenetic in-coupling element 163 coincides with the optical signal light path, when configuring the dichroic mirror, it can be configured based on the wavelength of the optogenetic light beam, so that it can reflect the optogenetic light beam to enter the objective module 140. Specifically, the optogenetic in-coupling element 163 is generally arranged between the collection lens 430 and the objective module 140.

[0149] In some embodiments, considering that different wavelengths of optogenetic light beams can be used in optogenetic stimulation (such as the use of two different light beams in the aforementioned brain imaging scenario), to match the application in this scenario, the optogenetic light source 161 can provide at least two optogenetic light beams.

[0150] At this time, the optogenetic light source 161 shown in FIG. 11A can be configured as a replaceable optogenetic laser. The replaceable optogenetic laser is detachably connected with the optogenetic light beam imaging probe 100, and is used to provide a corresponding optogenetic light beam. That is, when replacing the wavelength of the optogenetic light beam, the optogenetic light source 161 can be replaced by a replaceable optogenetic laser of a corresponding wavelength through the aforementioned detachable connection.

[0151] In some embodiments, to realize the emission of multiple optogenetic light beams, the aforementioned optogenetic light source 161 can also be configured as an optogenetic optical fiber and an optogenetic laser group. Among them, FIG. 11B reflects the structure when the optogenetic light source is configured as an optogenetic optical fiber, and FIG. 11C reflects the structure when the optogenetic light source is configured as an optogenetic laser group.

[0152] As shown in FIG. 11B, an optogenetic optical fiber 350 can also be arranged on the imaging probe 100, and the part of the optogenetic optical fiber 350 inside the imaging probe 100 can be abstracted as the aforementioned optogenetic light source 161. The optogenetic optical fiber 350 can be connected with multiple optogenetic lasers in the imaging host, and by controlling the corresponding optogenetic laser to work, the emission of an optogenetic light beam of a corresponding wavelength can be realized during optogenetic stimulation.

[0153] As shown in FIG. 11C, the light genetic laser group (i.e., multiple composite light genetic lasers) can also be directly arranged in the imaging probe 100. Specifically, the aforementioned light genetic light source 161 can include multiple light genetic lasers 1611 and one light genetic beam combining element 1612. Among them, the light genetic laser 1611 can release light genetic light beams of different wavelengths, and the light genetic beam combining element 1612 can combine the light genetic light beams released by each light genetic laser 1611 to a specified position. Among them, in actual application, the specific combining structure of the aforementioned light genetic beam combining element 1612 can refer to the related description of the aforementioned coupling module 340, which is the same in principle. For example, the aforementioned light genetic beam combining element 1612 can be configured as a dichroic mirror.

[0154] Exemplary coupling module

[0155] Considering the actual structure of the coupling module, the propagation direction of the light beam emitted from the output end of the beam combining element is consistent with the propagation direction of the light beam incident from the first incident end, that is, the light is transmitted between the first incident end and the output end of the beam combining element, and reflected / refracted between the second incident end and the output end, thereby combining at the output end.

[0156] To further illustrate the processing process of the coupling module for different waveband excitation light, the present application also provides optical path schematic diagrams of multiple coupling modules (FIGS. 7A-7D). The structures of various coupling modules will be described below in conjunction with FIGS. 7A-7D.

[0157] As shown in FIG. 7A, the coupling module shown in FIG. 7A can combine two wavelengths of laser light, and therefore can include one beam combining element 342 and one beam combining mirror 341 in the coupling module shown in FIG. 7A. Among them, one laser enters the beam combining element through the beam combining mirror, and the other laser directly enters the beam combining element, thereby realizing the beam combining of two wavelengths of laser light. Among them, in FIGS. 7A-7C, the beam combining element 342 can be directly presented as a dichroic mirror, that is, its style is similar to a mirror. The light from the side (second incident end) of the dichroic mirror reaches the dichroic mirror and is reflected (marked as the output end) by the dichroic mirror, but the light entering from the other side (i.e., the first incident end) of the dichroic mirror can pass through the dichroic mirror and be emitted from the output end. Among them, the light from the second incident end is generally at 45° with the dichroic mirror, thereby ensuring that its output direction coincides with the light entering from the first incident end.

