A two-photon microscope and its objective lens mechanical arm

Abstract

The present application provides a two-photon microscope and an objective lens manipulator thereof, belonging to the technical field of two-photon microscopes, wherein the objective lens manipulator comprises: a zoom lens, a 5-DOF optical path manipulator, a dichroic mirror, a photomultiplier tube, a laser rangefinder, an objective lens and a controller, wherein the controller is used to pre-collect multi-layer depth images of a target brain region as multi-layer depth reference images, trigger head motion compensation and cortical motion compensation; collect the displacement of the animal head relative to the objective lens in real time, and adjust the focal length of the zoom lens according to the displacement to achieve head motion compensation; collect the current depth image of the target brain region, and adjust the displacement of the objective lens in the X-axis, Y-axis and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation. The present application enables the objective lens to be freely adjusted at any angle, and can achieve head motion compensation and cortical motion compensation, greatly improving the convenience and imaging accuracy of the two-photon microscope.

Description

Technical Field

[0001] The present application relates to the technical field of two-photon microscopes, and in particular to a two-photon microscope and an objective lens robot arm thereof. Background Art

[0002] Two-photon microscopy is a new technology that combines laser scanning microscopy and two-photon excitation technology and can be used to image the animal cerebral cortex.

[0003] When used for imaging the cerebral cortex of awake experimental animals performing behavioral tasks, existing two-photon microscopes have several problems: existing two-photon microscopes usually have only four degrees of freedom (XYZR), and the animal's head needs to be rotated and translated to image any part of the animal's head. Since awake animal experiments require animals to perform training tasks, uncomfortable head position and angle will cause experimental difficulties and make it impossible to continue experimental research. There is a lack of head movement and cortical movement compensation systems, and the data collected after the image plane shifts during the experiment is invalid. Summary of the invention

[0004] The embodiment of the present invention provides a two-photon microscope and an objective lens mechanical arm thereof, so as to solve the problem of inconvenience in use of the existing two-photon microscope.

[0005] Therefore, the first aspect of the present application provides an objective lens mechanical arm of a two-photon microscope, comprising:

[0006] Zoom lens, 5-DOF optical path manipulator, dichroic mirror, photomultiplier tube, laser rangefinder, objective lens and controller;

[0007] The zoom lens is mounted on the light inlet of the 5-DOF optical path mechanical arm, and the objective lens is mounted on the light outlet of the 5-DOF optical path mechanical arm; the scanning laser of the two-photon microscope enters the 5-DOF optical path mechanical arm from the light inlet, enters the objective lens from the light outlet after passing through the refracted light path in the 5-DOF optical path mechanical arm, and is emitted from the objective lens to the target brain area of ​​the animal's head to generate fluorescence;

[0008] The dichroic mirror is arranged on the optical path between the objective lens and the light outlet; the dichroic mirror allows the scanning laser to pass through, and refracts the fluorescence collected by the objective lens from the dichroic mirror to the photomultiplier tube for imaging;

[0009] The laser rangefinder is installed close to the objective lens and is used to measure the displacement of the animal's head relative to the objective lens;

[0010] The controller is used to pre-collect multi-layer depth images of the target brain area as multi-layer depth reference images to trigger head motion compensation and cortical motion compensation; collect the displacement of the animal's head relative to the objective lens in real time, and adjust the focal length of the zoom lens according to the displacement to achieve head motion compensation; collect the current depth image of the target brain area, and adjust the displacement of the objective lens in the X-axis, Y-axis, and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation.

[0011] In a possible implementation, the 5-DOF optical path robotic arm includes:

[0012] The first rotation axis and the second rotation axis are orthogonally connected through the bogie, and the second rotation axis is connected to three orthogonal translation axes, namely the X-axis, the Y-axis, and the Z-axis; the first rotation axis and the second rotation axis are hollow rotation axes, the light inlet is arranged on the first rotation axis, and the objective lens is arranged on the Z-axis.

[0013] In a possible implementation, the 5-DOF optical path robotic arm further includes:

[0014] A plurality of reflecting mirrors are arranged on the refractive optical path in the 5-DOF optical path mechanical arm, so as to enable the scanning laser to enter the objective lens through the 5-DOF optical path mechanical arm.

