Wavefront correction-based two-photon holographic optogenetics stimulation method, system and equipment and medium

Abstract

The invention relates to a wavefront correction-based two-photon holographic optogenetics stimulation method, system and device and a medium, and the method comprises the steps: S1, selecting a target neuron from two-photon imaging, and obtaining a hologram corresponding to a three-dimensional space position of the target neuron; s2, selecting a target area needing wavefront distortion correction for the target neuron on the hologram, and performing wavefront distortion correction on the target neuron in the target area to obtain a phase diagram containing wavefront distortion information and position information; and S3, performing optical excitation on the target neurons based on the phase diagram containing the wavefront distortion information and the position information. Compared with the prior art, deep single cell resolution excitation with a large view field, lower excitation power and more neurons is realized, and the accuracy and safety of optogenetics stimulation imaging are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging, and in particular to a two-photon holographic optogenetic stimulation method, system, device and medium based on wavefront correction. Background Art

[0002] The rapid development of optogenetics has provided revolutionary tools for studying neuronal functions and their roles in neural circuits. However, existing neuronal control methods still have significant limitations in achieving specific, real-time, and non-invasive activation or inhibition of specific types of neurons.

[0003] Traditional electrophysiological methods, such as patch clamp technology, study the relationship between neural circuits and behavior by directly controlling neuronal potentials. However, these methods have problems such as large damage to cells, limited number of cells that can be operated simultaneously, and difficulty in achieving precise stimulation of single cells. Although chemical genetics technology has solved these problems to a certain extent, it also has limitations due to its slow regulation speed and inability to accurately deconstruct the temporal dynamics of rapid biological processes.

[0004] In 2010, the discovery of microbial rhodopsin protein made optogenetics an important means to activate or inhibit mammalian neurons, greatly promoting the progress of neuroscience research. Despite this, early optogenetics still lacked single-cell precision in controlling multiple neurons simultaneously. In recent years, researchers have explored a variety of closed-loop optogenetics methods to achieve non-invasive, multi-neuron, single-cell precision real-time control. However, these methods still face challenges in control accuracy and flexibility, especially the complex light scattering phenomenon in deep tissues, which makes it difficult to effectively solve the problems of excitation and photodamage of non-targeted neurons.

[0005] Therefore, there is an urgent need for a technology that can accurately control the activation or inhibition of deep and shallow neurons in three-dimensional space in real time without damaging the cells, especially to reduce the excitation of non-targeted neurons with single-cell precision. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and to provide a two-photon holographic optogenetic stimulation method, system, equipment and medium based on wavefront correction, so as to achieve deep single-cell resolution excitation of more neurons with a large field of view, lower excitation power, and significantly improve the accuracy and safety of optogenetic stimulation imaging.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] According to a first aspect of the present invention, a two-photon holographic optogenetic stimulation method based on wavefront correction is provided, comprising:

[0009] Step S1, selecting a target neuron from two-photon imaging, and obtaining a hologram corresponding to the three-dimensional spatial position of the target neuron;

[0010] Step S2, selecting a target area on the hologram that needs to be corrected for wavefront distortion for the target neuron, and performing wavefront distortion correction on the target neuron in the target area to obtain a phase map containing wavefront distortion information and position information;

[0011] Step S3: optically excite the target neurons based on the phase map containing the wavefront distortion information and the position information.

[0012] Preferably, in step S1, target neurons are selected from two-photon imaging, specifically: neurons that are activated after stimulation and whose difference between ΔF / F generated by stimulation and ΔF / F generated by spontaneous activity is greater than a set value are selected from two-photon imaging as target neurons, wherein F is the baseline intensity of neuronal activity, and ΔF is the change value of the baseline intensity of neuronal activity.

