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

Abstract

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

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] How to achieve multiphoton imaging technology with good imaging results based on micro-devices is a technical problem that urgently needs to be solved by those skilled in the art. In view of this, the embodiments of this application provide 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 problem.

[0006] In a first aspect, this application provides an imaging device based on multiphoton depth imaging, the imaging device including at least two lasers, a coupling module, an input optical fiber, an imaging probe, a beam splitting module, at least two optical detectors, and an imaging host.

[0007] The imaging host contains at least two lasers and a coupling module. The lasers are used to provide laser light that meets the imaging requirements, and the coupling module is used to couple the laser light emitted by the at least two lasers into a composite laser light. The laser light emitted by the at least two lasers has a different wavelength.

[0008] One end of the input optical fiber is connected to the coupling module, and the other end is connected to the imaging probe. It is used to transmit the composite laser to the imaging probe. The energy transmission loss of the input optical fiber within the target bandwidth meets the excitation requirements of the multiphoton effect, and the wavelengths of each laser in the composite laser are within the target bandwidth.

[0009] The imaging probe is internally provided with an imaging device based on multi-photon deep imaging, which 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 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.

[0010] 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.

[0011] The application provides a micro multi-photon based imaging device, which realizes a miniaturized imaging probe by adopting a split structure. During imaging, the imaging probe can be fixedly connected with an imaging object, and a multi-photon effect is excited in the imaging object by excitation light to collect the optical signal in the imaging object, so as to detect the internal condition of the imaging object. In particular, the input optical fiber connecting the imaging probe and the imaging host in the imaging device provided by the application has a target bandwidth meeting the multi-photon effect excitation requirement, so as to carry the composite laser in the target bandwidth to simultaneously excite the multi-photon effect at different wavelengths and observe different internal structures. In addition, the imaging device sets the coupling module of the laser outside the imaging probe, so that the composite laser is directly transmitted through the input optical fiber, thereby improving the integration of the imaging probe. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the 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 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.

[0013] Fig. 1 is an application scenario diagram of the imaging device provided by some embodiments of the application.

[0014] Fig. 2 is a schematic diagram of the transmission of the photoelectric signal in the imaging device provided by some embodiments of the application.

[0015] Fig. 3A and Fig. 3B are schematic diagrams of the structure and performance of the input optical fiber provided by some embodiments of the application.

[0016] Fig. 4 is a system block diagram of the imaging device provided by some embodiments of the application.

[0017] Fig. 5 is another system block diagram of an imaging device according to some embodiments of the present application.

[0018] Fig. 6 is a schematic diagram of an imaging probe according to some embodiments of the present application.

[0019] Fig. 7 is another schematic diagram of an imaging probe according to some embodiments of the present application.

[0020] Fig. 8 is a schematic diagram of the optical path of parallel excitation light in an imaging probe according to some embodiments of the present application.

[0021] Figs. 9A-9C are schematic diagrams of a scanning galvanometer with excitation light at an acute angle according to some embodiments of the present application.

[0022] Fig. 10 is a schematic diagram of a zoom imaging probe according to some embodiments of the present application.

[0023] Figs. 11A-11C are schematic diagrams of an optogenetic stimulation imaging probe according to some embodiments of the present application.

[0024] Reference Signs:

[0025] 10, imaging device; 20, imaging subject; 100, imaging probe; 200, imaging host; 300, photoelectric composite cable; 210, laser group; 220, coupling module; 230, computing device; 410, beam splitting module; 420, optical detector group; 310, input optical fiber; 320, control cable; 330, output optical fiber; 211, laser; 221, coupling mirror; 222, coupling dichroic mirror; 223, coupling sensor; 412, decoupling dichroic mirror; 413, decoupling mirror; 411, environmental filter; 414, beam splitting filter; 421, optical detector; 430, collection module; 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 condensing element; 163, optogenetic in-coupling element; 340, optogenetic optical fiber; 1611, optogenetic laser; 1612, beam combining element. DETAILED DESCRIPTION

[0026] As mentioned above, in multiphoton imaging, excitation light is often relied on to trigger multiphoton linearization, thereby obtaining corresponding optical signals. For example, second harmonic generation (SHG) is a second-order multiphoton phenomenon in which two photons of the same frequency interact with a nonlinear material to combine into a new photon of twice the frequency. As another example, three-photon excitation fluorescence (3PEF) requires three photons to be excited simultaneously to generate a fluorescence signal.

[0027] In practical applications, based on the imaging characteristics of multi-photons, it is often applied to depth imaging, that is, generally the excitation light containing multiple photons is focused at a certain depth to trigger the corresponding multi-photon effect to generate the corresponding optical signal. Therefore, in multi-photons, the frequency and power of the excitation light have certain requirements. In addition, further considering that multi-photons often depend on excitation light of a specific frequency band. Thus resulting in that in practical applications, multi-photon imaging can often be realized based on excitation light of a single frequency band. Especially in the process of miniaturized imaging devices, considering the complexity of the internal space, it cannot be propagated.

[0028] To further illustrate the actual application of the miniaturized device of the multi-photon imaging technology, the present application provides an application scene diagram of an imaging device (Figure 1). Among them, the application scene shown in Figure 1 can reflect the process of the imaging device performing optical brain imaging on a mouse.

[0029] As shown in Figure 1, in the application scene of the imaging device can include an imaging device 10 and an imaging object 20.

[0030] The imaging device 10 is the execution subject of multi-photon imaging (for example, the imaging device 10 can be configured as a multi-photon microscope), which can release excitation light to trigger multi-photon effect (such as 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.

[0031] Exemplarily, the imaging device 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 imaging, to obtain the fluorescence signal of the point. Then through scanning technology to change the focus position at the target focal depth to perform plane scanning, thereby obtaining the fluorescence signal of each place at the target focal depth inside the imaging object 20, to determine the fluorescence image.