[0015] In a possible implementation, the 5-DOF optical path robotic arm further includes:

[0016] Two angle sensors, respectively mounted on the first rotating shaft and the second rotating shaft, for measuring the rotation angles of the rotating shafts;

[0017] Three displacement sensors are respectively installed on the X-axis, Y-axis and Z-axis for measuring the displacement of the translation axis.

[0018] In a possible implementation, the variable focus lens is a liquid variable focus lens.

[0019] In a possible implementation, the controller collects the displacement of the animal's head relative to the objective lens in real time, and adjusts the focal length of the zoom lens according to the displacement to achieve head motion compensation, which specifically includes:

[0020] The liquid zoom lens is calibrated, and the calibration process is: the liquid zoom lens is installed in the optical path, the two-photon microscope is controlled to focus on a certain initial imaging plane, and the control current of the liquid zoom lens is changed in stages. I i , at each level of control current, the focal plane is moved back to the initial imaging plane through the translation stage, and the displacement of the translation stage is recorded as S i, each level of control current corresponds to a displacement; according to the data point ( I i ，S i ) Fitting the curve to get the function I = f(S) , at this time the calibration is completed;

[0021] When the head motion compensation trigger signal is received, the displacement of the animal's head relative to the objective lens is collected and the displacement is set as the zero point. S 0 ;

[0022] The displacement of the animal head relative to the objective lens is collected cyclically, and the displacement is recorded as the real-time displacement S. The amount of the real-time displacement offset from the zero point is recorded as ΔS = SS 0 , substitute into the function I = f(S) A control current of the liquid variable focus lens is obtained, and the liquid variable focus lens is controlled according to the control current, that is, the focal length of the variable focus lens is adjusted to achieve head movement compensation.

[0023] In a possible implementation, the frequency of the head motion compensation is set to be greater than 100 Hz.

[0024] In a possible implementation, the controller collects a current depth image of the target brain area, and adjusts the displacement of the objective lens in the X-axis, Y-axis, and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation, specifically including:

[0025] The depth reference image of each layer of the target brain area is recorded as R i , i Indicates the number of layers, i = -d~d, that is, d layers are collected along the Z axis at the target depth, and the depth interval is Δd ;

[0026] Collect the current depth image of the target brain area, denoted as F c ;

[0027] The depth reference image of each depth R i With the current depth image F c Perform two-dimensional cross-correlation calculation to obtain the cross-correlation diagram C i , C i The amplitude of the peak point on E i , E i The maximum value corresponds toR i Recorded as R m , which is the current depth image F c The depth position, at this time the compensation amount of the Z axis is Δz = -Δd*m ;

[0028] Will R m With the current depth image F c The cross-correlation graph is denoted as C m , C m The coordinates of the upper peak are marked as P m , the current depth image F c and R m When there is no offset in the X-axis and Y-axis directions, P m for C m The center point P 0 ; When there is an offset, according to P m Relative to the center point P 0 The displacement in the x and y directions obtains the current depth image F c The offset in the X-axis and Y-axis directions is recorded as Δx and Δy, and the compensation amounts of the X-axis and Y-axis are -Δx and -Δy.

[0029] In a possible implementation, the frequency of the cortical motion compensation is set to be less than 0.1 Hz.

[0030] The second aspect of the present application provides a two-photon microscope, comprising: a multi-channel objective lens robot arm of the two-photon microscope described in the first aspect.

[0031] The beneficial effects of this application are as follows:

[0032] The objective mechanical arm of the two-photon microscope provided by the present application includes: a zoom lens, a 5-DOF optical path mechanical arm, a dichroic mirror, a photomultiplier tube, a laser rangefinder, an objective lens and a controller, wherein the controller is used to pre-collect multi-layer depth images of the target brain area as multi-layer depth reference images, trigger head motion compensation and cortical motion compensation; real-time acquisition of the displacement of the animal head relative to the objective lens, and adjustment of the focal length of the zoom lens according to the displacement to achieve head motion compensation; acquisition of the current depth image of the target brain area, and adjustment of the displacement of the objective lens in the X-axis, Y-axis, and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation. Compared with the prior art, the objective mechanical arm of the present application allows the objective lens to be freely adjusted at any angle, and can achieve head motion compensation and cortical motion compensation, greatly improving the convenience and imaging accuracy of the two-photon microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] By reading the detailed description of the preferred embodiment below, various other advantages and benefits will become clear to those of ordinary skill in the art. The accompanying drawings are only used for the purpose of illustrating the preferred embodiment and are not to be considered as limiting the present application. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0034] Figure 1 A schematic structural diagram of an objective lens mechanical arm of a two-photon microscope provided by the present application is shown;