[0013] Preferably, the method further comprises, when the target neuron is optically excited, simultaneously imaging feedback to monitor the gray value change of the target neuron and measuring the ΔF / F change of the target neuron, specifically comprising:

[0014] Collect a signal from a target neuron that has not been stimulated by optogenetics, average the grayscale value changes in the neuron cell body area, and obtain the baseline intensity F of the target neuron activity;

[0015] Starting from a low excitation power setting, gradually increase the excitation power of each target neuron to a set value, not exceeding the maximum excitation power, and subtract the baseline intensity F from the gray value in the post-stimulation imaging to obtain the ΔF of the neuron activity;

[0016] Set the ΔF / F threshold of the target neuron. When the ΔF / F activity of the target neuron is greater than the set threshold, reduce the light stimulation weight corresponding to the target neuron. If the ΔF / F activity of the target neuron is less than the set threshold, increase the corresponding light stimulation weight until the ΔF / F activity of the target neuron reaches the set threshold.

[0017] Preferably, in step S1, a hologram corresponding to the three-dimensional spatial position of the target neuron is obtained by a WGS algorithm.

[0018] Preferably, in step S2, a target area requiring wavefront distortion correction is selected for the target neuron on the hologram, specifically: a circular area of ​​a set diameter is selected as the target area requiring wavefront distortion correction for the target neuron with the target neuron cell body as the center.

[0019] Preferably, in step S2, the wavefront distortion correction of the target neurons in the target area is performed using the Zernike coefficient one-by-one correction method, specifically:

[0020] During each traversal, the phase map of the current Zernike coefficient is superimposed on the spatial light modulator or digital micromirror device, and a photomultiplier tube is used to collect the image of the target area and calculate the average grayscale value;

[0021] Selecting the Zernike coefficient that produces the maximum average gray value as the optimal value, and loading the phase map corresponding to the optimal value onto the spatial light modulator or the digital micromirror device;

[0022] After looping through all Zernike coefficients, the final phase map is generated and loaded onto a spatial light modulator or a digital micromirror device to complete the detection and correction of the optical wavefront distortion of a single target neuron.

[0023] Preferably, the step S3 performs optical excitation on the target neuron based on the phase map including the wavefront distortion information and the position information, specifically:

[0024] The phase map containing wavefront distortion information and position information of all target neurons is loaded through the spatial light modulator, and the galvanometer is used to perform spiral excitation on all target neurons in the target area.

[0025] According to a second aspect of the present invention, there is provided a two-photon holographic optogenetic stimulation system with wavefront correction capability, wherein the system uses any one of the above-mentioned methods to optically excite target neurons.

[0026] According to a third aspect of the present invention, there is provided an electronic device, comprising a memory and a processor, wherein a computer program is stored in the memory, and when the processor executes the program, any one of the methods described above is implemented.

[0027] According to a fourth aspect of the present invention, there is provided a computer-readable storage medium having a computer program stored thereon, wherein when the program is executed by a processor, any one of the methods described above is implemented.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The two-photon holographic optogenetic stimulation method based on wavefront correction designed by the present invention obtains the wavefront distortion information of the target neuron area by using the adaptive optical wavefront correction method, superimposes the phase map that compensates the wavefront distortion with the phase map obtained from the three-dimensional position of the target neuron, and then projects the phase map to the target brain area by using the two-photon holographic optogenetic stimulation technology. This can significantly reduce the point spread function deterioration and light scattering caused by deep tissue heterogeneity, restore the focus near the diffraction limit, avoid non-specific neuronal excitation caused by scattering, and achieve deep single-cell resolution excitation of more neurons with a large field of view and lower excitation power, significantly improving the accuracy and safety of optogenetic research.