[0032] The imaging object 20 can refer to an object that needs to be multi-photon imaged. For example, the imaging object 20 can be an ex vivo sample, a living sample, etc. Exemplarily, the imaging object 20 in the present application can be a mouse, a rabbit, a bird, a non-human primate macaque, a marmoset, etc. Imaging object. Among them, the imaging object 20 can generally be used for imaging the internal structure (brain nerve, spinal nerve, etc.) of the imaging object.

[0033] As shown in FIG. 1, the imaging object 20 in FIG. 1 can be a mouse, and specifically, the brain of the mouse can be subjected to multiphoton imaging. The imaging device 10 can be fixedly connected to the brain of the imaging object 20, and the imaging object 20 can be free to move, and the imaging device 10 can periodically scan the imaging object 20 during its free movement to obtain optical signals at each position in a specific focal plane of the brain of the imaging object 20, so as to determine a time-series optical image of the position. The optical image obtained can generally include a synapse at the position.

[0034] It should be noted that before the imaging object 20 is fixedly connected to the imaging device 10, the imaging object 20 can be subjected to adaptive processing. For example, the fixed connection part between the imaging object 20 and the imaging device 10 can be subjected to skin preparation. For another example, for the mouse shown in FIG. 1, a skull window can be provided on the brain of the mouse during the fixed connection, so as to improve the imaging effect. For another example, a fluorescent probe can be injected into the imaging object 20 in advance, so as to perform multiphoton fluorescence imaging based on the fluorescent probe.

[0035] In order to miniaturize the pre-imaging device, the imaging device 10 provided in the specification can be provided in a split manner, so as to further include an imaging probe 100 and an imaging host 200, and the imaging probe 100 and the imaging host 200 can be communicatively connected based on an optoelectrical composite cable 300.

[0036] The imaging probe 100 can be a device for releasing excitation light and detecting optical signals. The imaging probe 100 can convert laser generated by a laser source into excitation light focused to a focal plane and having scanning capability through an optical assembly. In some embodiments, the imaging probe 100 can be a contact imaging probe.

[0037] The imaging host 200 can be a set of devices that cannot be integrated into the imaging probe 100, and specifically, can be used to provide laser and perform imaging based on optical signals. The imaging host 200 can generally include a laser, a fluorescence collection device, and a scanning and acquisition controller.

[0038] The optoelectrical composite cable 300 can be a communication cable between the imaging probe 100 and the imaging host 200, and can be used to realize data transmission of optical signals and electrical signals between the imaging host 200 and the imaging probe 100. The optoelectrical composite cable 300 can transmit excitation light generated by the imaging host 200 to the inside of the imaging probe 100, and transmit optical signals collected by the imaging probe 100 to the imaging host 200, so as to form an optical image.

[0039] Based on the foregoing imaging device 10, multi-photon imaging (such as multi-photon fluorescence imaging) inside the imaging object 20 can be achieved. However, considering that the spectral bandwidth of the optical fiber is limited at present, and the imaging device 10 often needs a highly stable and high-power laser, the optical fiber can usually only transmit a single-wavelength laser, as shown in FIG. 3. Therefore, the optical fiber for 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.

[0040] However, in practical applications, 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 neural synapses in the brain are all objects that need to be observed, but they often need different wavelengths of excitation light for excitation.

[0041] To achieve multiple wavelengths of excitation light, in the related art, multi-photon imaging of multiple wavelengths of excitation light can be achieved by switching laser sources. That is, when performing multi-photon scanning, the laser source connected with the optical fiber can be switched at the host computer to provide different wavelengths at different times, so as to achieve multi-photon imaging of multiple wavelengths.

[0042] However, when time division multiplexing is used for imaging, the optical signals corresponding to different wavelengths are not synchronized in time, and when analyzing specific parameters (such as calcium transient changes), this method cannot meet the actual needs. 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.

[0043] 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.

[0044] Therefore, to achieve multiple wavelengths of excitation light, the present application provides an imaging device based on micro multi-photon. In particular, the input optical fiber of the imaging device provided by the present application, which connects the imaging probe and the imaging host, has a target bandwidth that meets the excitation requirements of multi-photon effects, so as to carry composite laser within the target bandwidth to simultaneously excite multi-photon effects under different wavelengths and observe different internal structures. In addition, the coupling module of the imaging device is arranged outside the imaging probe, so that the composite laser is directly transmitted through the input optical fiber, improving the integration of the imaging probe.

[0045] The various non-limiting embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0046] Exemplary imaging device

[0047] To further illustrate the imaging device capable of realizing multi-wavelength simultaneous excitation provided in the present application, the present application further provides a schematic block diagram of the imaging device (Fig. 2) to illustrate the transmission of photoelectric signals (especially laser of various wavelengths) in the imaging device.

[0048] As shown in Fig. 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.

[0049] It should be noted that, considering that the imaging host and the imaging probe in the aforementioned 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 Fig. 2.

[0050] 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 a corresponding wavelength in response to the control signal and transmit 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 to the imaging device. The imaging device can convert the composite laser into excitation light capable of triggering a multi-photon effect and apply it to a certain depth of the imaging object, thereby triggering a multi-photon effect at the depth to collect a corresponding optical signal.

[0051] Considering that the multi-photon effect triggered by laser of different wavelengths generally presents optical signals of different wavelengths, the aforementioned excitation light containing different components generally produces optical signals containing different wavelengths when triggering a multi-photon 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 optical signal.

[0052] Considering that multi-photon depth imaging can generally only excite a single point, i.e., the imaging device often needs to focus the excitation light at a focal position inside the imaging object during imaging. To realize optical imaging of a specific region, the focal position can be changed in the present application by an electrically controlled device in the imaging device. Specifically, the scanning process of multi-photon depth imaging is generally horizontal scanning, i.e., the imaging device can generally focus the excitation light at a focal position of a focal plane inside the imaging object, thereby realizing scanning of each point on the focal plane by changing the focal position.

[0053] Thus, 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 to align with each position to render the optical image.