[0035] Figure 2 A schematic structural diagram of an objective lens mechanical arm of a specific two-photon microscope provided in the present application is shown;

[0036] Figure 3 A flow chart of an XYZ three-axis real-time correction algorithm provided by the present application is shown. DETAILED DESCRIPTION

[0037] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0038] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by technicians in the field to which this application belongs.

[0039] In addition, the terms "first" and "second" etc. are used to distinguish different objects, rather than to describe a specific order. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or units that are inherent to these processes, methods, products or devices.

[0040] This application designs and manufactures a high-precision 5-DOF adjustable two-photon microscope objective robot, which can realize free adjustment of the objective lens at any angle; it can measure and compensate for head movement in the Z-axis direction at high speed (>100 Hz) in real time, and measure and compensate for slow cortical drift (cortical movement) in the XYZ direction at low speed (0.1 Hz); it can realize two-photon microscopic imaging of any number of brain regions while keeping the animal's head still.

[0041] The objective lens mechanical arm of the two-photon microscope provided in the present application has a compact structure, flexible use, high control accuracy, simple operation, and easy expansion, and is very suitable for microscopic imaging of the head of awake large animals. Detailed description is given below with reference to the accompanying drawings.

[0042] Please refer to Figure 1 , Figure 1 A schematic structural diagram of an objective lens robot arm of a two-photon microscope provided in an embodiment of the present application is shown, wherein the objective lens robot arm comprises: a zoom lens 10, a 5-DOF optical path robot arm 20, a dichroic mirror 30, a photomultiplier tube 40, a laser rangefinder 50, an objective lens 60 and a controller 70. The controller 70 can control the zoom lens 10, the 5-DOF optical path robot arm 20, the photomultiplier tube 40 and the laser rangefinder 50.

[0043] The zoom lens 10 is installed at the light inlet of the 5-DOF optical path mechanical arm 20, and the objective lens 60 is installed at the light outlet of the 5-DOF optical path mechanical arm 20; the scanning laser of the two-photon microscope enters the 5-DOF optical path mechanical arm from the light inlet, enters the objective lens from the light outlet after passing through the refracted light path in the 5-DOF optical path mechanical arm, and is emitted from the objective lens to the target brain area of ​​the animal's head to generate fluorescence.

[0044] Figure 2 FIG. 1 is a schematic diagram showing the structure of a specific objective lens mechanical arm of a two-photon microscope provided in an embodiment of the present application. Figure 2 As shown, the 5-DOF optical path robot 20 comprises:

[0045] The first rotating axis 21 and the second rotating axis 22 are orthogonally connected by a bogie, and the second rotating axis 22 is connected to three orthogonal translation axes, namely X-axis, Y-axis and Z-axis; the first rotating axis 21 and the second rotating axis 22 are hollow rotating axes, the light inlet is arranged on the first rotating axis 21, and the objective lens 60 is arranged on the Z-axis of the above three orthogonal translation axes.

[0046] In some embodiments, the 5-DOF optical path robot 20 further includes: a plurality of reflectors, which are arranged on the refractive optical path in the 5-DOF optical path robot to enable the scanning laser to enter the objective lens through the 5-DOF optical path robot.

[0047] like Figure 2 As shown, three reflectors can be specifically arranged on the refracted light path, namely a first reflector 23, a second reflector 24, and a third reflector 25. The first reflector 23 is arranged at the intersection of two rotation axes, and the second reflector 24 and the third reflector 25 are arranged near three orthogonal translation axes. The scanning laser enters the objective lens 60 after being reflected by the three reflectors.