[0030] (2) During light stimulation, the present invention uses imaging feedback to monitor the grayscale value changes of target neurons, measures the ΔF / F changes of neurons, adjusts the light stimulation weight, and realizes adaptive optical wavefront correction. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a flow chart of the method of the present invention;

[0032] Figure 2 It is the experimental optical path diagram in the embodiment;

[0033] Figure 3 Schematic diagram of the wavefront distortion information correction process;

[0034] Figure 4 It is the process of combining the dot matrix phase image with the wavefront distortion information;

[0035] Figure numerals: 1-two-photon imaging laser, 2-two-photon optical stimulation laser, 3-emitting mirror, 4-emitting mirror, 5-lens, 6-lens, 7-emitting mirror, 8-spatial light modulator, 9-lens, 10-emitting mirror, 11-lens, 12-galvanometer, 13-lens, 14-emitting mirror, 15-lens, 16-dichroic mirror, 17-objective lens, 18-lens, 19-filter, 20-lens. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0037] Example

[0038] like Figure 1 As shown, this embodiment provides a two-photon holographic optogenetic stimulation method based on wavefront correction, comprising:

[0039] Step S1, selecting a target neuron from two-photon imaging, and obtaining a hologram corresponding to the three-dimensional spatial position of the target neuron;

[0040] Step S2, selecting a target area on the hologram that needs to be corrected for wavefront distortion for the target neuron, and performing wavefront distortion correction on the target neuron in the target area to obtain a phase map containing wavefront distortion information and position information;

[0041] Step S3: optically excite the target neurons based on the phase map containing the wavefront distortion information and the position information.

[0042] Next, the method of this embodiment is introduced in detail.

[0043] Step S1, selecting a target neuron from two-photon imaging, and obtaining a hologram corresponding to the three-dimensional spatial position of the target neuron.

[0044] like Figure 2 As shown, the two-photon imaging optical path uses the resonant imaging optical path. In order to ensure that the imaging field of view is equivalent to the stimulation field of view, the imaging system and the stimulation system field of view are matched. During the entire experiment, imaging is performed and saved, and light stimulation is manually triggered during the imaging process.

[0045] Two-photon imaging optical path: The laser emitted by the two-photon imaging laser 1 is reflected by the reflector 3, and then reflected again by the reflector 4, and then passes through the lens 5 and the lens 6. The output of the lens 6 is reflected by the transmitting mirror 7 and input to the spatial light modulator 8. The output of the spatial light modulator 8 passes through the lens 9, the transmitting mirror 10 and the lens 11 in sequence, and is input to the galvanometer 12. The output of the galvanometer 12 passes through the lens 13, the transmitting mirror 14, the lens 15 and the dichroic mirror 16 in sequence, and acts on the target through the objective lens 17.

[0046] Two-photon laser optical path: The laser emitted by the two-photon optical stimulation laser 2 passes through lenses 5 and 6. The output of lens 6 is reflected by the transmitting mirror 7 and input to the spatial light modulator 8. The output of the spatial light modulator 8 passes through lens 9, transmitting mirror 10 and lens 11 in sequence and is input to the galvanometer 12. The output of the galvanometer 12 passes through lens 13, transmitting mirror 14, lens 15 and dichroic mirror 16 in sequence and acts on the target through objective lens 17.

[0047] Through the above imaging optical path, the real-time activity status of different neurons at different depths of the target is obtained, and the imaging is displayed in real time through the GUI.

[0048] Through two-photon real-time imaging, the real-time activity status of different neurons at different depths of the target is obtained, and the target neurons related to the study are selected in the three-dimensional area, and the three-dimensional spatial position coordinates are obtained. In addition, neurons that are activated after stimulation and whose ΔF / F generated by stimulation is significantly greater than the ΔF / F of spontaneous activity are selected as much as possible. The hologram corresponding to the position of each target neuron is obtained through the WGS (Weighted Gerchberg-Saxton) algorithm. The weights of the holograms corresponding to each initial neuron position are the same. Specifically, move the Z axis, select the target neurons related to the study in different Z planes, record the three-dimensional spatial coordinates of each target neuron, and obtain the hologram corresponding to the position of each target neuron through the WGS (Weighted Gerchberg-Saxton) algorithm. Among them, the weights of the holograms corresponding to each initial neuron position are the same, and are all set to 1.