[0054] In this application, the focal plane can also be changed by the imaging device, at which time the processing module can also generate a depth control signal to generate a zoom control signal of the imaging device to control the imaging depth of the imaging device. In some embodiments, the optical images at different depths can be reconstructed in three dimensions to determine a three-dimensional image.

[0055] In this application, the processing module, signal collection module, scanning control module, laser group and coupling module in FIG. 2 can be integrated in the imaging host, and the imaging device can be integrated in the imaging probe. Considering the split design of the imaging probe and the imaging host, an output optical fiber can be provided between the imaging host and the imaging probe to realize the transmission of the composite laser.

[0056] Further considering the excitation requirement of the multi-photon effect, the energy transmission loss of the input optical fiber within the target bandwidth meets the excitation requirement of the multi-photon effect, and the wavelength of each laser in the composite laser is within the target bandwidth. Among them, the specific bandwidth of the input optical fiber can be determined according to the specific excitation light of the multi-photon effect.

[0057] To achieve the foregoing target bandwidth, the input optical fiber meets the excitation requirement of the multi-photon effect within the target bandwidth based on the anti-resonance principle. Among them, the anti-resonance principle optical fiber, especially the hollow core anti-resonance fiber (HC-ARF), is a microstructured optical fiber that uses anti-resonance effect to realize optical signal transmission. Its basic principle is to set a series of high and low refractive index thin layers around the fiber core to form a structure similar to a Fabry-Perot (F-P) resonant cavity. When the light wavelength meets the specific condition, the constraint of the optical fiber on the light becomes weak, and the light quickly transmits through the cladding and escapes to the outside, resulting in increased transmission loss; on the contrary, when the light wavelength does not meet the condition, the constraint of the optical fiber on the light is enhanced, and the light propagates along the core, at which time the transmission loss is reduced.

[0058] Based on the foregoing principle, the input optical fiber provided in the application can include a two-layer structure. Specifically, the input optical fiber can include a silicon core and a cladding surrounding the silicon core, and a plurality of air holes arranged at equal intervals in the cladding. Among them, the cladding forming the air holes can form the foregoing resonant cavity structure, thereby having a strong constraint on the excitation light within the target bandwidth.

[0059] In the field of traditional multi-photon imaging (such as multi-photon depth imaging), in order to ensure the excitation power of the excitation light, an air-core photonic bandgap fiber (PBG-HCF) is generally used for input fiber. Such a fiber only has good transmission capability (small energy loss and small dispersion) in a small range of specific wavelengths. Therefore, such a fiber cannot meet the transmission requirements of the composite laser of the present application.

[0060] To further illustrate the target bandwidth of the transmission light of the present application, taking multi-photon imaging as an example, the excitation light is generally 550nm-1800nm. Therefore, the input fiber provided by the present application can have good performance in the range of 550nm-1800nm.

[0061] In particular, considering the imaging requirements of mouse brain imaging, when depth imaging based on two-photon fluorescence excitation effect, the target bandwidth range of the transmission fiber provided by the present application can be 550nm-1100nm.

[0062] To further illustrate the performance of the input fiber provided by the present application, the present application also provides a transmission spectrum (Figure 3A). The chart shown in Figure 3A can reflect the energy loss of light of different wavelengths through different types of fibers. The abscissa can be the wavelength, and the ordinate can reflect the energy loss after passing through the corresponding fiber. In Figure 3A, the broken line with a wide bandpass range (the broken line with a white color) reflects the loss of the air-core anti-resonant fiber (AR-HCF), and the broken line with a narrow bandpass range (the broken line with a black color) reflects the loss of the air-core photonic bandgap fiber (PBG-HCF).

[0063] In addition, the structural diagram of the input fiber provided by the present application (i.e. AR-HCF in Figure 3) and the structural diagram of the traditional pulse fiber (i.e. PBG-HCF in Figure 3) are also shown in Figure 3A.

[0064] As can be seen from Figure 3A, the traditional pulse input fiber only has a bandpass at a specific wavelength (such as the bandpass of PBG-HCF in Figure 3 at about 920nm), while the input fiber provided by the present application (i.e. AR-HCF in Figure 3) has good performance in the range of 550nm-1100nm, so that the target bandwidth range thereof can be 550nm-1100nm.

[0065] As an exemplary illustration only, based on the aforementioned target bandwidth range, when two-photon fluorescence microscopic imaging is used in the field of mouse brain imaging, the aforementioned laser group includes at least one of a first laser for emitting 780nm laser, a second laser for emitting 920nm laser, and a third laser for emitting 1030nm laser.

[0066] The 780nm excitation light is used to excite the fluorescence information of amyloid plaques in the imaging object. The fluorescence probe of the 780nm excitation light can be Methoxy-X04, and the wavelength of the optical signal of the probe is generally 460±25nm. The 920nm excitation light is used to excite the fluorescence information of mitochondria and its calcium ion channels in the imaging object. The 920nm excitation light fluorescence probe can be AAV-mito-GCaMP6f+CaMKII-Cre, and the wavelength of the optical signal of the probe is generally 520±35nm. The 1030nm excitation light is used to excite the fluorescence information of neurons in the imaging object. The 1030nm excitation light fluorescence probe can be AAV-hSyn-jRGECO1a, and the wavelength of the optical signal of the probe is generally 625±45nm. For specific content of each fluorescence probe and its mechanism, please refer to the related technology, which will not be repeated here.

[0067] In addition, in actual application, the output optical fiber provided by the present application can withstand high power (multi-laser transmission, the power will be higher), and the high power output quality is not affected. Therefore, when multiple lasers are combined, the output power is higher, and the output optical fiber provided by the present application can also meet the transmission requirements in this case. In addition, after optical setting, the dispersion of the aforementioned output optical fiber is very small, and after autocorrelation instrument test, it is known that the dispersion of the aforementioned output optical fiber is basically consistent with that of the traditional single-wavelength pulse-type input optical fiber, and will not cause additional dispersion of the system.

