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

Abstract

The invention provides imaging equipment based on multi-photon depth imaging and an imaging probe thereof, and a miniaturized imaging probe is realized by adopting a split type structure. In the imaging process, the imaging probe can be fixedly connected with an imaging object, the multi-photon effect is excited in the imaging object through the exciting light, so that optical signals in the imaging object are collected, and the internal situation in the imaging object is detected. Particularly, a plurality of input optical fibers are arranged in the imaging probe, laser of multiple wavelengths is provided at the same time in the multi-photon imaging process, optical signals can comprise optical signal components responding to exciting light of different wavelengths at the same time, and different internal structures are excited and detected at the same time. Besides, in the probe, the laser of each wavelength is subjected to beam combination treatment through the coupling module to form composite laser, so that the subsequent treatment of the composite laser is similar to that of exciting light, and the internal structure of the imaging probe is further simplified.

Description

Technical Field

[0001] The present application relates to the field of multiphoton imaging technology, and in particular to an imaging device based on multiphoton depth imaging and an imaging probe thereof. Background Art

[0002] Multiphoton depth imaging is a technology for depth imaging based on the nonlinear optical effects of multiphotons. Specifically, multiphoton imaging uses various nonlinear phenomena and effects generated when multiple photons interact with matter simultaneously to generate 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, multiphoton imaging often relies on large-scale equipment because of the complex internal optical path of the imaging device. Therefore, how to implement multiphoton imaging technology based on micro-devices and present better imaging effects is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] In view of this, an embodiment of the present application provides an imaging device that realizes the miniaturization of the probe through the separate design of the probe and the host. At the same time, the input optical fiber used to transmit the excitation light has a target bandwidth that meets the requirements of multi-photon imaging, and can carry a composite laser containing components of different wavelengths, thereby improving the imaging effect of the probe, thereby solving the above-mentioned technical problems.

[0005] In a first aspect, the present application provides an imaging probe, which is applied to an imaging device based on multiphoton depth imaging. The imaging probe is connected to an imaging host of the imaging device via an optoelectronic composite cable. The imaging probe includes a shell, at least two input optical fibers, a coupling module, and an imaging device based on multiphoton depth imaging.

[0006] A first opening is provided at one end of the shell and is used for fixed connection with the life body to be tested, and a second opening is provided at the other end, and the first opening is communicated with the second opening respectively; wherein at least two input optical fibers and a coupling module are sequentially arranged in the second opening, and the imaging device is arranged at the first opening.

[0007] At least two input optical fibers, a coupling module, and an imaging device form an excitation light path, wherein the at least two input optical fibers are used to provide at least two lasers of different wavelengths, the coupling module is used to combine the at least two lasers into a composite laser, and the imaging device is used to convert the composite laser into excitation light; focus the excitation light on an internal focus position of an imaging object to trigger a multiphoton effect at the internal focus position; collect optical signals generated by the multiphoton effect; and change the internal focus position to determine the optical signals at various positions in the focal plane, wherein the optical signals at various positions in the focal plane are used to form an optical image of the imaging object at the focal plane.

[0008] In a second aspect, the present application provides an imaging device based on multi-photon depth imaging and an imaging probe thereof, wherein the imaging device includes an imaging host, at least two lasers, a beam splitting module, at least two optical detectors and the imaging probe described in the first aspect.

[0009] The imaging host at least accommodates at least two lasers, which are used to provide laser light that meets imaging requirements and transmit it to the imaging probe through the corresponding output optical fiber in the imaging probe, wherein the wavelengths of laser light released by each of the at least two lasers are different.

[0010] The imaging probe has a built-in coupling module and an imaging device based on multi-photon depth imaging, wherein the coupling module is used to convert multiple lasers into a composite laser, and the imaging device is used to convert the composite laser into an excitation light; focus the excitation light on the internal focus position of the imaging object to trigger the multi-photon effect at the internal focus position; collect the optical signal generated by the multi-photon effect; and change the internal focus position 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 form an optical image of the imaging object at the focal plane.

[0011] 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 detectors one-to-one, and the signal intensity of the optical signal reflects the pixel value of the corresponding pixel in the optical image.

[0012] The present application provides an imaging device based on multi-photon depth imaging and an imaging probe thereof. By adopting a split structure, a miniaturized imaging probe is realized. During imaging, the imaging probe can be fixedly connected to the imaging object, and the multi-photon effect is excited inside the imaging object by the excitation light to collect the optical signal inside the imaging object to detect the internal situation inside the imaging object. In particular, the present application is provided with multiple input optical fibers in the imaging probe, and multiple wavelengths of laser light are provided simultaneously during the multi-photon imaging process, so that the optical signal can simultaneously include optical signal components that respond to excitation lights of different wavelengths, and simultaneously excite and detect different internal structures. In addition, inside the probe, lasers of various wavelengths are combined by a coupling module to form a composite laser. Therefore, the subsequent processing of the composite laser is similar to the excitation light, which further simplifies the internal structure of the imaging probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is an application scenario diagram of an imaging device provided in some embodiments of the present application.

[0015] Figure 2 This is a schematic diagram of the transmission of photoelectric signals within an imaging device provided in some embodiments of the present application.

[0016] Figure 3 This is a system block diagram of an imaging device provided in some embodiments of the present application.

[0017] Figure 4 This is another system block diagram of an imaging device provided in some embodiments of the present application.

[0018] Figure 5 This is a schematic diagram of the structure of an imaging probe provided in some embodiments of the present application.

[0019] Figure 6 This is another structural schematic diagram of an imaging probe provided in some embodiments of the present application.

[0020] Figure 7A to Figure 7D This is a schematic diagram of the optical path of the coupling module provided in some embodiments of the present application.

[0021] Figure 8 This is a schematic diagram of the optical path of parallel excitation light in an imaging probe provided in some embodiments of the present application.

[0022] Figures 9A to 9C These are multiple structural schematic diagrams of the scanning galvanometer provided in some embodiments of the present application when an acute angle is formed between the scanning galvanometer and the excitation light.

[0023] Fig.10 It is a schematic diagram of the structure of a zoom imaging probe provided in some embodiments of the present application.

[0024] Figures 11A to 11C These are multiple structural schematic diagrams of imaging probes for optogenetic stimulation provided in some embodiments of the present application.

[0025] Among them, 10, imaging device; 20, imaging object; 100, imaging probe; 200, imaging host; 300, optoelectronic composite cable; 210, laser group; 220, computing device; 410, beam splitting module; 420, optical detector group; 340, coupling module; 310, input optical fiber group; 211, laser; 311, input optical fiber; 320, control cable; 330, output optical fiber; 341, beam combining mirror; 342, beam combining element; 412, decoupling dichroic mirror; 413, decoupling mirror; 411, environmental filter; 41 4. 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 reflector; 150. Electric zoom module; 160. Optogenetics module; 161. Optogenetics light source; 162. Optogenetics focusing element; 163. Optogenetics input element; 350. Optogenetics optical fiber; 1611. Optogenetics laser; 1612. Beam combining element; 3421. Polarization beam splitter; 3422. Half glass slide. DETAILED DESCRIPTION

[0026] As mentioned above, in multiphoton imaging, it is often necessary to rely on the excitation light to trigger the multiphoton linearity to obtain the corresponding optical signal. For example, subharmonic generation (SHG) is a second-order multiphoton phenomenon, when two photons of the same frequency interact with the nonlinear material and merge into a new photon with twice the frequency. For another example, three-photon excited fluorescence (3PEF) requires three photons to be excited at the same time to produce a fluorescence signal.

[0027] In practical applications, based on the imaging characteristics of multi-photons, it is often used for depth imaging, that is, it is generally necessary to focus the excitation light containing multiple photons at a certain depth to trigger the corresponding multi-photon effect to generate the corresponding optical signal. Therefore, there are certain requirements for the frequency and power of the excitation light in multi-photons. In addition, it is further considered that multi-photons often rely on excitation light of a specific frequency band. As a result, in practical applications, multi-photon imaging can often only be achieved based on excitation light of a single frequency band. Especially in the process of miniaturization of imaging equipment, considering the complexity of its internal space, it cannot be transmitted.

[0028] To further illustrate the practical application of miniaturized devices using multiphoton imaging technology, the present application provides a schematic diagram of an application scenario of an imaging device ( Figure 1 ).in, Figure 1 The application scenario shown can reflect the process of the imaging device performing optical brain imaging on mice.