[0049] Here, obtaining the hologram corresponding to the target neuron position through the WGS algorithm is only one way. Other feasible ways include optimizing the existing hologram generation algorithm, or combining deep learning to obtain the hologram.

[0050] Step S2: Select a target area on the hologram that needs to be corrected for the target neuron, and perform wavefront distortion correction on the target neuron in the target area to obtain a phase map containing wavefront distortion information and position information.

[0051] For a specific neuron target, the region of the target neuron that needs to correct the wavefront distortion is first selected. Since the diameter of a neuron is about 10 microns, in this embodiment, a circular region with a diameter of 20 microns and a neuron cell body as the center is set as the target region.

[0052] like Figure 3 and Figure 4 As shown, the wavefront distortion is corrected for the target area by correcting the Zernike coefficients one by one. During the traversal process, the phase map of each Zernike coefficient is superimposed on the spatial light modulator SLM or the digital micromirror device DMD, and the target area image is collected by a photomultiplier tube (PMT, H10770PA-40, Hamamatsu) (lens 18, filter 19 and lens 20 are arranged in the collection light path), and the average grayscale value of the target area is calculated. Finally, the Zernike coefficient that produces the maximum average grayscale value is selected as the optimal value, and the corresponding phase map is loaded onto the spatial light modulator SLM or the digital micromirror device DMD. Subsequent traversals of the Zernike coefficients are superimposed on the phase map generated by the optimal value of the previous coefficient. In this way, all Zernike coefficients are traversed in a loop until the measurement of the last coefficient is completed, and the final phase map is generated and loaded onto the SLM or DMD to complete the detection of the optical wavefront distortion information of a single target neuron.

[0053] In addition to the wavefront distortion detection and correction method of traversing the Zernike coefficients one by one, you can also use an independent optimization method for each Zernike coefficient. When retrieving the maximum gray value, you can use direct selection of the maximum value, Gaussian fitting curve to find the maximum value, gradient descent method, conjugate gradient method, etc. However, this may cause the obtained Zernike coefficients to fall into a local optimum. In order to achieve global optimality, you can use global optimization algorithms such as simulated annealing algorithm, particle swarm algorithm, genetic algorithm, etc., or combine deep learning to achieve fast and accurate acquisition of wavefront distortion information.

[0054] In addition to optimizing wavefront distortion detection and correction from the software algorithm, direct adaptive optics systems can also be used in the optical path to add wavefront detectors such as Shack-Hartmann wavefront sensors or Shearing interferometers to the optical path to directly detect wavefront distortion, thereby directly obtaining the wavefront compensation phase map of the target neuron area and loading it into the SLM or DMD to realize wavefront distortion measurement and correction of the target area.

[0055] The wavefront distortion information of all target neurons is obtained in the same way, and the obtained phase map containing the wavefront distortion information is superimposed with the position phase map of the corresponding neurons.

[0056] Step S3: optically excite the target neurons based on the phase map containing the wavefront distortion information and the position information.

[0057] A two-photon holographic microscope system is used to image and optically excite target neurons in three-dimensional space. The phase map containing wavefront distortion information and position information of all target neurons is loaded through SLM, and a pair of XY galvanometers are used to perform spiral excitation on the target area, synchronizing and accurately exciting all target neurons in three-dimensional space, providing a larger correction field of view, achieving the excitation of more and deeper target neurons with smaller excitation power, reducing the misexcitation of non-target neurons, and significantly reducing nonspecific damage caused by light scattering.

[0058] Specifically, the two-photon imaging laser 1 selects a femtosecond laser with a wavelength of 920nm, and the repetition frequency is set to 80MHz. The two-photon optical stimulation laser 2 selects a femtosecond laser with a wavelength of 1030nm and a repetition frequency of 1MHz. A two-photon adaptive optical holographic optogenetic system is used to image and optically excite the target neurons in three-dimensional space. The phase map containing wavefront distortion information and position information of all target neurons is loaded through the spatial light modulator SLM, and the target area is spirally excited by the X and Y galvanometers 12. One spiral scan is 20ms, and 20 spiral scans are performed for each round of stimulation. The time interval between two stimulations is not less than 20s to avoid damage caused by multiple stimulations in a short time as much as possible.