[0068] To further illustrate the transmission of lasers with different powers, the present application also provides an energy loss diagram of different power lasers based on the aforementioned input optical fiber (Figure 3B).

[0069] In Figure 3B, the abscissa can be the shift amount of the central frequency of the laser, and the ordinate can be the loss ratio. The curves with different depths in the figure can reflect lasers with different central frequencies, which are 780nm, 920nm and 1030nm respectively. Among them, the lasers with different central frequencies can be composed of multiple curves with different gray scales, and each curve reflects a different oscillation period (unit: fs). As shown in Figure 3B, the output optical fiber provided by the present application has good transmission effect for lasers with different wavelengths and different powers, which can meet the imaging requirements of brain deep imaging.

[0070] In some embodiments, the aforementioned beam splitting module and optical detector group 420 can be arranged in the imaging probe or the imaging host according to actual needs. Considering the differences caused by the two arrangement modes, the present application also provides two system block diagrams of the imaging equipment (Figures 4 and 5). Among them, Figure 4 can reflect the imaging equipment when the beam splitting module and the optical detector group 420 are integrated in the imaging host. Figure 5 can reflect the imaging equipment when the beam splitting module and the optical detector group 420 are integrated in the imaging host.

[0071] As shown in FIG. 4, 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, a coupling module 220, a computing device 230, 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 310 for connecting the laser group 210 and the imaging probe 100, a control cable 320, and an output optical fiber 330.

[0072] The laser group 210 can reflect a set of at least two lasers 211. Each laser 211 can emit laser beams of different wavelengths to meet the requirements of multi-photon imaging. The specific wavelengths of the lasers can be determined according to the actual selected multi-photon effect and the imaging purpose, which is not limited herein. 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.

[0073] The coupling module 220 can be a set of optical elements for coupling multiple lasers into a composite laser, i.e., the lasers emitted by the aforementioned lasers 211 are offset in space. In order to simultaneously excite different wavelength corresponding multi-photon effects, different lasers need to be coupled into one light beam. In some embodiments, the coupling module 220 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.

[0074] The computing device 230 can be a set of devices (such as processors, memories, etc.) with computing capability in the imaging host. The computing device 230 can generally include a processing module, a signal collection module, and a scanning control module. Since the computing device 230 is composed of computing devices, the wires related to the computing device are generally realized by electrical cables.

[0075] The imaging probe 100 can be internally provided with an imaging device based on multi-photon effect, which is used to convert the composite laser into excitation light and focus on the focal position in the focal plane inside the imaging object to trigger the multi-photon effect at the focal position when the imaging probe 100 is fixedly connected with the imaging object. Since the composite laser contains multiple wavelength lasers, the excitation light can simultaneously excite multi-photon effects of different wavelengths.

[0076] It should be noted that the specific structure and composition of the imaging device can be adaptively modified based on the adopted multi-photon effect. Since the nonlinear optical effect based on multi-photon generally excites when multiple photons are focused on the same position, the aforementioned excitation light can be a parallel light beam that meets the requirements of multi-photon imaging. When the parallel light beam converges at a point, the multi-photon effect can be triggered to generate an optical signal.

[0077] Exemplarily, the aforementioned imaging device can be constructed based on a second harmonic generation or high harmonic generation effect, and the aforementioned excitation light can be a parallel light beam of a specific frequency meeting the requirements of multi-photon imaging. When the parallel light beam converges at a point, the harmonic generation can be triggered to generate new photons of double / multiple frequency. In addition, the aforementioned imaging device can also be constructed based on multi-photon excitation fluorescence, coherent anti-Stokes Raman scattering (CARS) and other multi-photon effects. The specific structure can be adjusted according to the corresponding multi-photon effect. Subsequently, the imaging device based on two-photon excitation fluorescence effect will be described, and other multi-photon imaging effects can be adjusted adaptively.

[0078] 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 a control signal of a scanning galvanometer to the imaging probe 100, so as to control the focusing position to realize scanning of each position in the focusing plane. Specifically, considering that the multi-photon effect often needs to converge the excitation light to the focusing position when depth imaging, in order to obtain an optical image of a specific region, the imaging probe 100 can control the focusing position of the excitation light in the focusing plane of the imaging object during multi-photon imaging, and by scanning the focusing plane, the optical signals of each position in the focusing plane are 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.

[0079] The output optical fiber 330 is used to transmit the optical signals 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 signals generated by the multi-photon effect often judge the actual situation of the corresponding position by the presence or absence (such as the presence or absence of fluorescence and the presence or absence of high-frequency signals), the output optical fiber 330 can generally transmit the optical signals 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 signals include signals corresponding to lasers of various wavelengths, the output optical fiber 330 can transmit the optical signals. Preferably, in order to reduce the influence of the output optical fiber 330 on the free movement of the imaging object, the output optical fiber 330 can be configured as a collection of flexible optical fiber bundles (SFB).

[0080] The beam splitting module 410 can be an optical element-based light beam splitting module, which can be used to split the optical signals based on the nonlinear optical effect triggered by the multi-photon effect, so as 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 during the aforementioned multi-photon imaging can trigger the nonlinear optical effect of the corresponding wavelength / frequency band, so as to contain optical signals responding to different wavelengths. Therefore, by the beam splitting module 410, the responses of each wavelength of laser in the optical signals can be separated, so as to obtain a plurality of light signal splits.

[0081] 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 beam splitting and be arranged towards the corresponding light signal beam splitting for detecting the signal intensity of the corresponding light signal beam splitting. In the actual detection process, considering the fast transformation in the scanning process, each optical detector in the optical detector group 420 can be configured as a sensor based on an analog signal (such as a photomultiplier tube (PMT)), thereby outputting the time sequence intensity of each light signal beam splitting (denoted as time sequence light signal intensity).