[0029] like Figure 1 As shown, the application scenario of the imaging device may include an imaging device 10 and an imaging object 20 .

[0030] The imaging device 10 is the executor of multiphoton imaging (for example, the imaging device 10 can be configured as a multiphoton microscope), which can release excitation light to trigger a multiphoton effect (such as a multiphoton 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] For example, the imaging device 10 can focus multiple photons of the excitation light on a point at the target focus depth inside the imaging object 20 based on the principle of multiphoton imaging to obtain the fluorescence signal of the point. Then, the focus position at the target focus depth is changed by scanning technology to perform plane scanning, thereby obtaining the fluorescence signals at various locations inside the imaging object 20 at the target focus depth to determine the fluorescence image.

[0032] The imaging object 20 may refer to an object for multiphoton imaging as required. For example, the imaging object 20 may be an in vitro sample, a living sample, etc. Exemplarily, the imaging object 20 in the present application may be an imaging object such as a mouse, a rabbit, a bird, a non-human primate macaque, a marmoset, etc. Among them, the imaging object 20 can generally be used to image the internal structure (brain nerves, spinal nerves, etc.) of the imaging object.

[0033] like Figure 1 As shown, Figure 2 The imaging object 20 in the method can be a mouse, and specifically, multiphoton imaging can be performed on the brain of the mouse. The imaging device 10 can be fixedly connected to the brain of the imaging object 20, and the imaging object 20 can move freely. The imaging device 10 can periodically scan the imaging object 20 during its free movement to obtain optical signals at various positions within a specific focal plane of the brain, thereby determining a time-series optical image of the position. The collected optical image can generally include the synapses 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 may be subjected to adaptation treatment. For example, the fixed connection between the imaging object 20 and the imaging device 10 may be subjected to skin preparation treatment. Figure 1For the mouse shown in , a cranial window can be set in the mouse brain during the fixed connection to improve the imaging effect. For another example, the imaging object 20 can be injected with a fluorescent probe in advance to perform multiphoton fluorescence imaging based on the fluorescent probe.

[0035] As a miniaturized front-end imaging device, the imaging device 10 provided in this specification may be configured in a split manner, further comprising an imaging probe 100 and an imaging host 200 , wherein the imaging probe 100 and the imaging host 200 may be communicatively connected based on an optoelectronic composite cable 300 .

[0036] The imaging probe 100 may be a device that releases excitation light and detects optical signals. The imaging probe 100 may convert the laser light generated by the laser source into excitation light that is focused on a focal plane and has scanning capability through an optical component. In some embodiments, the imaging probe 100 may be a contact imaging probe.

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

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

[0039] Based on the aforementioned imaging device 10, multiphoton imaging (such as multiphoton fluorescence presentation) can be achieved inside the imaging object 20. However, considering that the spectral bandwidth of the current optical fiber is limited, and the imaging device 10 often requires a highly stable and high-power laser, the optical fiber can usually only transmit a single wavelength of laser. For details, see Figure 3 Therefore, the optical fiber transmitting the excitation light in the imaging device 10 generally transmits only 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 to image multiple substances simultaneously. For example, when performing multiphoton imaging of the mouse brain, calcium ions, plaques, and synapses in the brain are all objects that need to be observed, but they often require excitation light of different wavelengths for excitation.

[0041] In order to realize excitation light of multiple wavelengths, multi-photon imaging of excitation light of multiple wavelengths can be realized by switching the laser source in the related art. That is, when performing multi-photon scanning, the laser source connected to the optical fiber can be switched at the host, so as to provide different wavelengths at different times to realize two-photon imaging of multiple wavelengths.

[0042] However, when time multiplexing is used for imaging, the optical signals corresponding to different wavelengths are not synchronized in time. When analyzing specific parameters (such as calcium transient changes), this method cannot meet actual needs. At the same time, considering the bandwidth of the optical fiber, when the above method is executed, it may affect the excitation efficiency of photons, so that some fluorescent substances are not excited under the optimal excitation conditions, affecting the imaging quality.

[0043] In addition, in traditional fluorescence imaging, there is also a technology that transmits different fluorescence light sources in one cable at the same time. Specifically, composite optical fibers can be used to transmit different optical signals to form a composite light field. However, the principle of conventional fluorescence imaging is completely different from that of multiphoton depth imaging. The transmission of the aforementioned composite optical fiber can only provide the field light source required for conventional fluorescence imaging, and cannot meet the requirements of multiphoton imaging.

[0044] Therefore, in order to realize excitation light of multiple wavelengths, the present application provides an imaging device based on miniature multiphotons. In particular, the present application is provided with multiple input optical fibers in the imaging probe, and multiple wavelengths of laser light are provided simultaneously during the multiphoton imaging process, so that the optical signal can simultaneously include optical signal components that respond to excitation light of different wavelengths, and simultaneously excite and detect different internal structures. In addition, inside the probe, lasers of various wavelengths are combined through a coupling module to form a composite laser, so that the subsequent processing of the composite laser is similar to the excitation light, further simplifying the internal structure of the imaging probe.

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

[0046] Exemplary Imaging Devices To further illustrate the imaging device capable of achieving multi-wavelength simultaneous excitation provided by the present application, the present application also provides a schematic block diagram of the interior of the imaging device ( Figure 2 ) to illustrate the transmission of optoelectronic signals (especially lasers of various wavelengths) within the imaging device.

[0047] like Figure 2 As shown, the actual functional modules in the imaging device may 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.

[0048] It should be noted that, considering that the imaging host and the imaging probe in the aforementioned imaging device are actually a receiving structure for receiving components / modules, Figure 2 The imaging host and the imaging probe are not shown in the illustrated structure.

[0049] like Figure 2 As shown, 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 affect the release of lasers of a wavelength corresponding to the control signal and send it to the coupling module. The coupling module can perform spatial coupling processing on each laser so that it can be combined into a composite laser and transmitted to the imaging device. The imaging device can convert the composite laser into an excitation light that can trigger a multiphoton effect, and apply it to a certain depth of the imaging object, thereby triggering a multiphoton effect at this depth to collect the corresponding optical signal.

[0050] Taking into account that the multiphoton effect triggered by lasers of different wavelengths generally presents itself as optical signals of different wavelengths, the aforementioned excitation light containing different components, when triggering the multiphoton effect, generates an optical signal that also often contains different wavelengths. Therefore, after 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 multiple optical signal beams. The optical 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 optical 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 based on the received optical signal.

[0051] Considering that multiphoton depth imaging can generally only excite a single point, that is, during imaging, the imaging device often needs to focus the excitation light on the focal position inside the imaging object. In order to achieve optical imaging of a specific area, the focal position can be changed by an electronic control device in the imaging device in the present application. Specifically, the scanning process of multiphoton depth imaging is generally horizontal scanning, that is, the imaging device can often focus the excitation light on the focal position of the focal plane inside the imaging object, thereby achieving scanning of each point on the focal plane by changing the focal position.

[0052] Therefore, if Figure 2 As shown, the processing module can generate a scanning control signal during imaging and convert it into a galvanometer control module through the scanning control module and apply it to the imaging device to change the position of the excitation light in the focal plane. In the processor, the intensity of the timing light signal can be determined according to the scanning control signal to align with each position to render the optical image.

[0053] In the present application, the focal plane can also be changed by the imaging device. At this time, the processing module can also generate a depth control signal to send to the scanning control module to generate a zoom control signal of the imaging device to control the imaging depth of the imaging device. In some embodiments, three-dimensional reconstruction can be performed based on optical images at different depths to determine a three-dimensional image.

[0054] In this application, Figure 2 The processing module, signal collection module, scanning module and laser group can be integrated in the imaging host, and the imaging device and coupling module can be integrated in the imaging probe. Considering the separate design of the imaging probe and the imaging host, multiple output optical fibers can be set between the imaging host and the imaging probe to realize the transmission of each laser.

[0055] As an exemplary illustration only, in the practical application of two-photon fluorescence microscopy in the field of brain imaging of mice, the aforementioned laser group includes at least one of a first laser for releasing 780 nm laser, a second laser frequency band component for releasing 920 nm laser, and a third laser for releasing 1030 nm (or 1064 nm) laser. Then the input fiber group for connecting the imaging host and the imaging probe may include three corresponding optical fibers. Among them, each optical fiber in the input fiber group can have a higher transmission effect (such as low spherical aberration and high power transmission efficiency) for the corresponding wavelength. Exemplarily, each optical fiber in the input fiber group can be configured as a bandgap hollow-core photonic crystal fiber or an antiresonant hollow-core photonic crystal fiber, and has a better transmission effect at the corresponding wavelength after optical design.