[0158] In practical applications, the input optical fiber in the aforementioned imaging probe is usually arranged along the propagation direction of the excitation light, so as to avoid disorder of the internal light path. The direction of the composite laser output by the aforementioned coupling module 340 can be consistent with the direction of the laser of the input optical fiber 311. Specifically, the input optical fiber group can include a beam combining optical fiber and an edge optical fiber, wherein the propagation direction of the laser in the beam combining optical fiber is consistent with the propagation direction of the composite laser. The edge optical fiber is an optical fiber distributed on the other side relative to the beam combining optical fiber. Based on the aforementioned beam combining module, the first incident end of the beam combining element corresponding to the beam combining optical fiber is directed towards the beam combining optical fiber, the second incident end is directed towards the intermediate beam combining direction, the first incident end of the beam combining element corresponding to the other optical fiber of the at least two input optical fibers is directed towards the intermediate beam combining direction, and the second incident end is directed towards the corresponding optical fiber.

[0159] For example, the propagation direction of the laser in the beam combining optical fiber can be the second direction in the figure, and the intermediate beam combining direction is generally perpendicular to the second direction and can be the positive or negative direction of the first direction. The subsequent description takes the negative direction of the first direction as an example.

[0160] To provide a coupling module that meets the above requirements, as shown in FIG. 7B, in the structure shown in FIG. 7B, n-1 beam combining elements and n-1 beam combining mirrors can be provided for n excitation lights, wherein the first incident end of each beam combining element is directed towards the beam combining optical fiber (the outgoing end is directed towards the first incident end of the downstream beam combining element), and the second incident end is directed towards the beam combining mirror. The beam combining mirror corresponds to the other optical fiber of each non-beam combining optical fiber and is used to emit the corresponding laser to the second incident end of the beam combining element. In this way, the lasers of each non-beam combining optical fiber are staggered in space and converge to the laser propagation direction of the beam combining optical fiber.

[0161] However, the above structure needs to be staggered in space and still occupies a large space. To further reduce the occupied space, the present application also provides a structure as shown in FIG. 7C. The structure shown in FIG. 7C is similar to the coupling module in FIG. 3, except that the direction of the beam combining element corresponding to the beam combining optical fiber is different. Specifically, in FIG. 7C, the beam combining mirror is used to reflect the laser of the edge optical fiber to the intermediate beam combining direction and into the first incident end of the beam combining element. The first incident end of the beam combining element corresponding to at least one propagation optical fiber (an optical fiber located between the beam combining optical fiber and the edge optical fiber) is directed towards the intermediate beam combining direction, the second input end is directed towards the excitation light of the corresponding propagation optical fiber, and the second incident end of the beam combining element corresponding to the beam combining optical fiber is directed towards the intermediate beam combining direction.

[0162] In some embodiments, each beam combining element can be implemented by a dichroic mirror. Considering the structural requirements of the dichroic mirror, in the structure shown in FIG. 7C, the dichroic mirror corresponding to the beam combining optical fiber is used to transmit the laser of the beam combining optical fiber corresponding to a certain wavelength range and refract the laser of other wavelength ranges. The dichroic mirror corresponding to the propagation optical fiber is used to refract the laser of the propagation optical fiber corresponding to a certain wavelength range and transmit the laser of other wavelength ranges.

[0163] As an alternative embodiment, the aforementioned beam combining element can also be implemented based on a polarization beam splitter. Specifically, the beam combining element comprises a polarization beam splitter and a half-wave plate, wherein the polarization beam splitter is configured to transmit a light beam of a first polarization direction and reflect a light beam of a second polarization direction, and the half-wave plate is configured to adjust the polarization direction, and the first polarization direction is perpendicular to the second polarization direction. Specifically, the coupling module based on the aforementioned polarization beam splitter can be referred to FIG. 7D.

[0164] As shown in FIG. 7D, the structure shown in FIG. 7D reflects the beam combining process of two lasers. Different from FIG. 7C, the beam combining element used in FIG. 7D is a polarization beam splitter 3421 and a half-wave plate 3422, wherein the polarization beam splitter 3421 can transmit a light beam of a first polarization direction and reflect a light beam of a second polarization direction. Specifically, the initial state of the laser can be the first polarization direction, and the laser becomes the second polarization direction after passing through a half-wave plate 3422, thereby the beam combining can also be implemented based on the polarization beam splitter 3421.

