Two-photon holographic optogenetic stimulation method, system, device and medium based on wavefront correction

Abstract

The present invention relates to a two-photon holographic optogenetic stimulation method, system, device, and medium based on wavefront correction. The method comprises: 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 region on the hologram requiring wavefront distortion correction for the target neuron, and performing wavefront distortion correction on the target neuron in the target region to obtain a phase map containing wavefront distortion information and position information; and step S3, optically exciting the target neuron based on the phase map containing wavefront distortion information and position information. Compared with existing technologies, the present invention achieves deep single-cell resolution excitation of more neurons with a large field of view, lower excitation power, and significantly improves the accuracy and safety of optogenetic stimulation imaging.

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 function and its role 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 neuronal types.

[0003] Traditional electrophysiological methods, such as patch clamp techniques, directly manipulate neuronal potentials to investigate the relationship between neural circuits and behavior. However, these methods suffer from significant cell damage, a limited number of cells that can be manipulated simultaneously, and difficulty achieving precise single-cell stimulation. While chemical genetics has addressed these issues to some 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 proteins made optogenetics an important means of activating or inhibiting mammalian neurons, greatly promoting the progress of neuroscience research. Despite this, early optogenetics still lacked the single-cell precision required to simultaneously control multiple neurons. In recent years, researchers have explored a variety of closed-loop optogenetic 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 due to the complex light scattering phenomenon in deep tissue, which makes it difficult to effectively address the excitation of non-targeted neurons and photodamage.

[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 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 existing technologies and 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: Select a target neuron from two-photon imaging and obtain a hologram corresponding to the three-dimensional spatial position of the target neuron;

[0010] Step S2: selecting a target area on the hologram that requires wavefront distortion correction 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 light-exciting the target neuron, simultaneously performing imaging feedback to monitor the grayscale value change of the target neuron and measuring the ΔF / F change of the target neuron, specifically comprising:

[0014] Collect signals from a target neuron that has not been stimulated by optogenetics, average the grayscale value changes in the neuron cell body area, and calculate 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. Subtract the baseline intensity F from the grayscale value in the post-stimulation imaging to obtain the ΔF of the neuronal 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 using 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 the image of the target area is collected using a photomultiplier tube and the average grayscale value is calculated;

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

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

[0023] Preferably, step S3 performs optical excitation on the target neurons based on the phase map including wavefront distortion information and 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, a two-photon holographic optogenetic stimulation system with wavefront correction capability is provided, wherein the system uses any one of the above methods to optically excite target neurons.

[0026] According to a third aspect of the present invention, an electronic device is provided, 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, a computer-readable storage medium is provided, on which a computer program is stored, and 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 uses the adaptive optical wavefront correction method to obtain the wavefront distortion information of the target neuron area, superimposes the phase map that compensates for 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 through the two-photon holographic optogenetic stimulation technology. It can significantly reduce the deterioration of the point spread function and light scattering caused by deep tissue heterogeneity, restore the focus near the diffraction limit, avoid nonspecific 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) The present invention monitors the grayscale value changes of target neurons through imaging feedback during light stimulation, 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 This is the experimental light 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 photostimulation 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 mirror, 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 clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts 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: Select a target neuron from two-photon imaging and obtain a hologram corresponding to the three-dimensional spatial position of the target neuron;

[0040] Step S2: selecting a target area on the hologram that requires wavefront distortion correction 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: Select a target neuron from two-photon imaging and obtain 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 a resonant imaging optical path. 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. Images are taken and saved throughout the entire experiment, and light stimulation is manually triggered during imaging.