[0056] The 780 nm excitation light is used to excite the fluorescence information of amyloid plaques in the imaging object. The fluorescent probe of the 780 nm excitation light can be Methoxy-X04, and the wavelength of the optical signal of this probe is generally 460±25 nm. The 920 nm excitation light is used to excite the fluorescence information of mitochondria and their calcium ion channels in the imaging object. The 920 nm excitation light fluorescent probe can be AAV-mito-GCaMP6f+CaMKll-Cre, and the wavelength of the optical signal of this probe is generally 520±35 nm. The 1030 nm excitation light is used to excite the fluorescence information of neurons in the imaging object. The 1030 nm excitation light fluorescent probe can be AAV-hSyn-jRGECO1a, and the wavelength of the optical signal of this probe is generally 625±45 nm. For the specific content of each fluorescent probe and its mechanism of action, please refer to the relevant technology and will not be repeated here.

[0057] In some embodiments, the aforementioned beam splitting module and optical detector group can be set in the imaging probe or imaging host according to actual needs. Considering the differences caused by the two settings, the present application also provides two system block diagrams of the imaging device ( Figure 3, 4 ).in, Figure 3 It can reflect the imaging device when the beam splitting module and the optical detector group are integrated in the imaging host. Figure 4 It can reflect the imaging device when the beam splitting module and the optical detector group are integrated in the imaging host.

[0058] like Figure 3 As shown, the imaging device 10 may specifically include an imaging probe 100, an imaging host 200, and an optoelectronic composite cable 300. The imaging host 200 may include at least a laser group 210, a computing device 220, a beam splitting module 410, and an optical detector group 420. The optoelectronic composite cable 300 may include at least an input optical fiber group 310 for connecting the laser group 210 with a coupling module 340 in the imaging probe 100. Considering that the coupling module 340 is integrated in the imaging probe 100, in order to ensure the stability of its position, the input optical fiber group 310 is generally fixedly connected to the imaging probe 100, and it can also be understood as a part of the imaging probe 100.

[0059] The laser group 210 may reflect a collection of at least two lasers 211. Each laser 211 may release lasers of different wavelengths that meet the requirements of multiphoton imaging. The specific wavelength of the laser may be determined based on the multiphoton effect actually selected and its imaging purpose, and is not limited here. In some embodiments, the laser 211 in the present application may be a femtosecond laser. For example, the laser 211 may be a fiber femtosecond laser, a titanium sapphire femtosecond laser, or the like.

[0060] In some embodiments, after each laser in the laser group 210 releases laser light, it may pass through multiple optical elements and then enter the corresponding input fiber group 310. For example, the optical element may be a position sensor, a power sensor, etc., so as to detect the stability of the laser light.

[0061] The computing device 220 may be a collection of devices with computing capabilities in the imaging host. The computing device 220 may generally include a processing module, a signal processing module, and a scanning control module. Considering that the computing device 220 is composed of computing devices, the connection related to the computing device is generally realized through electrical cables.

[0062] The input fiber group 310 may specifically include a plurality of input fibers (referred to as input fibers 311 in the subsequent figures). The number of input fibers in the input fiber group 310 corresponds to the aforementioned laser, and is used to transmit the corresponding laser. In the application, each input fiber may be constructed based on the femtosecond value and wavelength of the laser it transmits, so as to be configured as a pulse fiber within the corresponding wavelength bandwidth, thereby having a good transmission effect (high transmission power and low spherical aberration) for the laser of the wavelength.

[0063] The coupling module 340 may be a set of optical elements for coupling multiple lasers into a composite laser, that is, the lasers input into the imaging probe 100 by the aforementioned input optical fiber group 310 are staggered in space, and in order to achieve the multi-photon effect corresponding to different wavelengths of simultaneous excitation, it is necessary to couple different lasers into one light beam. In some embodiments, the coupling module 340 may construct a coupling optical path through conventional beam combining elements (such as dichroic mirrors) and related optical elements, so that multiple lasers enter the coupling optical path to form a composite laser.

[0064] The imaging probe 100 may be built with an imaging device based on the multiphoton effect, which is used to convert the composite laser into excitation light when fixedly connected to the imaging object and focus it on the focal position in the focal plane inside the imaging object to trigger the multiphoton effect at the focal position. Considering that the composite laser contains lasers of multiple wavelengths, the excitation light can simultaneously excite multiphoton effects of different wavelengths.

[0065] It should be noted that the specific structure and composition of the imaging device can be adaptively modified based on the multiphoton effect adopted. Considering that the nonlinear optical effect based on multiphotons is generally activated based on multiple photons focusing on the same position at the same time, the aforementioned excitation light can be a parallel light beam that meets the requirements of multiphoton imaging. When the parallel light beams converge at one point, the multiphoton effect can be triggered to generate an optical signal.

[0066] Exemplarily, the aforementioned imaging device can be constructed based on the second harmonic generation or high harmonic generation effect, then the aforementioned excitation light can be a parallel light beam of a specific frequency that meets the requirements of multiphoton imaging, and when the parallel light beams converge at one point, harmonic generation can be triggered to generate new photons of twice the frequency / multiple frequencies. In addition, the aforementioned imaging device can also be constructed based on multiphoton effects such as multiphoton excited fluorescence and coherent anti-Stokes Raman scattering (CARS), and the specific structure can be adjusted according to the corresponding multiphoton effect. The imaging device based on the two-photon excited fluorescence effect will be described later, and other multiphoton imaging effects can be adaptively adjusted.

[0067] The control cable 320 can be used to transmit electrical signals between the imaging probe 100 and the imaging host 200. Among them, the control cable 320 can transmit the control signal of the scanning galvanometer to the imaging probe 100, so as to control the focusing position to realize the scanning of various positions in the focal plane. Specifically, considering that the multi-photon effect often requires the excitation light to be converged to the focusing position during deep imaging, in order to obtain the optical image of a specific area, the imaging probe 100 can control the focusing position of the excitation light in the focal plane inside the imaging object during the multi-photon imaging process, and collect the optical signals of various positions in the focal plane by scanning the focal plane. In addition, the control signals of other controllable devices of the imaging probe 100 (such as the electric zoom module) can also be transmitted by the control cable.

[0068] The output optical fiber 330 is used to transmit the optical signal generated by the multi-photon effect between the imaging probe 100 and the imaging host 200. Different from the composite laser, considering that the optical signal generated by the multi-photon effect is often determined by the actual situation of the corresponding position (such as the presence or absence of fluorescence, high-frequency signal, etc.), the output optical fiber 330 can generally transmit the optical signal of the corresponding frequency band, and there are no excessive requirements for its energy loss and other performance. Among them, further considering that the optical signal includes the signals corresponding to the lasers of each wavelength, the output optical fiber 330 can transmit the optical signal. Preferably, in order to reduce the influence of the output optical fiber 330 on the free movement of the imaging object, the output optical fiber 330 can be configured as a collection flexible fiber bundle (SFB).

[0069] The beam splitting module 410 can be a light beam splitting module constructed based on optical elements, which can be used to split the optical signal based on the nonlinear optical effect triggered by multi-photons to determine multiple optical signal beams of different wavelengths. Considering that the aforementioned composite laser includes at least two lasers of different wavelengths, the lasers of each wavelength can trigger the nonlinear optical effect of the corresponding wavelength / frequency band during the aforementioned multi-photon imaging, thereby including optical signals with different wavelength responses. Therefore, the response of the optical signal to the lasers of each wavelength can be separated by the beam splitting module 410, thereby obtaining multiple optical signal beams.

[0070] The optical detector group 420 may be a collection of optical detectors in the imaging host 200. The optical detector may correspond to the optical signal beam and be arranged toward the corresponding optical signal beam to detect the signal intensity of the corresponding optical signal beam. In the actual detection process, considering that the scanning process changes quickly, each optical detector in the optical detector group 420 may be configured as an analog signal-based sensor (such as a photomultiplier tube (PMT)), so as to output the time-series intensity of each optical signal beam (recorded as the time-series optical signal intensity).