[0165] It is particularly pointed out that the aforementioned half-wave plate 3422 can be set according to the actual polarization direction of the laser. For example, in FIG. 7D, both of the two lasers need to pass through the half-wave plate 3422, so as to adjust the polarization direction. In other cases, when the polarization direction of the laser itself meets the requirements, the half-wave plate 3422 can not be set. Specifically, the elements can be adjusted and increased or decreased according to the actual situation, which will not be described here.

[0166] All the optional technical solutions described above can be combined to form optional embodiments of the present application, which will not be described one by one here.

[0167] Those skilled in the art can realize that the units and algorithm steps of the examples described in combination with the embodiments disclosed herein can be realized by electronic hardware or a combination of computer software and electronic hardware. Whether the functions are realized by hardware or software depends on the specific application and design constraints of the technical solutions. Those skilled in the art can use different methods to realize the described functions for each specific application, but such implementation should not be considered beyond the scope of the present application.

[0168] Those skilled in the art can clearly understand that, for the convenience and brevity of the description, the specific working process of the system, device and unit described above can refer to the corresponding process in the foregoing method embodiments, which will not be described here.

[0169] In several embodiments provided in the present application, it should be understood that the disclosed system, device and method can be implemented in other manners. For example, the division of the above-described device embodiment is only a logical function division, and there can be another division manner for actual implementation, for example, a plurality of units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the displayed or discussed mutual couplings or direct couplings or communication connections between different units, or the among different units, can be indirect couplings or communication connections through some interfaces, devices or units, and can be in electrical, mechanical or other forms.

[0170] The units described as separated components can or can not be physically separated, and the components displayed as units can or can not be physical units, i.e., can be located in one place, or can be distributed on a plurality of network units. Some or all of the units can be selected according to actual needs to achieve the purposes of the embodiments.

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

[0172] It should be noted that, in the present application, "miniaturization" means that the multi-photon microscopic system has less influence on the activity of the living body during observation of the living body, for example, when the multi-photon microscopic system is fixed on the living body to be observed, the living body to be observed can still move freely.

[0173] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An imaging probe, characterized in that, The imaging probe is applied to an imaging device based on multi-photon depth imaging, and is connected to an imaging host of the imaging device through a photoelectric composite cable. One end of the shell is provided with a first opening and is used for fixed connection with a to-be-measured living body, and the other end is provided with a second opening, and the first opening is in communication with the second opening; wherein the at least two input optical fibers, the coupling module are sequentially arranged in the second opening, and the imaging device is arranged at the first opening; The at least two input optical fibers, the coupling module and the imaging device form an excitation light path, wherein the at least two input optical fibers are used to provide at least two laser beams of different wavelengths, the coupling module is used to combine the at least two laser beams into a composite laser, and the imaging device is used to convert the composite laser into excitation light; the excitation light is focused on an internal focus position of an imaging object, so as to trigger a multi-photon effect at the internal focus position; an optical signal generated by the multi-photon effect is collected; and the internal focus position is changed to determine the optical signal of each position in the focal plane, wherein the optical signal of each position in the focal plane is used to generate an optical image of the imaging object at the focal plane.

2. The imaging probe of claim 1, wherein, The at least two input optical fibers include a beam combining optical fiber and an edge optical fiber, wherein the propagation direction of the excitation light in the beam combining optical fiber is consistent with the propagation direction of the composite laser; The coupling module includes at least one beam combining mirror and at least one beam combining element, wherein the beam combining element corresponds to each input optical fiber except the edge optical fiber in the at least two input optical fibers one by one; The beam combining element includes a first incident end, a second incident end and an output end, wherein the light beams entering the beam combining element from the first incident end and the second incident end are emitted from the output end when the beam combining condition is met, and the propagation direction of the light beam entering the beam combining element from the first incident end is unchanged in the beam combining element; The first incident end of the beam combining element corresponding to the beam combining optical fiber is directed to the beam combining optical fiber, and the second incident end is directed to an intermediate beam combining direction, and the first incident end of the beam combining element corresponding to other optical fibers in the at least two input optical fibers is directed to the intermediate beam combining direction, and the second incident end is directed to the corresponding optical fiber.