[0082] The processing module in the computing device 230 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 beam splitting can be rendered by different colors in the light signal image, and the pixel value of each light signal beam splitting 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 beam splitting. The light signal image can be obtained by superimposing the images of each beam splitting.

[0083] In some embodiments, the aforementioned computing device 230 or its processing module can further process the light signal image. For example, the light signal image can be continuously obtained during actual detection, and subsequent analysis can be performed based on the time sequence light signal image. For example, in this application, the transient change curve of calcium ions in the mouse body can be determined based on the aforementioned intracranial imaging of the mouse. For another example, the aforementioned processing module can also perform three-dimensional spatial reconstruction on the optical signal corresponding to the corresponding signal acquisition based on the depth control signal and the imaging probe to determine the corresponding three-dimensional model.

[0084] As shown in FIG. 4, the laser group 210 can include 4 lasers 211, wherein the wavelengths of the lasers emitted by each laser 211 are different, and the specific wavelengths can be determined according to the imaging requirements of specific nonlinear optical effects. In addition, the number of lasers 211 can be adjusted according to actual needs. For example, in the aforementioned content, the number of lasers in the mouse brain imaging can be 3, the number of lasers 211 in FIG. 4 can be 4, and the number of lasers 211 in FIG. 5 can be 2.

[0085] In some embodiments, in order to realize the coupling of the lasers emitted by different lasers 211, a plurality of coupling elements can be included in the aforementioned coupling module 220. Among them, the plurality of coupling elements are arranged along the direction of the corresponding laser, and are used to form a coupling light path for outputting a composite laser. When the laser of each wavelength enters the coupling light path, it will overlap in space to form a composite laser.

[0086] In practical applications, the coupling elements can generally include reflecting elements (such as mirrors) for changing the light propagation path and beam combining elements (such as dichroic mirrors, PBSs, etc.) for beam combining. Specifically, for the four lasers 211, the coupling module 220 can include one coupling mirror 221 and three coupling dichroic mirrors 222. The coupling mirror 221 is arranged towards the edge of the lasers 211, and the coupling dichroic mirrors 222 of the middle lasers 211 can reflect the corresponding laser light and project the laser light upstream, so that the laser light converges downstream and is consistent with the propagation direction of the composite laser light. The coupling dichroic mirror corresponding to the laser 211 consistent with the propagation direction of the composite laser light can project the laser light of the corresponding laser 211, so that the laser light of each laser 211 converges at the same position, thereby forming the composite laser light.

[0087] It should be noted that the foregoing is only an exemplary structure of the coupling optical 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.

[0088] In some embodiments, considering that the coupling module 220 of the present application is arranged in the imaging host 200, the size and function of the coupling module 220 are not limited by the environment. In order to ensure the coupling accuracy, the foregoing coupling optical path can be adjusted based on the coupling of the composite laser light to ensure high-precision coupling. Therefore, the foregoing coupling module 220 can also include a coupling sensor 223, wherein the coupling sensor 223 is arranged at least at the output end of the composite laser light in the imaging host 200, for detecting the position data and / or power data of the corresponding light beam.

[0089] Exemplarily, the coupling sensor 223 can split part of the light beam from the composite laser light through a dichroic mirror or other beam splitting elements, so as to detect the power and position of the part of the light beam, thereby reflecting the power and position of the composite laser light.

[0090] In some embodiments, in response to the detection result of the coupling sensor 223, the foregoing coupling elements (such as the coupling mirror 221 and the coupling dichroic mirror 222) can be arranged on a moving mechanism (such as a deformable mirror frame), so as to adjust the coupling optical path based on the position data and / or power data, so that the excitation light meets the excitation requirements of the nonlinear optical effect. In practical applications, this process can be configured as a negative feedback process, that is, the moving mechanism can be controlled based on the difference between the target power and the actual power and / or the difference between the target position and the actual position to eliminate the difference.

[0091] Similar to the coupling module 220, the aforementioned beam splitting module 410 (which can also be 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 properties), similar to the coupling module 220. 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 220, which will not be repeated here.

[0092] 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.

[0093] 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.

[0094] In some embodiments, considering the heat accumulation caused by the 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.

[0095] 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.

[0096] In some embodiments, considering that the optical signal collected in the imaging probe 100 can be divergent with the propagation of the optical path, so as to be unable to be transmitted through the aforementioned output optical fiber 330 (or unable to enter the beam splitting module 410 in FIG. 5), a collecting device (see the collecting lens 430 in the subsequent FIG. 6 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.

[0097] 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.

[0098] 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 FIG. 5.

[0099] As shown in FIG. 5, 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).

[0100] 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 230 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.

[0101] 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.

[0102] Thus, the imaging equipment based on multi-photon depth imaging shown in FIGS. 1-5 achieves a miniaturized imaging probe by adopting a split structure. 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 situation of the imaging object. In particular, the input optical fiber connecting the imaging probe and the imaging host of the imaging equipment provided in the present application has a target bandwidth that meets the demand for multi-photon effect excitation, thereby being capable of carrying the composite laser in the target bandwidth to simultaneously excite the multi-photon effect under different wavelengths to observe different internal structures. In addition, the imaging equipment sets the coupling module of the laser outside the imaging probe, so that the composite laser is directly transmitted through the input optical fiber, thereby improving the integration of the imaging probe.

[0103] Exemplary imaging probe

[0104] To further illustrate the light path propagation in the process of multi-photon imaging, the application further provides a structural diagram of an imaging probe (Figures 6 and 7). Among them, Figures 6 and 7 can present a cross-sectional schematic diagram of the imaging probe 100. Figure 6 can reflect the imaging probe when the aforementioned beam splitting module 410 and the optical detector group 420 are arranged in the imaging host, and Figure 7 can reflect the imaging probe when the beam splitting module 410 and the optical detector group 420 are arranged in the imaging probe.

[0105] As shown in Figure 6, there is an excitation light path in the body of the imaging probe 100 that connects the first end and the second end of the imaging probe 100, wherein 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.