[0071] The processing module in the computing device 220 can obtain the aforementioned time-series optical signal intensity through the signal collection module, and process the aforementioned time-series optical signal intensity according to the scanning control signal sent by the processing module through the scanning control module, thereby determining the optical signal image. Among them, in the optical signal image, optical signal beams of different wavelengths can be rendered by different colors, and the pixel value of each optical signal beam in the pixel of the image can be determined based on the signal intensity at the corresponding moment. That is, the intensity of the time-series optical signal on the focal plane is converted into the intensity distribution at the focal plane based on the scanning timing in the scanning control signal, and then converted into gray / color value based on the intensity to determine the image under the optical signal beam, and the images of each beam are superimposed to obtain the optical signal image.

[0072] In some embodiments, the aforementioned computing device 220 or its processing module may further process the optical signal image. For example, during actual detection, the optical signal image may be continuously acquired, and subsequently analyzed based on the time-series optical signal image. Exemplarily, in the present application, the transient change curve of calcium ions in the mouse body may be determined based on the aforementioned mouse intracranial imaging involving multiple lasers. For another example, the aforementioned processing module may also perform three-dimensional spatial reconstruction based on the depth control signal and the optical signal corresponding to the corresponding signal acquisition of the imaging probe to determine the corresponding three-dimensional model.

[0073] like Figure 4 As shown, the laser group 210 may include four lasers 211, wherein the wavelength of the laser light released by each laser 211 is different, and the specific wavelength can be determined according to the imaging requirements of the specific nonlinear optical effect. In addition, the number of lasers 211 can be adjusted according to actual needs. For example, in the above content, the number of lasers in mouse brain imaging can be three, Figure 4 The number of lasers 211 can be 4. Figure 5 There may be two lasers 211 .

[0074] In some embodiments, in order to achieve coupling of lasers released by different lasers 211, a plurality of coupling elements may be included in the aforementioned coupling module 340. The plurality of coupling elements are arranged along the corresponding laser direction to form a coupling optical path for outputting a composite laser. When lasers of different wavelengths enter the coupling optical path, they will overlap in space to form a composite laser.

[0075] In practical applications, coupling elements may generally include a reflective element (such as a reflector) for changing the propagation path of light and a beam combining element (such as a dichroic mirror, PBS, etc.) for beam combining. Specifically, for four lasers 211, the coupling module 340 may include a beam combining reflector 341 and three beam combining elements 342. The beam combining reflector 341 is arranged toward the laser 211 at the edge, and the beam combining element 342 of the middle laser 211 can reflect the corresponding laser and project the upstream laser, so that the laser is gathered downstream, so that the lasers of each laser 211 are gathered at the same position, thereby forming a composite laser.

[0076] It should be noted that the above is only an exemplary structure of a coupling optical path, and its specific structure can be adjusted according to actual needs, and its active coupling elements can also be selected according to needs. For example, PBS and lenses can also be used for beam coupling. For details, please refer to Figures 7A to 7D Related description.

[0077] Similar to the aforementioned coupling module 340, considering the reversibility of light propagation, the aforementioned beam splitting module 410 (also referred to as a decoupling module) can also be constructed based on similar optical elements to decouple different wavelength components in the optical signal. That is, the beam splitting module 410 can include multiple decoupling elements similar to the coupling module 340 to achieve decoupling. For example, Figure 4 As shown, the beam splitting module 410 may include a decoupling dichroic mirror 412 and a decoupling reflector 413. The optical path in the beam splitting module 410 is similar to that in the coupling module 340, and will not be described in detail herein.

[0078] In some embodiments, to ensure the purity of the optical signal beam splitting, a filter for filtering other light may be included in the aforementioned beam splitting module 410. Specifically, it may include an environmental filter 411 for filtering ambient light and a beam splitting filter 414 for filtering light other than the optical signal beam splitting.

[0079] Similar to the aforementioned laser group 210, the aforementioned optical detector group 420 may also be provided with a plurality of optical detectors 421. It should be noted that the wavelengths collected by the optical detectors 421 and the number of settings may be adjusted according to the actual nonlinear optical effects and imaging requirements, and are not strictly consistent with the number of lasers 211.

[0080] In some embodiments, considering the heat accumulation caused by the long-term operation of the optical detector 421 , a temperature control device is further provided in the imaging host 200 , wherein the temperature control device is at least used to reduce the operating temperature of the optical detector group 420 .

[0081] In some embodiments, the aforementioned temperature control device may be disposed in the space where the optical detector 421 is located, so as to achieve heat dissipation of the optical detector 421 by controlling the temperature of the space where the optical detector 421 is located. In some embodiments, the temperature control device may also directly contact each optical detector 421, so as to directly dissipate heat of the optical detector 421. For example, the temperature control device may be integrated inside the optical detector 421.

[0082] In some embodiments, considering that the optical signal collected in the imaging probe 100 may diverge along the propagation of the optical path, it is impossible to transmit through the aforementioned output optical fiber 330 (or cannot enter Figure 4 In the beam splitting module 410), a collecting device may also be provided in the imaging probe 100 (see the following Figure 5 The collecting device may be constructed based on a lens, so as to perform convergence processing on the optical signal transmitted through the collecting device.

[0083] Specifically, the lens constituting the aforementioned collecting device can be configured to reduce the focus of the optical signal on the surface of the output optical fiber 330 to 1 mm by adopting an aspherical lens, thereby allowing the use of a thinner output optical fiber 330 diameter to minimize obstruction to the movement of the imaging object.

[0084] 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, for details, see Figure 5 .

[0085] like Figure 5 As shown, the beam splitting module 410 and the optical detector group 420 can be directly integrated into the imaging probe 100. In this case, the imaging probe 100 does not need to be provided with the aforementioned output optical fiber 330, and the optical signal is transmitted to the beam splitting module 410 through the internal optical path. It is only necessary to transmit the time-series optical signal intensity of the optical detector group 420 to the signal collection module through a cable (referred to as a transmission cable).

[0086] Considering that the multi-photon imaging device in the imaging probe 100 in the present application also needs to be connected to the aforementioned computing device 220 via a cable, the cable can be reused so that the transmission cable can be used as a control cable 320 to transmit the control signal of the multi-photon imaging device, and can also transmit the sensor signal of the optical detector group 420 to the outside.

[0087] In some embodiments, considering that the imaging probe 100 itself has a small volume, each optical detector in the optical detector group 420 integrated in the imaging probe 100 can be configured as a micro-detector capable of collecting light signal intensity, so as to reduce the volume of the imaging probe 100. Among them, the micro-detector can include but is not limited to a silicon photomultiplier tube (SiPM), a micro photomultiplier tube, a photodiode (PD), an avalanche photodiode (APD), a single photon avalanche diode (SPAD), etc.

[0088] Therefore, the aforementioned Figure 1 to Figure 5 The imaging device based on multiphoton deep imaging shown in the figure realizes a miniaturized imaging probe by adopting a split structure. During imaging, the imaging probe can be fixedly connected to the imaging object, and the multiphoton effect is excited inside the imaging object by the excitation light to collect the optical signal inside the imaging object to detect the internal situation inside the imaging object. In particular, the present application is provided with multiple input optical fibers in the imaging probe, and multiple wavelengths of lasers are provided simultaneously during the multiphoton imaging process, so that the optical signal can simultaneously include optical signal components that respond to excitation lights of different wavelengths, and simultaneously excite and detect different internal structures. In addition, inside the probe, lasers of various wavelengths are combined by a coupling module to form a composite laser, whereby the subsequent processing of the composite laser is similar to the excitation light, further simplifying the internal structure of the imaging probe.

[0089] Exemplary Imaging Probes To further illustrate the light path propagation during multiphoton imaging, the present application also provides a structural schematic diagram of an imaging probe ( Figure 5 , Figure 6 ).in, Figure 5 , Figure 6 A schematic cross-sectional view of the imaging probe 100 may be presented. Figure 5 It can reflect the imaging probe when the aforementioned beam splitting module and the optical detector group are arranged in the imaging host. Figure 6 It can reflect the imaging probe when the beam splitting module and the optical detector group are arranged in the imaging probe.

[0090] like Figure 5 As shown, there is an excitation light path connecting the first end and the second end of the imaging probe 100 in the body of the imaging probe 100, wherein the first end is used to be fixedly connected to the imaging object, and the input optical fiber is connected to the imaging probe at the second end for releasing the composite laser at the second end.