[0048] In some embodiments, Figure 2 As shown, the 5-DOF optical path robot 20 further includes: two angle sensors 26 and three displacement sensors 27 .

[0049] Specifically, two angle sensors 26 are respectively installed on the first rotation axis and the second rotation axis to measure the rotation angle of the rotation axis; three displacement sensors 27 are respectively installed on the X axis, Y axis, and Z axis to measure the displacement of the translation axis. The setting of two angle sensors and three displacement sensors can improve the control accuracy of each degree of freedom of the robot arm.

[0050] The present application adds the above-mentioned 5-DOF optical path mechanical arm at the light exit hole of the two-photon microscope, so that the objective lens can move with multiple degrees of freedom without affecting the imaging, and realizes that the objective lens can be freely adjusted at any angle, so that imaging of any part of the animal's head can be achieved without rotating and translating the object's head, thereby greatly improving the convenience of using the two-photon microscope.

[0051] The dichroic mirror 30 is disposed on the optical path between the objective lens 60 and the light outlet of the 5-DOF optical path mechanical arm 20. The dichroic mirror 30 allows the scanning laser to pass through, and refracts the fluorescence collected by the objective lens 60 from the dichroic mirror to the photomultiplier tube 40 for imaging. A filter is also disposed at the light inlet of the photomultiplier tube 40 in the fluorescence optical path to filter out stray light other than the fluorescence signal.

[0052] By converting the fluorescent signal into an electrical signal through the photomultiplier tube 40, quantitative analysis and image acquisition of the target brain area can be achieved.

[0053] The laser rangefinder 50 is installed close to the objective lens 60 and is used to measure the displacement of the animal's head relative to the objective lens 60 .

[0054] A specific implementation of the above objective lens mechanical arm is as follows:

[0055] like Figure 2 As shown, an existing two-photon microscope can be connected via a cantilever 28. The scanning laser of the two-photon microscope is emitted from its tube lens and enters the light inlet of the 5-DOF optical path mechanical arm 20 provided in the present application. A zoom lens 10 is installed on the light inlet, and the zoom lens 10 can be set as a liquid zoom lens. Directly below the light inlet is a hollow rotating shaft (first rotating shaft 21), and the first rotating shaft 21 directly below the light through hole and another hollow rotating shaft (second rotating shaft 22) orthogonal thereto are connected by a bogie. Angle sensors 26 are installed on both rotating shafts, and a first reflector 23 is set at the intersection of the two rotating shafts. The laser entering from the light inlet reaches the first reflector 23 along the first rotating shaft 21, and enters the second rotating shaft 22 after reflection. The second rotating shaft 22 is followed by three orthogonal translation axes, and the three translation axes are equipped with a high-precision electric displacement stage 29 (repeatability 1 micron) and a displacement sensor 27. The laser light enters the objective lens 60 after being reflected by the second reflecting mirror 24 and the third reflecting mirror 25 on three orthogonal axes.

[0056] A dichroic mirror box is arranged behind the objective lens 60, and a dichroic mirror 30 is built in, which allows the laser to pass through and reflects the fluorescence excited by the two-photon effect from the side of the dichroic mirror box to the photomultiplier tube 40. A high-speed micron-level laser rangefinder 50 is installed above the dichroic mirror box to measure the displacement of the animal head relative to the objective lens. The optical axis in the objective lens manipulator passes through the center of two rotating axes and three orthogonal axes. This layout allows the light to always enter the objective lens during the adjustment of the five axes, without adjusting the optical path after each adjustment of the angle and position of the objective lens. In addition, relay lenses can be set on the empty positions in the five axes to ensure that the scanning range of the laser remains unchanged.

[0057] The controller 70 is used to pre-collect multi-layer depth images of the target brain area as multi-layer depth reference images to trigger head motion compensation and cortical motion compensation. Head motion is the displacement of the animal head relative to the objective lens 60 measured by the laser rangefinder 50, that is, the movement in the Z-axis direction. Cortical motion is the movement of the cortex at a certain depth of the target brain area, which can move in the XYZ axis.

[0058] In the present application, the controller 70 can achieve high-speed Z-axis compensation through real-time, high-speed and high-precision measurement of the movement of the animal's head relative to the objective lens through a zoom lens, and can also achieve low-speed XYZ-axis slow drift motion compensation through image matching feedback.