[0106] Based on the aforementioned excitation light path, the imaging device of the imaging probe 100 can include a collimation 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. Among them, the collimation module 110, the scanning module 120 and the lens module 130 and the input optical fiber can be integrated in the second port of the second end, and the objective lens module 140 can be arranged in the first port of the first end.

[0107] In the excitation light path, the collimation module 110 can be used to convert the composite laser into excitation light. Among them, 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 internal focal plane of the imaging object.

[0108] Specifically, considering the specific principle of multi-photon depth imaging, i.e. multiple photons can be focused to the same position during imaging, thereby exciting the nonlinear optical effect (such as fluorescence effect) of the position. In the aforementioned multi-photon imaging device, the collimation 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.

[0109] As shown in FIG. 4, when the beam splitting module 410 and the optical detector set 420 are arranged in the imaging host, the imaging probe 100 only needs to collect the corresponding optical signals and transmit them to the output optical fiber 330. That is, the imaging probe 100 can further include a collection light path connecting the first end and the second end, and the output optical fiber 330 is arranged at the opening of the collection light path at the second end, so that the optical signals collected by the objective lens module 140 are directly transmitted to the output optical fiber 330 through the collection light path. Among them, the collection lens 430 can be arranged in the collection light path to focus the optical signals to the surface of the output optical fiber 330.

[0110] Specifically, the output optical fiber 330 can be arranged at the third opening, wherein the third opening is in communication with the first opening arranged at the first end to form a collection light path, thereby transmitting the optical signals to the output optical fiber 330.

[0111] In the aforementioned light path, the collimating module 110, the scanning module 120, the lens module 130, and the objective lens module 140 can each be a collection of optical elements capable of achieving corresponding functions.

[0112] The collimating 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 collimating 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, which is approximately a point source, into a beam of approximately parallel collimated light (i.e., the excitation light of the aforementioned parallel light beam).

[0113] 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.

[0114] 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.

[0115] In some embodiments, considering that the parallel light beams with different exit angles in the scanning module 120 have different focusing 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.

[0116] 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 focusing position is excited, it can enter the imaging probe 100 along the objective module 140.

[0117] 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 collimation module 110, lens module 130, and objective module 140. Among them, the doublet lens is an optical lens composed of two lenses glued together, 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.

[0118] Specifically, in order to realize multi-color excitation, several sets of achromatic doublet lenses are introduced into the collimation module 110, lens module 130, and 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).

[0119] As shown in FIG. 6, when the beam splitting module 410 and the optical detector group 420 are arranged in the imaging host, the imaging probe 100 only needs to collect the corresponding optical signals and transmit them to the output optical fiber 330. That is, the aforementioned imaging probe 100 can also include a collection optical path connected between the first end and the second end, and the output optical fiber 330 is arranged at the opening (denoted as the third opening) of the collection optical path at the second end, so that the optical signals collected by the aforementioned objective 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 onto the surface of the output optical fiber 330.

[0120] 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 arranged in the aforementioned objective module 140 to multiplex the optical paths without cross talk.

[0121] That is, in FIG. 6, the optical signal collected by the objective lens 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.

[0122] For further illustration, the beam splitting module 410 and the optical detector group 420 are integrated in the imaging probe, please refer to FIG. 7. In FIG. 7, the beam splitting module 410 and the optical detector group 420 are integrated in the imaging probe 100.

[0123] 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, and the corresponding 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.

[0124] In addition, considering that the transmission of the optical signal does not need to be realized through the output optical fiber, in FIG. 7, 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 lens module 140 is directly transmitted to the beam splitting module. Among them, in FIG. 7, 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.

[0125] 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. 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.

[0126] In some embodiments, in order to adjust the imaging effect, the aforementioned objective lens 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.

[0127] 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).

[0128] 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.

[0129] 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.

[0130] 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 smaller 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 smaller 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 larger than the field of view range of the U objective, which is larger than the field of view range of the LF objective.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] In some embodiments, the aforementioned various candidate objective lenses can also be designed based on the spherical aberration elimination design 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), as the imaging depth in the brain tissue increases, the spherical aberration also increases. 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] As shown in FIG. 9B, the aforementioned scanning module 120 can only include a scanning galvanometer 121. The input optical fiber 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 optical 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.

[0140] In combination with the arrangements of FIG. 9A and FIG. 9B, in FIG. 9C, the aforementioned scanning mirror 122 is arranged, and the input optical fiber 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.

[0141] 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.

[0142] 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 stage or the like, however, considering the size of the imaging probe itself, the displacement stage 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] In the actual application of mouse brain imaging, based on the aforementioned electric zoom module, the focal plane change of 0 μm-850 μm can be realized, 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 the three-dimensional modeling of amyloid plaques, mitochondria, calcium ion channels, and neurons in mice within 0 μm-850 μm.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] For example, during the aforementioned two-photon brain imaging, the ChR2 inhibitory optogenetic protein stimulation can be performed using a 635 nm LED light source. At this time, the two-photon fluorescence excitation can use a 920 nm light beam to perform GCaMP green fluorescence indicator imaging. In addition, in order 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.

[0154] For another example, during the aforementioned two-photon brain imaging, the NpHR excitatory optogenetic protein stimulation can be performed using a 488 nm LED light source. At this time, the two-photon uses a 1030 nm light beam to perform jRGECO red fluorescence indicator imaging. In addition, in order 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.

[0155] In order to realize the aforementioned optogenetic stimulation, the aforementioned imaging probe 100 can also be provided with an optogenetic module. In order to illustrate the specific structure of the optogenetic module, the application also provides various structural diagrams (such as FIGS. 11A-11C).

[0156] 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, and 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, in order 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.

[0157] In some embodiments, in order 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 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 (for example: a lens or a lens group with converging function) is used to converge the optogenetic light beam in the optogenetic light fiber, and the optogenetic converging element 163 is based on the objective module 140 and is used to input the optogenetic light beam into the objective module 140.

[0158] Considering 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.