[0091] Based on the aforementioned excitation optical path, the imaging device of the imaging probe 100 may include a plurality of input optical fibers 311 arranged in sequence at the second end along the propagation direction of the composite laser, a coupling module 340, a collimation module 110, a scanning module 120 and a lens module 130, an objective lens module 140 arranged at the first end, and a collection module 430.

[0092] In the excitation light path, multiple input optical fibers 311 can provide lasers of different wavelengths and are coupled into a composite laser through a coupling module 340. The collimation module 110 can be used to convert the composite laser into an excitation light. Among them, the excitation light can release multiple photons at the same time, which can be understood as a parallel light beam with the same wavelength component. The scanning module 120 can be used to control the emission angle of the excitation light, and 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 a focal position corresponding to the emission angle in the internal focal plane of the imaging object.

[0093] Specifically, considering the specific principle of multi-photon depth imaging, that is, multiple photons can be used to focus on the same position during imaging, thereby stimulating the nonlinear optical effect (such as fluorescence effect) at that position. In the aforementioned multi-photon imaging device, the collimation module 110 can convert the composite laser into an excitation light containing parallel beams, wherein each parallel 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 beam is only used to illustrate that the excitation light provides multiple photons at the same time. In actual applications, the specific split beams of the excitation light can be configured with different refractive powers, resulting in it being non-parallel.

[0094] Based on the aforementioned optical path, it can be known that the excitation light including multiple wavelengths of excitation light passes through the collimation module 110, the lens module 130 and the objective lens module 140. Considering that different wavelengths may have differences when passing through optical elements, the aforementioned collimation module 110, the lens module 130 and the objective lens module 140 may each include at least one set of doublet lenses. Among them, the doublet lens is an optical lens composed of two lenses glued together, which is used to correct spherical aberration, coma and chromatic aberration. That is, the propagation differences of excitation light of different wavelengths in the composite laser can be eliminated by adjusting the parameters of the dual-core lens.

[0095] Specifically, in order to achieve multi-color excitation, several groups of achromatic doublet lenses are introduced in the collimation module 110, the lens module 130 and the objective lens module 140. And by using various optical material combinations with different dispersions, the system achieves achromaticity in the 780nm-1030nm band.

[0096] like Figure 5 As shown, when the beam splitting module and the optical detector group are arranged in the imaging host, the imaging probe 100 only needs to collect the corresponding optical signal and transmit it to the output optical fiber 330. That is, the aforementioned imaging probe 100 may also include a collection optical path connecting the first end and the second end, and the output optical fiber 330 is arranged at the opening of the collection optical path at the second end, so that the optical signal collected by the aforementioned objective lens module 140 is directly transmitted to the output optical fiber 330 through the collection optical path. Among them, the aforementioned collection module 430 can be arranged in the middle of the collection optical path to focus the optical signal on the surface of the output optical fiber 330.

[0097] In addition, considering that the excitation light path and the collection light path multiplex the aforementioned objective lens module 140, a beam splitting element such as a dichroic mirror can be set in the aforementioned objective lens module 140 to achieve multiplexing of the light path without crosstalk. For more information about the aforementioned coupling module 340, please refer to Figure 7A to Figure 7D Related description.

[0098] For further explanation, the beam splitting module 410 and the optical detector group 420 are integrated in the imaging probe, see Figure 6 .exist Figure 6 In the embodiment, the beam splitting module 410 and the optical detector group 420 are integrated in the imaging probe 100 .

[0099] 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 two optical detectors can be configured. 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 sensor signal to the computing device.

[0100] In addition, considering that there is no need to transmit optical signals through output optical fibers, Figure 6 In the embodiment, the collection optical path may not be connected at the second end, but only connected to the interior of the imaging probe 100, so that the optical signal collected by the objective lens module 140 is directly transmitted to the beam splitting module. Figure 6 In the embodiment, 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 .

[0101] In some embodiments, considering that only two optical detectors are involved in FIG. 7 , the beam splitting module in FIG. 7 may directly include a decoupling dichroic mirror 412 for beam splitting. To filter optical signals of other wavelengths, the beam splitting module may further include an environmental filter 411 for filtering ambient light and a beam splitting filter 414 for filtering light other than the optical signal beam splitting. Specifically, considering that the beam splitting filter 414 may allow the optical signal beam splitting to pass through, the aforementioned environmental filter 411 may also be omitted.

[0102] In some embodiments, to adjust the imaging effect, the objective lens module 140 may include a plurality of candidate objective lenses, wherein each of the plurality of candidate objective lenses has a different focusing position for the excitation light of the same propagation angle, so as to present a different display field of view. To assemble each candidate objective lens, the first end of the imaging probe 100 may be detachably connected to each candidate objective lens, wherein the candidate objective lens currently assembled at the first end may be recorded as the target objective lens.

[0103] To further illustrate the specific implementation of different field of view ranges, the present application also provides a schematic diagram of the optical path of the excitation light in the imaging probe based on different candidate objective lenses ( Figure 8 ).

[0104] like Figure 8 As shown, the optical path schematic diagram may include a scanning galvanometer 121, a lens module 130, and a plurality of candidate objective lenses (respectively denoted as HR objective lens, U objective lens, and LF objective lens). Figure 8 As shown, the scanning galvanometer 121 can adjust the emission angle of the excitation light, that is, Figure 8 The light of each color has different exit angles and enters different candidate objective lenses through the same lens module 130.

[0105] In the excitation light of each color in the figure, the angle between the red light and the first direction (i.e., the propagation angle) is 0, and the propagation angle of the green light is smaller than that of the blue light. In the focal plane of each candidate objective lens, the red light is focused on the center point of the focal plane, and the focus position of the green light is closer to the center point than that of the blue light.

[0106] Among different candidate objective lenses, the larger the field of view, the smaller the angle displacement on the focal plane. Figure 8 As shown, the distance of the blue light of the HR objective lens relative to the center point is smaller than the distance of the blue light of the U objective lens relative to the center point, which is smaller than the distance of the blue light of the LF objective lens relative to the center point. Therefore, the corresponding field of view of the HR objective lens is larger than the field of view of the U objective lens, which is larger than the field of view of the LF objective lens.

[0107] In some embodiments, the parfocal distances of the candidate objective lenses may be the same, thereby ensuring that the focal plane positions of the candidate objective lenses for the excitation light are the same. In some embodiments, an objective lens length adapter ring and / or a length adjustment device are provided on the candidate objective lens or the first end, wherein the objective lens length adapter ring and / or the length adjustment device are used to match the parfocal distances of different objective lenses.

[0108] In some embodiments, in order to ensure the performance of the candidate objective lens and reduce the parameter adjustment process, the candidate objective lens includes a common structure and an adjustment structure, wherein the common structure is shared by multiple candidate objective lenses, the adjustment element is used to adjust the focus position of the excitation light, and the common element at least includes a doublet lens. Figure 8 The lens types contained in each candidate objective are the same, and they are only adjusted in size.

[0109] Based on the aforementioned doublet isochromatic setup, the remaining lateral chromatic aberration is less than 0.5 microns at half the field of view of each candidate objective. The remaining axial chromatic aberration is about 1 micron in both the 780-920 nm and 920-1030 nm bands for the HR and U objectives, and about 1 micron in the 780-920 nm band and 3 microns in the 920-1030 nm band for the LF objective. In practical applications, the chromatic aberration is so small that it can be considered as the excitation of nonlinear optical effects at the same position.

[0110] In some embodiments, the aforementioned candidate objective lenses can also be designed to eliminate spherical aberration based on actual applications. Specifically, in the actual deep brain imaging process, since the refractive index of the cortex (about 1.38) is higher than the refractive index of water (about 1.33), the spherical aberration increases with the increase of the imaging depth in the brain tissue. Therefore, the optical path of the HR / U / LF candidate objective lenses is optimized so that the spherical aberration introduced in the cortical depth range of 0-800 microns 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 targets is expanded to 0.74, 0.67 and 0.4 respectively, so that it can cooperate with the collection module 430 in the figure, so that the focus of the optical signal on the surface of the output optical fiber 330 is reduced 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.

[0111] In practical applications, considering that in the scanning module 120, the excitation light entering the scanning galvanometer at a large angle may cause scanning distortion, the propagation direction can be adjusted by a reflector (referred to as a scanning reflector) to reduce the angle of the excitation light entering the scanning galvanometer to be as acute as possible.

[0112] In order to achieve the excitation light entering the scanning galvanometer at an acute angle, there are two technical directions inside the imaging probe. First, at least one reflector (referred to as a scanning reflector) can be set between the scanning galvanometer and the collimation module to adjust 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 tilted so that the excitation light itself has a certain inclination angle, thereby entering the scanning galvanometer at an acute angle.