[0059] Figure 3This is a flow chart of an XYZ three-axis real-time correction algorithm provided by the present application. Specific implementation process: the controller 70 first starts the image acquisition thread, which first acquires multi-layer depth reference images of the target brain area as a template for subsequent correction. Then a trigger signal is generated to trigger the high-speed Z-axis compensation thread and the low-speed XYZ-axis compensation thread to run.

[0060] Specifically, the controller 70 collects the displacement of the animal's head relative to the objective lens in real time, and adjusts the focal length of the zoom lens according to the displacement to achieve head movement compensation.

[0061] The specific process of the controller 70 performing head motion compensation is as follows:

[0062] High-speed Z-axis compensation is achieved through a liquid zoom lens. The deformation of the liquid zoom lens is determined by the control current and is nonlinear, so it needs to be calibrated in advance.

[0063] The liquid zoom lens is calibrated. The calibration process is as follows: the liquid zoom lens is installed in the optical path, the two-photon microscope is controlled to focus on a certain initial imaging plane, and the control current of the liquid zoom lens is changed in stages. I i , at each level of control current, the focal plane is moved back to the initial imaging plane through the translation stage, and the displacement of the translation stage is recorded as S i , each level of control current corresponds to a displacement; according to the data point ( I i ，S i ) Fitting the curve to get the function I = f(S) , at this time the calibration is completed;

[0064] When the head motion compensation trigger signal is received (the high-speed Z-axis compensation thread receives the trigger signal of the image acquisition thread), the displacement of the animal's head relative to the objective lens is collected and the displacement is set as the zero point S 0 , that is, the value measured by the current laser rangefinder is set as the zero point; the displacement of the animal head relative to the objective lens is cyclically collected, and the displacement is recorded as the real-time displacement S, and the amount of the real-time displacement offset from the zero point is ΔS = SS 0 , substitute into the function I = f(S) The control current of the liquid zoom lens is obtained, and the liquid zoom lens is controlled according to the control current, that is, the focal length of the liquid zoom lens is adjusted to achieve head motion compensation. Specifically, since the animal's head moves quickly, the frequency of the head motion compensation is set to be greater than 100 Hz.

[0065] Specifically, the controller 70 collects a current depth image of the target brain area, and adjusts the displacement of the objective lens in the X-axis, Y-axis, and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation.

[0066] In the present application, low-speed XYZ-axis cortical motion compensation is achieved by comparing the current depth image (denoted as Fc) with the multi-layer depth reference image R.

[0067] The specific process of the controller 70 performing cortical motion compensation is as follows:

[0068] The depth reference image of each layer of the target brain area is recorded as R i , i Indicates the number of layers, i = -d~d, that is, d layers are collected along the Z axis at the target depth, and the depth interval is Δd ; For example, the image at each depth of the multi-layer depth reference image is recorded as R i ( i =-5~5), target depth plane R 0 There are 11 planes in total, including 5 planes at the top and bottom. Generally, the interval between two depths is Δd Take 2 microns.

[0069] Acquire the current depth image of the target brain area F c ;

[0070] The depth reference image of each depth R i With the current depth image F c Perform two-dimensional cross-correlation calculation to obtain the cross-correlation diagram C i , C i The amplitude of the peak point on E i , E i The maximum value corresponds to R i Recorded as R m , which is the current depth image F c The depth position, at this time the compensation amount of the Z axis is Δz = -Δd*m ; m express E i The number of layers corresponding to the maximum value i .

[0071] Will R m With the current depth image F c The cross-correlation graph is denoted as C m , C m The coordinates of the upper peak are P m , the current depth image F c and R m When there is no offset in the X-axis and Y-axis directions, P m for C m The center point P 0 ; When there is an offset, according to P m Relative to the center point P 0 The displacement in the x and y directions obtains the current depth image F c The offset in the X-axis and Y-axis directions is recorded as Δx and Δy, and the compensation amounts of the X-axis and Y-axis are -Δx and -Δy.