[0059] In addition, the present embodiment also includes closed-loop control and real-time adjustment, specifically: imaging feedback is performed simultaneously during stimulation to monitor the grayscale value changes of the target neuron and measure the ΔF / F changes of the neuron. First, a signal of a neuron that has not been stimulated by optogenetics is collected, and the grayscale value changes in the neuron cell body area are averaged to obtain the baseline intensity F of the neuron activity. Then, starting from a smaller excitation power, 5mW is gradually increased on each neuron, and the maximum does not exceed 20mW to avoid damaging the neuron. The ΔF of the neuron activity is obtained by subtracting the baseline intensity F from the grayscale value in the stimulated imaging. The neuron ΔF / F threshold is set to 1. When the ΔF / F activity of the neuron is greater than the set threshold, the light stimulation weight corresponding to the neuron is reduced. If the ΔF / F activity of the neuron is less than this threshold, the corresponding light stimulation weight needs to be increased until the ΔF / F activity of the neuron reaches the set threshold, and the light stimulation weight is no longer adjusted.

[0060] This embodiment also provides a two-photon holographic optogenetic stimulation system with wavefront correction capability, which uses the above method to optically excite target neurons.

[0061] The electronic device of the present invention includes a central processing unit (CPU), which can perform various appropriate actions and processes according to computer program instructions stored in a read-only memory (ROM) or loaded from a storage unit into a random access memory (RAM). In the RAM, various programs and data required for device operation can also be stored. The CPU, ROM and RAM are connected to each other via a bus. An input / output (I / O) interface is also connected to the bus.

[0062] Multiple components in the device are connected to the I / O interface, including: input units, such as keyboards, mice, etc.; output units, such as various types of displays, speakers, etc.; storage units, such as disks, optical disks, etc.; and communication units, such as network cards, modems, wireless communication transceivers, etc. The communication unit allows the device to exchange information / data with other devices through computer networks such as the Internet and / or various telecommunication networks.

[0063] The processing unit performs the various methods and processes described above, such as methods S1 to S3. For example, in some embodiments, methods S1 to S3 may be implemented as a computer software program, which is tangibly contained in a machine-readable medium, such as a storage unit. In some embodiments, part or all of the computer program may be loaded and / or installed on the device via a ROM and / or a communication unit. When the computer program is loaded into the RAM and executed by the CPU, one or more steps of methods S1 to S3 described above may be performed. Alternatively, in other embodiments, the CPU may be configured to execute methods S1 to S3 in any other appropriate manner (e.g., by means of firmware).

[0064] The functions described above herein may be performed at least in part by one or more hardware logic components. For example, without limitation, exemplary types of hardware logic components that may be used include: field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), application specific standard products (ASSPs), systems on chip systems (SOCs), load programmable logic devices (CPLDs), and the like.

[0065] The program code for implementing the method of the present invention can be written in any combination of one or more programming languages. These program codes can be provided to a processor or controller of a general-purpose computer, a special-purpose computer or other programmable data processing device, so that the program code, when executed by the processor or controller, enables the functions / operations specified in the flow chart and / or block diagram to be implemented. The program code can be executed entirely on the machine, partially on the machine, partially on the machine as a stand-alone software package and partially on a remote machine, or entirely on a remote machine or server.

[0066] In the context of the present invention, a machine-readable medium may be a tangible medium that may contain or store a program for use by or in conjunction with an instruction execution system, device, or equipment. A machine-readable medium may be a machine-readable signal medium or a machine-readable storage medium. A machine-readable medium may include, but is not limited to, electronic, magnetic, optical, electromagnetic, infrared, or semiconductor systems, devices, or equipment, or any suitable combination of the foregoing. A more specific example of a machine-readable storage medium may include an electrical connection based on one or more lines, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing.