[0113] In practical applications, the above technical directions can be combined to form three implementation methods. Figures 9A to 9C These three implementations are reflected.

[0114] like Fig. 9A As shown, the scanning module 120 may include a scanning galvanometer 121 and a scanning reflector 122. The scanning reflector 122 is disposed 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 setting angle of the scanning reflector 122 may be adjusted based on the position of the scanning galvanometer 121 and the excitation light so that the excitation light propagates in the direction shown in the figure.

[0115] like Fig. 9BAs shown, the scanning module 120 may only include the scanning galvanometer 121. The input fiber group 310 has a second angle with the second direction (i.e., the vertical direction, the objective lens direction) at the second end, so that the composite laser enters the imaging probe along the second angle. Correspondingly, the collimation module is arranged downstream of the connection point of the second end of the input fiber along the second angle, and is used to convert the composite laser along the second angle into the excitation light along the second angle. Fig. 9B As shown, the second angle can be set directly based on the setting direction of the scanning galvanometer mirror 121, so that the excitation light enters the scanning galvanometer mirror 121 at an acute angle.

[0116] Combined with the above Fig. 9A as well as Fig. 9B The settings, in Fig. 9C In the embodiment, the scanning reflector 122 is provided, and the input optical fiber group 310 has a second 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 reflector 122 on the propagation, Fig. 9C The angle direction of the second angle in Fig. 9B different.

[0117] In particular, the included angle of the scanning galvanometer 121 along the first direction may also produce scanning distortion. Preferably, the included angle between the scanning galvanometer and the first direction is 20°, wherein the first direction is the extension direction of the lens module 130 .

[0118] In some embodiments, in practical applications, multiphoton depth imaging can also change the focal plane position to obtain optical images at different depths, thereby performing three-dimensional modeling. In practical applications, the aforementioned change in focal plane position is often achieved through a device such as a translation stage. However, considering the size of the imaging probe itself, when changing the focal plane position, it is not possible to use a translation stage to achieve the relative position of the imaging probe and the imaging object to achieve the change in the focal plane position.

[0119] In order to achieve zooming of the imaging probe, the present application creatively changes the internal optical path of the excitation light in the imaging probe. Fig.10 shown.

[0120] like Fig.10 As shown, the imaging probe 100 provided in the present application may be provided with an electric zoom module 150 between the aforementioned collimation module 110 and the scanning module 120 .

[0121] The electric zoom module 150 may be an optical device for changing the diopter of the excitation light. For example, the electric zoom module 150 may be constructed by an electric zoom device. For example, a liquid zoom lens, a piezoelectric zoom lens, etc. may be used.

[0122] That is, considering the principle of multi-photon depth imaging, the excitation light is often configured as a parallel beam, and the electric zoom module 150 can change the diopter of the parallel beam. The diopter of the parallel beam refers to the refraction ability of the optical system to the incident parallel beam, which is used to indicate the degree of focusing or divergence of the beam after passing through the optical element.

[0123] As mentioned above Figure 8 As shown, when the excitation light is a parallel beam, the objective lens module 140 will converge the parallel light to the corresponding position in the focal plane. When the aforementioned parallel beams diverge or converge in advance, based on the unchanged refractive power of the objective lens module 140 itself, the corresponding excitation light will converge later or in advance, thereby changing the depth of the focal plane.

[0124] In some embodiments, the focal plane depth changes caused by each diopter can be calibrated in advance. Therefore, in actual control, the electric zoom module can be connected to 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 correspondence, thereby adjusting the focal plane depth.

[0125] In the actual application of mouse brain imaging, the electric zoom module can achieve a focal plane change of 0μm~850μm. In the above scanning process, after completing the scanning of one position, the focal plane depth can be changed by the electric zoom module to determine the fluorescent images of multiple depths. Thus, the region can be three-dimensionally modeled based on the fluorescent images of each depth. For example, the present application can achieve three-dimensional modeling of amyloid plaques, mitochondria, calcium ion channels and neurons in mice within 0μm~850μm.

[0126] In summary, the imaging device based on the aforementioned design can realize multi-color excitation, imaging depth extension, and switching between targets of different magnifications to achieve an expandable field of view.

[0127] In some embodiments, considering that there may be photosensitive proteins (such as ChR2 and NpHR) in the imaging object, the imaging light weighing of the aforementioned multiphoton depth imaging (especially multiphoton fluorescence excitation) can be used to perform optogenetic stimulation on the photosensitive proteins, thereby activating or inhibiting specific neurons. Among them, optogenetic stimulation generally refers to the use of a light beam of a specific wavelength to stimulate the corresponding photosensitive protein.

[0128] Therefore, considering that the present application can detect optical images of brain nerves (such as mouse brain nerves), an optogenetic light beam is applied simultaneously during imaging to achieve optogenetic stimulation.

[0129] For example, during the aforementioned two-photon brain imaging, a 635nm LED light source can be used to stimulate the inhibitory optogenetic protein ChR2. At this time, two-photon fluorescence excitation can use 920nm to image the GCaMP green fluorescent indicator. In addition, to avoid the influence of the aforementioned LED light source and the collection of fluorescence signals, a filter (such as the aforementioned environmental filter 411) can be used at the beam splitting module to filter out the 635nm LED light.

[0130] For another example, during the aforementioned two-photon brain imaging, a 488nm LED light source can be used to stimulate the excitatory optogenetic protein NpHR. At this time, two-photon imaging of the jRGECO red fluorescent indicator is performed using 1030nm. In addition, to avoid the influence of the aforementioned LED light source and the collection of the fluorescent signal, a filter (such as the aforementioned environmental filter 411) can be used at the beam splitting module to filter out the 488nm LED light.

[0131] To achieve the aforementioned optogenetic stimulation, an optogenetic module may also be provided in the aforementioned imaging probe 100. To illustrate the specific structure of the optogenetic module, the present application also provides a variety of structural schematic diagrams (eg Figures 11A to 11C ).

[0132] See also Fig.11A , the aforementioned imaging probe 100 may also include an optogenetic module 160. Among them, the optogenetic module 160 is used to release the optogenetic light beam and merge it into the excitation light path at the objective lens 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 simultaneously with the excitation light. Taking into account the implementation principle of optogenetic stimulation, in order to ensure that each position in the focal plane is optogenetically stimulated, the aforementioned optogenetic stimulation area can include the focal plane area formed by the excitation light.

[0133] In some embodiments, to achieve the generation of the aforementioned optogenetic light beam, the aforementioned optogenetic module 160 may include an optogenetic light source 161, an optogenetic focusing element 162, and an optogenetic input element 163. Among them, the optogenetic light source 161 is used to release the optogenetic light beam, the optogenetic focusing element 162 is used to converge the optogenetic optical fiber, and the optogenetic input element 163 is arranged based on the objective lens module 140 to input the optogenetic light beam into the objective lens module 140.

[0134] Considering that the optogenetic light beam covers the focal area of ​​the excitation light at the focal plane, the aforementioned optogenetic light source can generally be configured as a surface light source. Fig.11AIn the embodiment, the optogenetic light source 161 may be an optogenetic laser that can release an optogenetic light beam. For example, the optogenetic light source 161 may be an LED light source of a specific wavelength. Further considering the divergence of the surface light source, the optogenetic focusing element 162 may focus the optogenetic optical fiber to meet the optogenetic stimulation requirements.

[0135] The aforementioned optogenetics input element 163 can be constructed based on a conventional light beam combining element. Fig.11A As shown, the aforementioned optogenetics input element 163 can be configured as a dichroic mirror. Considering that the optogenetics input element 163 and the optical signal optical path overlap, the dichroic mirror can be configured based on the wavelength of the optogenetics light beam so that it can reflect the optogenetics light beam to enter the objective lens module 140. Specifically, the optogenetics input element 163 is generally disposed between the collection module 430 and the objective lens module 140.

[0136] In some embodiments, taking into account that optogenetic light beams of different wavelengths may be used in optogenetic stimulation (such as the aforementioned brain imaging scenario using two different light beams), in order to match the application in this scenario, the optogenetic light source 161 can provide at least two optogenetic light beams.