[0159] 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.

[0160] 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.

[0161] At this time, the optogenetic light source 161 shown in FIG. 11A can be configured as a replaceable optogenetic laser. Among them, the replaceable optogenetic laser is detachably connected with the optogenetic light beam imaging probe 100, 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.

[0162] In some embodiments, to realize the release 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.

[0163] As shown in FIG. 11B, an optogenetic optical fiber 340 can also be arranged on the imaging probe 100, and the part of the optogenetic optical fiber 340 in the imaging probe 100 can be abstracted as the aforementioned optogenetic light source 161. The optogenetic optical fiber 340 can be connected with multiple optogenetic lasers in the imaging host, and by controlling the corresponding optogenetic laser to work, the release of the optogenetic light beam of a corresponding wavelength can be realized during optogenetic stimulation.

[0164] As shown in FIG. 11C, a group of optogenetic lasers (i.e., multiple compound optogenetic lasers) can also be directly arranged in the imaging probe 100. Specifically, the aforementioned optogenetic light source 161 can include multiple optogenetic lasers 1611 and one optogenetic beam combining element 1612. Among them, the optogenetic lasers 1611 can emit optogenetic light beams of different wavelengths, and the optogenetic beam combining element 1612 can combine the optogenetic light beams emitted by each optogenetic laser 1611 to a designated position. Among them, in actual application, the specific combining structure of the aforementioned optogenetic beam combining element 1612 can refer to the related description of the aforementioned coupling module 220, which is the same in principle. For example, the aforementioned optogenetic beam combining element 1612 can be configured as a dichroic mirror.

[0165] All the optional technical solutions described above can be combined to form optional embodiments of the present application, which will not be repeated here.

[0166] Those skilled in the art can appreciate 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 in hardware or software depends on the specific application and design constraints of the technical solution. Professional technicians can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of the present application.

[0167] Those skilled in the art can clearly understand that, for the convenience and brevity of the description, the specific working processes of the above-described system, device and unit can refer to the corresponding processes in the foregoing method embodiments, which will not be repeated here.

[0168] In several embodiments provided in the present application, it should be understood that the disclosed system, device and method can be realized by other ways. For example, the device embodiments described above are only schematic, for example, the division of the units is only a logical function division, and actual implementation can have another division manner, for example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the coupling or direct coupling or communication connection between the units shown or discussed can be indirect coupling or communication connection through some interface, device or unit, and can be electrical, mechanical or other forms.

[0169] The units described as separate components can or can not be physically separate, and the components shown as units can or can not be physical units, i.e., they can be located in one place or distributed on multiple network units. According to actual needs, part or all of the units can be selected to achieve the purpose of the embodiment.

[0170] 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.

[0171] It should be noted that "miniaturization" in the present application means that the multi-photon microscopic system has less effect on the activity of the living body to be observed 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.

[0172] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An imaging apparatus based on multi-photon depth imaging, characterized by, The imaging device comprises at least two lasers, a coupling module, an input optical fiber, an imaging probe, a beam splitting module, at least two optical detectors, and an imaging host; The imaging host contains at least the at least two lasers and the coupling module, the lasers are used to provide laser light meeting the imaging requirements, and the coupling module is used to couple each laser light released by the at least two lasers into composite laser light, wherein the wavelengths of each laser light released by the at least two lasers are different; One end of the input optical fiber is connected with the coupling module, and the other end is connected with the imaging probe, and is used to transmit the composite laser light to the imaging probe, wherein the energy transmission loss of the input optical fiber within a target bandwidth meets the excitation requirement of a multi-photon effect, and the wavelengths of each laser light in the composite laser light are within the target bandwidth; The imaging probe is internally provided with an imaging device based on multi-photon depth imaging, which is used to convert the composite laser light into excitation light, focus the excitation light on an internal focus position of an imaging object, trigger the multi-photon effect at the internal focus position, collect optical signals generated by the multi-photon effect, and change the internal focus position to determine the optical signals of each position in a focal plane, wherein the optical signals of each position in the focal plane are used to obtain an optical image of the imaging object at the focal plane; The beam splitting module is used to split the optical signals into at least two optical signal beams, and the optical detectors are used to receive 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 signals generated by the multi-photon effect at each wavelength, the optical signal beams correspond to the optical detectors one by one, and the signal intensity of the optical signals reflects the pixel value of a corresponding pixel in the optical image.

2. The imaging device of claim 1, wherein, The input optical fiber comprises a silicon core and a cladding surrounding the silicon core, a plurality of air holes arranged at equal intervals are arranged in the cladding, and the input optical fiber meets the excitation requirement of the multi-photon effect within the target bandwidth based on the anti-resonance principle.

3. The imaging device of claim 2, wherein, The imaging device is configured to be based on a two-photon fluorescence effect, and the target bandwidth of the input optical fiber is 550 nm-1800 nm.

4. The imaging device of claim 3, wherein, The imaging device is configured to be a two-photon microscope, and the at least two lasers comprise at least one of a laser for releasing 780 nm laser light, a laser for releasing 920 nm laser light, and a laser for releasing 1030 nm laser light; The 780 nm laser light is used to excite the fluorescence information of amyloid plaques in a to-be-tested living body, the 920 nm laser light is used to excite the fluorescence information of mitochondria and calcium ion channels in the to-be-tested living body, and the 1030 nm laser light is used to excite the fluorescence information of neurons in the to-be-tested living body.

5. The imaging device of claim 1, wherein, The imaging probe has an excitation light path connecting a first end and a second end of the imaging probe, wherein the first end is used to be fixedly connected with the imaging object, the input optical fiber is communicated with the imaging probe at the second end, and is used to release the excitation light at the second end; The imaging device comprises a collimation module, a scanning module and a lens module arranged in sequence along the propagation direction of the excitation light at the second end, and an objective module arranged at the first end, wherein in the excitation light path, the collimation 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 module, and the objective module is used to focus the excitation light to a focal position corresponding to the exit angle in the focal plane inside the imaging object. The collimation module, the lens module and the objective module each comprise at least one set of doublet lenses for eliminating the propagation difference of lasers of different wavelengths in the excitation light.