3. The imaging probe of claim 2, wherein, The at least two input optical fibers further include at least one propagation optical fiber between the beam combining optical fiber and the edge optical fiber, and the beam combining element corresponding to the propagation optical fiber is used to combine the light beams of the propagation optical fiber to the intermediate beam combining direction.

4. The imaging probe of claim 2, wherein, The beam combining element is a dichroic mirror, wherein the dichroic mirror corresponding to the beam combining optical fiber is used to transmit the excitation light of the wavelength range corresponding to the beam combining optical fiber and refract the excitation light of other wavelength ranges, and the dichroic mirror corresponding to other optical fibers in the at least two input optical fibers is used to refract the excitation light of the wavelength range corresponding to the corresponding optical fiber and transmit the excitation light of other wavelength ranges.

5. The imaging probe of claim 2, wherein, The beam combining element comprises a polarization beam splitter and a half-wave plate, wherein the polarization beam splitter is used to transmit a light beam of a first polarization direction and reflect a light beam of a second polarization direction, and the half-wave plate is used to adjust the polarization direction, the first polarization direction being perpendicular to the second polarization direction.

6. The imaging probe of claim 1, wherein, The imaging device comprises a collimating module, a scanning module and a lens module arranged in the second through hole in sequence along the propagation direction of the excitation light, and an objective lens module arranged at the first end of the imaging probe. In the excitation light path, the collimating module is used to convert the composite laser into the excitation light, the scanning module is used to control the exit angle of the excitation light, the lens module is used to transmit the excitation light to the objective lens module, and the objective lens module is used to focus the excitation light at a focal position in the internal focal plane of the imaging object corresponding to the exit angle. The collimating module, the lens module and the objective lens module each comprise at least one set of doublet lenses, which are used to eliminate the propagation difference of different wavelengths of laser in the excitation light.

7. The imaging probe of claim 6, wherein, The objective lens module is detachably connected to the first end. The objective lens module comprises a plurality of candidate objective lenses, each of which has a different focal position for the excitation light of the same propagation angle to present different display fields of view.

8. The imaging probe of claim 7, wherein, The candidate objective lens or the first end of the imaging probe is provided with a parfocal distance adjusting device, wherein the parfocal distance adjusting device comprises an objective lens length adapter ring and / or a length adjusting device, and the focal planes of the candidate objective lenses during imaging are the same.

9. The imaging probe of claim 6, wherein, The imaging probe further comprises a collection lens arranged at the end of the objective lens module away from the imaging object, which is used to converge the optical signals collected by the objective lens module.

10. The imaging probe of claim 6, wherein, The scanning module comprises a scanning galvanometer, wherein the scanning galvanometer is used to control the exit angle of the excitation light, and the included angle between the excitation light at the scanning galvanometer and the scanning galvanometer is an acute angle. The scanning module further comprises a scanning mirror, wherein the scanning mirror is arranged between the collimating module and the scanning galvanometer and is used to change the propagation direction of the excitation light; and / or The input optical fiber has a second included angle with a second direction at the second through hole, so that the composite laser enters the imaging probe along the second included angle, and the collimating module is arranged at the communication between the second through hole and the first through hole along the second included angle, and is used to convert the composite laser into excitation light along the second included angle, wherein the second direction is the extension direction of the objective lens module. The included angle between the scanning galvanometer and a first direction is 15°-30°, wherein the first direction is the extension direction of the lens module.

11. The imaging probe of claim 10, wherein, The imaging device further comprises an electric zoom module arranged between the collimating module and the scanning module, which is used to change the diopter of the excitation light, adjust the focal plane depth of the excitation light, and determine optical signals of different focal plane depths, wherein the optical signals of different focal plane depths are used to determine a three-dimensional model.