[0072] The compensation values ​​-Δx, -Δy, and Δz are sent to the X, Y, and Z axis translation stages respectively to compensate for the cortical drift in the x, y, and z directions. Specifically, the cortical drift occurs relatively slowly, and the frequency of the cortical motion compensation is set to be less than 0.1 Hz.

[0073] The objective lens robot of the two-photon microscope provided in the present application allows the scanning laser of the two-photon microscope to reach the objective lens through five flexible and adjustable axes, thereby achieving adjustment of the angle and position of the objective lens without affecting imaging, and can also compensate for imaging deviations caused by head movement and cortical drift, thereby facilitating two-photon microscopic imaging of the head and brain regions of awake large animals.

[0074] The present application also discloses a two-photon microscope, which includes a multi-channel objective lens mechanical arm of the above two-photon microscope. For example, please refer to Figure 2 , an N-way objective lens robotic arm can be set up to facilitate two-photon microscopic imaging of multiple brain regions in the head of awake large animals.

[0075] The two-photon microscope provided in the present application can achieve two-photon microscopic imaging of any number of brain regions while keeping the animal's head still, which can solve the problem that existing two-photon microscopes cannot achieve synchronous imaging of multiple brain regions and cannot carry out synchronous experimental research on multiple brain regions.

[0076] It should be noted that the flowcharts and block diagrams in the accompanying drawings show the possible architecture, functions and operations of the systems, methods and computer program products according to multiple embodiments of the present application. In this regard, each box in the flowchart or block diagram can represent a module, a program segment or a part of a code, and the module, a program segment or a part of a code contains one or more executable instructions for implementing a specified logical function. It should also be noted that in some alternative implementations, the functions marked in the box can also occur in a different order from the order marked in the accompanying drawings. For example, two consecutive boxes can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram and / or flowchart, and the combination of the boxes in the block diagram and / or flowchart can be implemented with a dedicated hardware-based system that performs a specified function or action, or can be implemented with a combination of dedicated hardware and computer instructions.

[0077] 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.

[0078] In the several embodiments provided in the present application, it should be understood that the disclosed devices and methods can be implemented in other ways. The device embodiments described above are merely schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation. For example, 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 communication interfaces, and the indirect coupling or communication connection of devices or units can be electrical, mechanical or other forms.

[0079] 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.

[0080] In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit.

[0081] If the functions are implemented in the form of software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present application, or the part that contributes to the prior art or the part of the technical solution, can be embodied in the form of a software product. The computer software product is stored in a storage medium, including several instructions to enable a computer device (which can be a personal computer, server, or network device, etc.) to execute all or part of the steps of the method described in each embodiment of the present application. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk, and other media that can store program codes.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application, and they should all be included in the scope of the claims and specification of the present application.

Claims

1. An objective lens mechanical arm of a two-photon microscope, characterized in that: include: Zoom lens, 5-DOF optical path manipulator, dichroic mirror, photomultiplier tube, laser rangefinder, objective lens and controller; The zoom lens is mounted on the light inlet of the 5-DOF optical path mechanical arm, and the objective lens is mounted on the light outlet of the 5-DOF optical path mechanical arm; the scanning laser of the two-photon microscope enters the 5-DOF optical path mechanical arm from the light inlet, enters the objective lens from the light outlet after passing through the refracted light path in the 5-DOF optical path mechanical arm, and is emitted from the objective lens to the target brain area of ​​the animal's head to generate fluorescence; The dichroic mirror is arranged on the optical path between the objective lens and the light outlet; The dichroic mirror allows the scanning laser to pass through, and refracts the fluorescence collected by the objective lens from the dichroic mirror to the photomultiplier tube for imaging; The laser rangefinder is installed close to the objective lens and is used to measure the displacement of the animal's head relative to the objective lens; The controller is used to pre-collect multi-layer depth images of the target brain area as multi-layer depth reference images to trigger head motion compensation and cortical motion compensation; collect the displacement of the animal's head relative to the objective lens in real time, and adjust the focal length of the zoom lens according to the displacement to achieve head motion compensation; collect the current depth image of the target brain area, and adjust the displacement of the objective lens in the X-axis, Y-axis, and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation; The 5-DOF optical path robotic arm comprises: A first rotation axis and a second rotation axis are orthogonally connected through a bogie, and the second rotation axis is connected to three orthogonal translation axes, namely an X axis, a Y axis, and a Z axis; the first rotation axis and the second rotation axis are hollow rotation axes, the light inlet is arranged on the first rotation axis, and the objective lens is arranged on the Z axis; The controller collects a current depth image of the target brain area, and adjusts the displacement of the objective lens in the X-axis, Y-axis, and Z-axis directions according to the current depth image and the multi-layer depth reference image to achieve cortical motion compensation, specifically including: The depth reference image of each layer of the target brain area is recorded as R i , i Indicates the number of layers, i = -d~d, that is, d layers are collected along the Z axis at the target depth, and the depth interval is Δd ; Collect the current depth image of the target brain area, denoted as F c ; The depth reference image of each depth R i With the current depth image F c Perform two-dimensional cross-correlation calculation to obtain the cross-correlation diagram C i , C i The amplitude of the peak point on E i , E i The maximum value corresponds to R i Recorded as R m , which is the current depth image F c The depth position, at this time the compensation amount of the Z axis is Δz = -Δd*m ; Will R m With the current depth image F c The cross-correlation graph is denoted as C m , C m The coordinates of the upper peak are P m , the current depth image F c and R m When there is no offset in the X-axis and Y-axis directions, P m for C m The center point P 0; when there is an offset, according to P m Relative to the center point P 0 displacement in the x and y directions to obtain the current depth image F c The offset in the X-axis and Y-axis directions is recorded as Δx and Δy, and the compensation amounts of the X-axis and Y-axis are -Δx and -Δy.