6. The imaging device of claim 5, wherein, The objective module is detachably connected to the first end. The objective module comprises a plurality of candidate objective lenses, each of which has a different focal position for excitation light of the same propagation angle to present different display fields of view.

7. The imaging device of claim 6, wherein, The first end of the imaging probe or the candidate objective lens is provided with a parfocal distance adjusting device, wherein the parfocal distance adjusting device comprises an objective length adapter ring and / or a length adjusting device, and the focal planes of the candidate objective lenses during imaging are the same.

8. The imaging device of claim 5, wherein, The imaging probe further comprises a collection module arranged at the end of the objective module away from the imaging object, which is used to converge the optical signals collected by the objective module.

9. The imaging device of claim 5, 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 collimation 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 end, so that the composite laser enters the imaging probe along the second included angle, and the collimation module is arranged downstream of the communication of the input optical fiber at the second end along the second included angle, and is used to convert the composite excitation light into excitation light along the second included angle, wherein the second direction is the extension direction of the objective module.

10. The imaging device of claim 9, wherein, The included angle between the scanning galvanometer and a first direction is 20°, wherein the first direction is the extension direction of the lens module.

11. The imaging device of claim 5, wherein, The imaging device further comprises an electric zoom module arranged between the collimation module and the scanning module, which is used to change the refractive power 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 device of claim 5, wherein, The imaging probe further comprises an optogenetic module, wherein the optogenetic module is used to release an optogenetic light beam and merge into the excitation light path at the objective module, and form an optogenetic stimulation area at the focal depth of the focal plane, and the optogenetic stimulation area covers the focal plane. The optical genetic module comprises an optical genetic light source, an optical genetic converging element and an optical genetic in-coupling element arranged in sequence, wherein the optical genetic light source is configured to release the optical genetic light beam, the optical genetic converging element is configured to converge the optical genetic light beam, and the optical genetic in-coupling element is configured to input the optical genetic light beam into the objective module based on the objective module.

13. The imaging device of claim 12, wherein, The optical genetic light source can provide at least two optical genetic light beams, and the optical genetic light source is configured as one of an optical genetic optical fiber, a replaceable optical genetic laser and an optical genetic laser group. When the optical genetic light source is configured as the optical genetic optical fiber, the optical genetic optical fiber is connected with at least two optical genetic lasers in the imaging host for transmitting at least two optical genetic light beams. When the optical genetic light source is configured as the replaceable optical genetic laser, the replaceable optical genetic laser is detachably connected with the imaging probe for providing a corresponding optical genetic light beam. When the optical genetic light source is configured as the optical genetic laser group, the optical genetic laser group comprises at least two optical genetic lasers and at least one beam combining element.

14. The imaging device of claim 12, wherein, The beam splitting module comprises a filter for filtering the optical genetic light beam.

15. The imaging device of claim 1, wherein, The beam splitting module and the at least two optical detectors are arranged inside the imaging host, and the imaging device further comprises an output optical fiber. The output optical fiber is in communication with the second end of the imaging probe and the beam splitting module respectively, and is configured to transmit optical signals collected by the imaging probe to the beam splitting module. The imaging host further comprises a temperature control device, wherein the temperature control device is configured to reduce the working temperature of the at least two optical detectors.

16. The imaging device of claim 1, wherein, The beam splitting module and the at least two optical detectors are arranged inside the imaging probe. The optical detectors are configured as micro detectors, and the imaging probe further comprises a transmission cable connected with each optical detector, and the transmission cable is configured to transmit the signal intensity of each optical signal beam splitting at the current time.

17. The imaging device of claim 16, wherein, The transmission cable is integrated in a control cable, wherein the control cable connects the imaging probe and the imaging host, and is configured to transmit a control signal to a controllable device in the imaging probe.

18. The imaging device of claim 1, wherein, The coupling module comprises a plurality of coupling elements and a coupling sensor. The coupling elements are arranged along the corresponding laser directions to form a coupling light path for outputting the composite laser, and the coupling sensor is arranged at least at the output end of the composite laser in the imaging host to detect position data and / or power data of the corresponding light beam. The coupling elements are arranged on a moving mechanism, and are configured to adjust the coupling light path based on the position data and / or power data to make the excitation light meet the excitation requirements of the multi-photon effect.

19. An imaging probe, characterized by The imaging probe is applied to an imaging device based on multi-photon depth imaging, and the imaging probe comprises an imaging device based on multi-photon depth imaging and an input optical fiber connected with the imaging probe. The input optical fiber is used to transmit a composite laser provided by the imaging host to the imaging probe, wherein energy transmission loss of the input optical fiber within a target bandwidth meets excitation requirements of a multi-photon effect, and the composite laser includes a plurality of lasers with wavelengths within the target bandwidth. The imaging device is used to convert the composite laser into excitation light, focus the excitation light at an internal focus position of an imaging object, trigger the multi-photon effect at the internal focus position, collect optical signals generated by the multi-photon effect, and change the internal focus position to determine optical signals at each position in a focal plane, wherein the optical signals at each position in the focal plane are used to generate an optical image of the imaging object at the focal plane. The optical signals are subjected to beam splitting to form at least two optical signal beams, and the signal strengths of the optical signal beams are determined after being received by corresponding optical detectors, wherein the optical signal beams reflect signal components of the optical signals generated by the multi-photon effect at each wavelength, and the signal strengths of the optical signals reflect pixel values of corresponding pixels in the optical image.

20. The imaging probe of claim 19, wherein, The input optical fiber includes a silicon core and a cladding surrounding the silicon core, a plurality of air holes arranged at equal intervals are arranged in the cladding, and the input optical fiber meets excitation requirements of the multi-photon effect within the target bandwidth based on the anti-resonance principle.

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