[0067] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A two-photon holographic optogenetic stimulation method based on wavefront correction, characterized in that: include: Step S1, selecting a target neuron from two-photon imaging, and obtaining a hologram corresponding to the three-dimensional spatial position of the target neuron; Step S2, selecting a target area on the hologram that needs to be corrected for wavefront distortion for the target neuron, and performing wavefront distortion correction on the target neuron in the target area to obtain a phase map containing wavefront distortion information and position information; Step S3: optically excite the target neurons based on the phase map containing the wavefront distortion information and the position information.

2. The method according to claim 1, characterized in that In the step S1, target neurons are selected from two-photon imaging, specifically: neurons that are activated after stimulation and whose difference between ΔF / F generated by stimulation and ΔF / F generated by spontaneous activity is greater than a set value are selected from two-photon imaging as target neurons, wherein F is the baseline intensity of neuronal activity, and ΔF is the change value of the baseline intensity of neuronal activity.

3. The method according to claim 2, characterized in that The method also includes, when the target neuron is optically excited, simultaneously imaging feedback to monitor the gray value change of the target neuron and measuring the ΔF / F change of the target neuron, specifically including: Collect a signal from a target neuron that has not been stimulated by optogenetics, average the grayscale value changes in the neuron cell body area, and obtain the baseline intensity F of the target neuron activity; Starting from a low excitation power setting, gradually increase the excitation power of each target neuron to a set value, not exceeding the maximum excitation power, and subtract the baseline intensity F from the gray value in the post-stimulation imaging to obtain the ΔF of the neuron activity; Set the ΔF / F threshold of the target neuron. When the ΔF / F activity of the target neuron is greater than the set threshold, reduce the light stimulation weight corresponding to the target neuron. If the ΔF / F activity of the target neuron is less than the set threshold, increase the corresponding light stimulation weight until the ΔF / F activity of the target neuron reaches the set threshold.

4. The method according to claim 1, characterized in that: In the step S1, a hologram corresponding to the three-dimensional spatial position of the target neuron is obtained by using a WGS algorithm.

5. The method according to claim 1, characterized in that In the step S2, a target area requiring wavefront distortion correction is selected for the target neuron on the hologram, specifically: a circular area with a set diameter is selected as the target area requiring wavefront distortion correction for the target neuron with the target neuron cell body as the center.

6. The method according to claim 1, characterized in that In step S2, the wavefront distortion of the target neuron is corrected in the target area using the Zernike coefficient correction method, specifically: During each traversal, the phase map of the current Zernike coefficient is superimposed on the spatial light modulator or digital micromirror device, and a photomultiplier tube is used to collect the image of the target area and calculate the average grayscale value; Selecting the Zernike coefficient that produces the maximum average gray value as the optimal value, and loading the phase map corresponding to the optimal value onto the spatial light modulator or the digital micromirror device; After looping through all Zernike coefficients, the final phase map is generated and loaded onto a spatial light modulator or a digital micromirror device to complete the detection and correction of the optical wavefront distortion of a single target neuron.

7. The method according to claim 1, characterized in that The step S3 performs optical excitation on the target neuron based on the phase map including the wavefront distortion information and the position information, specifically: The phase map containing wavefront distortion information and position information of all target neurons is loaded through the spatial light modulator, and the galvanometer is used to perform spiral excitation on all target neurons in the target area.

8. A two-photon holographic optogenetic stimulation system with wavefront correction capability, characterized in that: The system uses the method described in any one of claims 1 to 7 to optically excite the target neurons.

9. An electronic device comprising a memory and a processor, wherein a computer program is stored in the memory, wherein: When the processor executes the program, the method according to any one of claims 1 to 7 is implemented.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the program is executed by a processor, the method according to any one of claims 1 to 7 is implemented.

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