12. The imaging probe of claim 6, wherein, ​ 13. The imaging probe of claim 6, wherein, The imaging device further comprises a optogenetic module, wherein the optogenetic module is configured to release an optogenetic light beam and converge the optogenetic light beam at the objective module, and form an optogenetic stimulation area at a focal depth of the focal plane, the optogenetic stimulation area covering the focal plane; The optogenetic module comprises an optogenetic light source, an optogenetic converging element and an optogenetic converging element arranged in sequence, wherein the optogenetic light source is configured to release the optogenetic light beam, the optogenetic converging element is configured to converge the optogenetic light beam, and the optogenetic converging element is configured to input the optogenetic light beam into the objective module based on the objective module.

14. The imaging probe of claim 13, wherein, The optogenetic light source can provide at least two optogenetic light beams, and the optogenetic light source is configured as one of an optogenetic optical fiber, a replaceable optogenetic laser and an optogenetic laser group; When the optogenetic light source is configured as the optogenetic optical fiber, the optogenetic optical fiber is connected with at least two optogenetic lasers in the imaging host for transmitting at least two optogenetic light beams; When the optogenetic light source is configured as the replaceable optogenetic laser, the replaceable optogenetic laser is detachably connected with the imaging probe for providing a corresponding optogenetic light beam; When the optogenetic light source is configured as the optogenetic laser group, the optogenetic laser group comprises at least two optogenetic lasers and at least one optogenetic beam combining element.

15. The imaging probe of claim 13, wherein, The imaging probe further comprises a beam splitting module arranged along the propagation direction of the optical signal, and the beam splitting module comprises a filter for filtering the optogenetic light beam.

16. The imaging probe of claim 1, wherein, The imaging probe further comprises a beam splitting module, at least two optical detectors and a transmission cable arranged in sequence along the propagation direction of the optical signal, wherein the transmission cable is connected with each optical detector, the beam splitting of the beam splitting module corresponds to the optical detector one by one, and the optical detector is configured as a micro detector. After the optical signal is collected by the imaging device, the optical signal enters the beam splitting module and is processed by the beam splitting module to form a plurality of optical signal beams, each optical signal beam enters the corresponding optical detector to determine the signal intensity of the optical signal beam, and the signal intensity of each optical signal beam at the current time is transmitted through the transmission cable.

17. The imaging probe of claim 16, wherein, The transmission cable and a control cable are integrated into an electrical cable group, and the control cable is connected between the imaging probe and the imaging host for transmitting a control signal to a controllable device in the imaging probe.

18. The imaging probe of claim 1, wherein, The photoelectric composite cable of the imaging device comprises an output optical fiber arranged in a third port, wherein the third port is in communication with the first port for transmitting an optical signal to the input optical fiber and transmitting the optical signal to the imaging host through the output optical fiber.

19. The imaging probe of claim 1, wherein, The at least two input optical fibers are uniformly distributed at the second port along the first direction.

20. An imaging device based on multi-photon depth imaging, characterized in that The imaging device comprises an imaging host, at least two lasers, a beam splitting module, at least two optical detectors and the imaging probe of claim 1. The imaging host contains at least the at least two lasers, which are used to provide laser light meeting the imaging requirements and transmit to the imaging probe through the corresponding output optical fiber in the imaging probe, wherein the wavelength of the laser light released by each of the at least two lasers is different; The imaging probe is internally provided with a coupling module and an imaging device based on multi-photon depth imaging, the coupling module is used to convert multiple laser lights into composite laser light, and the imaging device is used to convert the composite laser light into excitation light; the excitation light is focused on an internal focus position of an imaging object, so as to trigger the multi-photon effect at the internal focus position; an optical signal generated by the multi-photon effect is collected; and the internal focus position is changed to determine the optical signal of each position in the focal plane, wherein the optical signal of each position in the focal plane is used to generate an optical image of the imaging object at the focal plane; The beam splitting module is used to split the optical signal into at least two optical signal beams, and the optical detector is used to receive the corresponding optical signal beams and detect the signal intensity of the corresponding optical signal beams, wherein the optical signal beams reflect the signal components of the optical signal generated by the multi-photon effect at each wavelength, the optical signal beams correspond to the optical detector one by one, and the signal intensity of the optical signal reflects the pixel value of the corresponding pixel in the optical image.

Images