[0137] at this time, Fig.11A The optogenetic light source 161 shown can be configured as a replaceable optogenetic laser. The replaceable optogenetic laser is detachably connected to the optogenetic beam imaging probe 100 to provide a corresponding optogenetic beam. That is, when the wavelength of the optogenetic beam is changed, the optogenetic light source 161 can be replaced with a replaceable optogenetic laser of the corresponding wavelength through the aforementioned detachable connection.

[0138] In some embodiments, in order to achieve 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. Fig. 11B Reflects the structure when the optogenetic light source is configured as an optogenetic fiber, Fig. 11C Reflects the structure when the optogenetic light source is configured as an optogenetic laser group.

[0139] like Fig. 11B As shown, an optogenetic fiber 350 may also be provided on the imaging probe 100, and the portion of the optogenetic fiber 350 in the imaging probe 100 may be abstracted as the aforementioned optogenetic light source 161. The optogenetic fiber 350 may be connected to a plurality of optogenetic lasers in the imaging host, and during optogenetic stimulation, the optogenetic light beam of the corresponding wavelength may be released by controlling the operation of the corresponding optogenetic laser.

[0140] like Fig. 11CAs shown, an optogenetic laser group (i.e., a plurality of composite optogenetic lasers) can also be directly disposed in the imaging probe 100. Specifically, the aforementioned optogenetic light source 161 can include a plurality of optogenetic lasers 1611 and an optogenetic beam combining element 1612. Among them, the optogenetic laser 1611 can release optogenetic light beams of different wavelengths, and the optogenetic beam combining element 1612 can combine the optogenetic light beams released by each optogenetic laser 1611 to a designated position. Among them, in actual application, the specific beam combining structure of the aforementioned optogenetic beam combining element 1612 can refer to the relevant description of the aforementioned coupling module 340, which is the same in principle. For example, the aforementioned optogenetic beam combining element 1612 can be configured as a dichroic mirror.

[0141] Exemplary Coupling Modules Taking into account the actual structure of the coupling module, the propagation direction of the light beam emitted from the output end of the beam combining element is consistent with the propagation direction of the light beam incident from the first incident end, that is, the light is transmitted between the first incident end and the output end of the beam combining element, and reflected / refracted between the second incident end and the output end, thereby combining the light beams at the output end.

[0142] To further illustrate the processing of the coupling module for the excitation light of different wavelength bands, the present application also provides a schematic diagram of the optical path of multiple coupling modules ( Figure 7A to Figure 7D ). The following is combined Figure 7A to Figure 7D The structures of various coupling modules are described.

[0143] like Fig. 7A As shown, in Fig. 7A The coupling module shown can combine the excitation light of two wavelengths. Fig. 7A The coupling module shown may include a beam combining element 342 and a beam combining reflector 341, wherein one excitation light enters the beam combining element through the beam combining reflector, and the other excitation light directly enters the beam combining element, thereby achieving beam combining of two wavelengths of excitation light. Figure 7A to Figure 7C In the embodiment, the beam combining element 342 can be directly presented as a dichroic mirror, that is, its presentation style is similar to that of a reflector, and the light from one side (the second incident end) of the dichroic mirror will be reflected by the dichroic mirror (recorded as the output end) when it reaches the dichroic mirror, but the light entering from the other side (the first incident end) can pass through the dichroic mirror and be emitted from the output end. Among them, the light at the second incident end is generally 45° with the dichroic mirror, so as to ensure that its output direction coincides with the light entering from the first incident end.

[0144] In practical applications, considering that the input optical fiber in the aforementioned imaging probe is often arranged along the propagation direction of the excitation light, the disorder of the internal optical path is avoided, and the direction of the composite laser output by the aforementioned coupling module 340 can be consistent with the laser direction of the input optical fiber 311. Specifically, the input optical fiber group may include a beam combining optical fiber and an edge optical fiber, wherein the propagation direction of the laser in the beam combining optical fiber is consistent with the propagation direction of the composite laser. The edge optical fiber is an optical fiber distributed on the other side relative to the beam combining optical fiber. Based on the aforementioned beam combining module, the first incident end of the beam combining element corresponding to the beam combining optical fiber is toward the beam combining optical fiber, and the second incident end is toward the intermediate beam combining direction, and the first incident end of the beam combining element corresponding to the other optical fibers in at least two input optical fibers is toward the intermediate beam combining direction, and the second incident end is toward the corresponding optical fiber.

[0145] Exemplarily, the propagation direction of the laser in the combined optical fiber may be the second direction in the figure, and the intermediate combining direction is generally perpendicular to this direction and may be the positive and negative directions of the first direction. The following description will be made taking the negative direction of the first direction as an example.

[0146] To provide a coupling module that meets the above requirements, such as Figure 7B As shown, in Figure 7B In the structure shown, n-1 beam combining elements and n-1 beam combining reflectors can be provided for n excitation lights, wherein the first incident end of each beam combining element faces the beam combining fiber (the exit end faces the first incident end of the downstream beam combining element), and the second incident end faces the beam combining reflector, and the beam combining reflector corresponds to other optical fibers of each non-beam combining optical fiber, and is used to emit the corresponding excitation light to the second incident end of the beam combining element, so that the excitation light of each non-beam combining optical fiber is staggered in space and converged to the propagation direction of the excitation light of the beam combining optical fiber.

[0147] However, the above structures need to be staggered in space and still occupy a large space. In order to further reduce the space occupied by them. Figure 7C .in, Figure 7C With the aforementioned Figure 3 The coupling modules are similar, except that the orientation of the beam combining elements corresponding to the beam combining optical fibers is different. Figure 7C In the embodiment, the beam combining reflector is used to reflect the excitation light of the edge optical fiber to the intermediate beam combining direction and enter the first incident end of the beam combining element, the first incident end of the beam combining element corresponding to at least one propagation optical fiber (the optical fiber located between the beam combining optical fiber and the edge optical fiber) is arranged toward the intermediate beam combining direction, the second input end is arranged toward the excitation light of the corresponding propagation optical fiber, and the second incident end of the beam combining element corresponding to the beam combining optical fiber is arranged toward the intermediate beam combining direction.

[0148] In some embodiments, each of the aforementioned beam combining elements can be implemented by a dichroic mirror. Considering the structural requirements of the dichroic mirror, Figure 7CIn the embodiment, the dichroic mirror corresponding to the beam combining fiber is used to transmit the excitation light in the wavelength range corresponding to the beam combining fiber and refract the excitation light in other wavelength ranges, and the dichroic mirror corresponding to the propagation fiber is used to refract the excitation light in the wavelength range corresponding to the propagation fiber and transmit the excitation light in other wavelength ranges.

[0149] As an alternative embodiment, the aforementioned beam combining element may also be implemented based on a polarization beam splitter. Specifically, the beam combining element includes a polarization beam splitter and a half glass plate, wherein the polarization beam splitter is used to transmit a light beam in a first polarization direction and reflect a light beam in a second polarization direction, and the half glass plate is used to adjust the polarization direction, and the first polarization direction is perpendicular to the second polarization direction. Specifically, the coupling module constructed based on the aforementioned polarization beam splitter can be referred to. Fig.7D .

[0150] like Fig.7D As shown, Fig.7D The structure shown reflects the process of combining two excitation lights, which is different from Fig.7D , Fig.7D The beam combining elements used are a polarization beam splitter 3421 and a half glass slide 3422, wherein the polarization beam splitter 3421 can pass a light beam in a first polarization direction and reflect a light beam in a second polarization direction. Specifically, the initial state of the excitation light can be the first polarization direction, and when the excitation light passes through a half glass slide 3422, it changes to the second polarization direction, thus, beam combining can also be achieved based on the polarization beam splitter 3421.

[0151] It is particularly noted that the arrangement of the aforementioned half glass slide 3422 can be arranged according to the polarization direction of the actual excitation light, for example, Fig.7D Both excitation lights need to pass through the half glass slide 3422 to adjust their polarization directions. In other cases, when the polarization direction of the excitation light itself meets the requirements, the half glass slide 3422 may not be provided. The specific adjustment and addition and subtraction of components may be made according to the actual situation, which will not be described in detail here.

[0152] All the above optional technical solutions can be arbitrarily combined to form optional embodiments of the present application, and will not be described one by one here.

[0153] Those of ordinary skill in the art will appreciate that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0154] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0155] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms.

[0156] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0157] It should be noted that, in the description of this application, the terms "first", "second", "third", etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance. In addition, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0158] It should be noted that "miniaturization" in the present application means that the multiphoton microscopy system has little impact on the activities of the living body to be observed during the observation of the living body. For example, when the multiphoton microscopy system is fixed on the living body to be observed, the living body to be observed can still move freely.