2. The objective lens mechanical arm of the two-photon microscope according to claim 1, characterized in that: The 5-DOF optical path robotic arm further comprises: A plurality of reflecting mirrors are arranged on the refractive optical path in the 5-DOF optical path mechanical arm, so as to enable the scanning laser to enter the objective lens through the 5-DOF optical path mechanical arm.

3. The objective lens mechanical arm of the two-photon microscope according to claim 1, characterized in that: The 5-DOF optical path robotic arm further comprises: Two angle sensors, respectively mounted on the first rotating shaft and the second rotating shaft, for measuring the rotation angles of the rotating shafts; Three displacement sensors are respectively installed on the X-axis, Y-axis and Z-axis for measuring the displacement of the translation axis.

4. The objective lens mechanical arm of the two-photon microscope according to claim 1, characterized in that: The zoom lens is a liquid zoom lens.

5. The objective lens mechanical arm of the two-photon microscope according to claim 4, characterized in that: The controller collects the displacement of the animal's head relative to the objective lens in real time, and adjusts the focal length of the zoom lens according to the displacement to achieve head movement compensation, specifically including: The liquid zoom lens is calibrated, and the calibration process is: the liquid zoom lens is installed in the optical path, the two-photon microscope is controlled to focus on a certain initial imaging plane, and the control current of the liquid zoom lens is changed in stages. I i , at each level of control current, the focal plane is moved back to the initial imaging plane through the translation stage, and the displacement of the translation stage is recorded as S i , each level of control current corresponds to a displacement; according to the data point ( I i ，S i ) Fitting the curve to get the function I = f(S) , at this time the calibration is completed; When the head motion compensation trigger signal is received, the displacement of the animal's head relative to the objective lens is collected and the displacement is set as the zero point. S 0; The displacement of the animal head relative to the objective lens is collected cyclically, and the displacement is recorded as the real-time displacement S. The amount of the real-time displacement offset from the zero point is recorded as ΔS = SS 0, substitute into the function I = f(S) A control current of the liquid variable focus lens is obtained, and the liquid variable focus lens is controlled according to the control current, that is, the focal length of the variable focus lens is adjusted to achieve head movement compensation.

6. The objective lens mechanical arm of the two-photon microscope according to claim 5, characterized in that: The frequency of the head motion compensation is set to be greater than 100 Hz.

7. The objective lens mechanical arm of the two-photon microscope according to claim 1, characterized in that: The frequency of the cortical motion compensation is set to less than 0.1 Hz.

8. A two-photon microscope, characterized in that: include: An objective lens mechanical arm for a two-photon microscope according to any one of claims 1 to 7.

Images