[0159] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An imaging probe, characterized in that: The imaging probe is applied to an imaging device based on multiphoton depth imaging, the imaging probe is connected to an imaging host of the imaging device through an optoelectronic composite cable, and the imaging probe includes a housing, at least two input optical fibers, a coupling module, and an imaging device based on multiphoton depth imaging; One end of the housing is provided with a first opening for fixed connection with the life body to be tested, and the other end is provided with a second opening, the first opening is communicated with the second opening respectively; wherein the at least two input optical fibers and the coupling module are sequentially arranged in the second opening, and the imaging device is arranged at the first opening; The at least two input optical fibers, the coupling module, and the imaging device form an excitation light path, wherein the at least two input optical fibers are used to provide at least two lasers of different wavelengths, the coupling module is used to combine the at least two lasers into a composite laser, and the imaging device is used to convert the composite laser into excitation light; focus the excitation light on an internal focusing position of an imaging object to trigger a multiphoton effect at the internal focusing position; collect optical signals generated by the multiphoton effect; and change the internal focusing position to determine optical signals at various positions within a focal plane, wherein the optical signals at various positions within the focal plane are used for an optical image of the imaging object at the focal plane.

2. The imaging probe according to claim 1, characterized in that: The at least two input optical fibers include a beam combining optical fiber and an edge optical fiber, wherein a propagation direction of the excitation light in the beam combining optical fiber is consistent with a propagation direction of the composite laser; The coupling module comprises at least one beam combining reflector and at least one beam combining element, wherein the beam combining element corresponds to at least each input optical fiber other than the edge optical fiber among the at least two input optical fibers on a one-to-one basis; The beam combining element comprises a first incident end, a second incident end and an output end, wherein the light rays entering the beam combining element from the first incident end and the second incident end are emitted from the output end when the beam combining condition is satisfied, and the propagation direction of the light beam entering from the first incident end in the beam combining element remains unchanged; The first incident end of the beam combining element corresponding to the beam combining optical fiber faces the beam combining optical fiber, and the second incident end faces the intermediate beam combining direction; the first incident end of the beam combining element corresponding to the other optical fibers among the at least two input optical fibers faces the intermediate beam combining direction, and the second incident end faces the corresponding optical fiber.

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

4. The imaging probe according to claim 2, characterized in that: The beam combining element includes a polarization beam splitter and a half glass plate, wherein the polarization beam splitter is used to transmit a light beam in a first polarization direction and reflect a light beam in a second polarization direction, and the half glass plate is used to adjust the polarization direction, and the first polarization direction is perpendicular to the second polarization direction.

5. The imaging probe according to claim 1, characterized in that: The imaging device comprises a collimation module, a scanning module and a lens module which are sequentially arranged in the second opening along the propagation direction of the excitation light, and an objective lens module which is 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 emission angle of the excitation light, the lens module is used to transmit the excitation light to the objective lens module, and the objective lens module is used to focus the excitation light on a focal position corresponding to the emission angle in the internal focal plane of the imaging object; The collimation module, the lens module and the objective lens module each include at least one set of doublet lenses, which are used to eliminate the propagation difference of laser beams with different wavelengths in the excitation light.

6. The imaging probe according to claim 5, characterized in that: The objective lens module is detachably connected to the first end; The objective lens module includes a plurality of candidate objective lenses, each of which has a different focusing position for the excitation light with the same propagation angle, so as to present a different display field of view.

7. The imaging probe according to claim 6, characterized in that: The first end of the candidate objective lens or the imaging probe is provided with a parfocal distance adjustment device, wherein the parfocal distance includes an objective lens length adapter ring and / or a length adjustment device, and the focal planes of the candidate objective lenses during imaging are the same.

8. The imaging probe according to claim 5, characterized in that: The scanning module comprises a scanning galvanometer, wherein the scanning galvanometer is used to control the emission angle of the excitation light, and the angle between the excitation light at the scanning galvanometer and the scanning galvanometer is an acute angle; The scanning module further includes a scanning reflector, wherein the scanning reflector is arranged between the collimating module and the scanning galvanometer, and is used to change the propagation direction of the excitation light; and / or The input optical fiber has a second angle with the second direction at the second end, so that the excitation light enters the imaging probe along the second angle, and the collimation module is arranged downstream of the connection point of the input optical fiber at the second end along the second angle, for converting the excitation light into excitation light along the second angle, wherein the second direction is the extension direction of the objective lens module.

9. The imaging probe according to claim 8, characterized in that: The included angle between the scanning galvanometer and the first direction is 20°, wherein the first direction is the extension direction of the lens module.

10. The imaging probe according to claim 5, characterized in that: The imaging device also includes an electric zoom module arranged between the collimation module and the scanning module, and the electric zoom module is used to change the refractive power of the excitation light and adjust the focal plane depth of the excitation light to determine optical signals at different focal plane depths, wherein the optical signals at different focal plane depths are used to determine a three-dimensional model.

11. The imaging probe according to claim 5, characterized in that: The imaging device further comprises an optogenetic module, wherein the optogenetic module is used to release an optogenetic light beam and merge it into the excitation light path at the objective lens 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 optogenetic module includes an optogenetic light source, an optogenetic focusing element, and an optogenetic input element which are arranged in sequence, wherein the optogenetic light source is used to release the optogenetic light beam, the optogenetic focusing element is used to converge the optogenetic light beam, and the optogenetic input element is arranged based on the objective lens module to input the optogenetic light beam into the objective lens module.

12. The imaging probe according to claim 11, characterized in that: The optogenetic light source can provide at least two optogenetic light beams, and the optogenetic light source is configured as one of an optogenetic optical fiber, a replaceable optogenetic laser, and an optogenetic laser group; When the optogenetic light source is configured as the optogenetic optical fiber, the optogenetic optical fiber is connected to at least two optogenetic lasers in the imaging host for transmitting at least two optogenetic light beams; When the optogenetic light source is configured as the replaceable optogenetic laser, the replaceable optogenetic laser is detachably connected to the imaging probe to provide a corresponding optogenetic light beam; When the optogenetic light source is configured as the optogenetic laser group, the optogenetic laser group includes at least two optogenetic laser groups and at least one optogenetic beam combining element.

13. The imaging probe according to claim 1, characterized in that: The imaging probe further comprises a beam splitting module, at least two optical detectors and a transmission cable which are sequentially arranged along the propagation direction of the optical signal, wherein the transmission cable is connected to each optical detector, the optical signal beam splitting of the beam splitting module corresponds to the optical detector one by one, and the optical detector is configured as a micro detector; After being collected by the imaging device, the optical signal enters the beam splitting module, and is processed by the beam splitting module to form multiple optical signal beams. Each optical signal beam enters a corresponding optical detector to determine the signal strength of the optical signal beam, and then the signal strength of each optical signal beam at the current moment is transmitted through the transmission cable.

14. An imaging device based on multiphoton depth imaging and an imaging probe thereof, characterized in that: The imaging device comprises an imaging host, at least two lasers, a beam splitting module, at least two optical detectors and an imaging probe according to any one of claims 1 to 13; The imaging host at least accommodates the at least two lasers, and the lasers are used to provide laser light that meets the imaging requirements and transmit the laser light to the imaging probe through the corresponding output optical fiber in the imaging probe, wherein the wavelength of laser light released by each of the at least two lasers is different; The imaging probe has a built-in coupling module and an imaging device based on multi-photon depth imaging, wherein the coupling module is used to convert multiple lasers into a composite laser, and the imaging device is used to convert the composite laser into an excitation light; focusing the excitation light on an internal focus position of an imaging object to trigger the multi-photon effect at the internal focus position; collecting optical signals generated by the multi-photon effect; and changing the internal focus position to determine optical signals at various positions within a focal plane, wherein the optical signals at various positions within the focal plane are used for an optical image of the imaging object at the focal plane; The beam splitting module is used to split the optical signal into at least two optical signal beams, and the optical detector is used to receive the corresponding optical signal beams and detect the signal intensity of the corresponding optical signal beams, wherein the optical signal beams reflect the signal components of the optical signal generated by the multi-photon effect at each wavelength, the optical signal beams correspond one-to-one to the optical detectors, and the signal intensity of the optical signal reflects the pixel value of the corresponding pixel in the optical image.

Images