[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. 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 by the WGS (Weighted Gerchberg-Saxton) algorithm. The weights of the holograms corresponding to each neuron position are initially the same. Specifically, the Z axis is moved, and the target neurons related to the study are selected in different Z planes. The three-dimensional spatial coordinates of each target neuron are recorded, and the hologram corresponding to the position of each target neuron is obtained by the WGS (Weighted Gerchberg-Saxton) algorithm. Among them, the weights of the holograms corresponding to each neuron position are initially 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 methods 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 requires wavefront distortion correction 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 where wavefront distortion needs to be corrected is first selected. Since the diameter of a neuron is approximately 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 of the target area is corrected 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 digital micromirror device (DMD). A photomultiplier tube (PMT, H10770PA-40, Hamamatsu) is used to capture the target area image (lens 18, filter 19, and lens 20 are provided in the acquisition optical 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 digital micromirror device (DMD). Subsequent Zernike coefficient traversals are superimposed on the phase map generated by the optimal value of the previous coefficient. This cycle traverses all Zernike coefficients until the last coefficient is measured. The final phase map is generated and loaded onto the SLM or DMD, completing 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 iterating over the Zernike coefficients one by one, independent optimization methods for each Zernike coefficient can also be used. When searching for the maximum grayscale value, methods such as direct maximum selection, Gaussian curve fitting, gradient descent, and conjugate gradient methods can be used. However, this may cause the obtained Zernike coefficients to fall into a local optimum. To achieve a global optimum, global optimization algorithms such as simulated annealing, particle swarm optimization, and genetic algorithms can be used, or combined with deep learning to quickly and accurately obtain wavefront distortion information.

[0054] In addition to optimizing wavefront distortion detection and correction through software algorithms, 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 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. A phase map containing wavefront distortion and position information of all target neurons is loaded through the SLM, and a pair of XY galvanometers is used to perform spiral excitation of the target area, synchronously and precisely targeting all target neurons in three-dimensional space. This provides a larger corrected field of view, enables the excitation of more and deeper target neurons with less excitation power, reduces the false excitation of non-target neurons, and significantly reduces nonspecific damage caused by light scattering.

[0058] Specifically, the two-photon imaging laser 1 selects a femtosecond laser with a wavelength of 920nm and a repetition frequency of 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 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 using 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, this embodiment also includes closed-loop control and real-time adjustment, specifically: imaging feedback is used simultaneously during stimulation to monitor the grayscale value changes of the target neurons and measure the ΔF / F changes of the neurons. First, a signal of a neuron that has not been optogenetically stimulated is collected, and the grayscale value changes in the neuronal cell body area are averaged to obtain the baseline intensity F of the neuronal 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 neurons. The grayscale value in the post-stimulation imaging is subtracted from the baseline intensity F to obtain the ΔF of the neuronal activity. 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 the 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-mentioned 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] Many components in a device are connected to the I / O interface, including: input units, such as a keyboard and mouse; output units, such as various types of displays and speakers; storage units, such as magnetic disks and optical disks; and communication units, such as network cards, modems, and wireless communication transceivers. The communication unit allows the device to exchange information / data with other devices via 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 by any other appropriate means (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, and 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 chips (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. Such program code 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 when the program code is executed by the processor or controller, the functions / operations specified in the flow chart and / or block diagram are implemented. The program code can be executed entirely on the machine, partially on the machine, as a stand-alone software package, partially on the machine and partially on a remote machine, or entirely on a remote machine or server.

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

[0067] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A two-photon holographic optogenetic stimulation method based on wavefront correction, characterized in that: include: Step S1: Select a target neuron from two-photon imaging and obtain a hologram corresponding to the three-dimensional spatial position of the target neuron; Step S2: selecting a target area on the hologram that requires wavefront distortion correction 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 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, where 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 further includes, when light-exciting the target neuron, simultaneously providing imaging feedback to monitor grayscale value changes of the target neuron and measuring ΔF / F changes of the target neuron, specifically comprising: Collect signals from a target neuron that has not been stimulated by optogenetics, average the grayscale value changes in the neuron cell body area, and calculate 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. Subtract the baseline intensity F from the grayscale value in the post-stimulation imaging to obtain the ΔF of the neuronal 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, wherein In step S1, a hologram corresponding to the three-dimensional spatial position of the target neuron is obtained by using the WGS algorithm.

5. The method according to claim 1, wherein In 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 neurons in the target area is corrected using the Zernike coefficient one-by-one 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 the image of the target area is collected using a photomultiplier tube and the average grayscale value is calculated; Selecting the Zernike coefficient that produces the maximum average grayscale value as the optimal value, and loading the phase map corresponding to the optimal value onto a spatial light modulator or a digital micromirror device; After looping through all Zernike coefficients, the final phase map is generated and loaded onto a spatial light modulator or 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 is to perform optical excitation on the target neuron based on the phase map containing 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 according to any one of claims 1 to 7 to perform optical excitation on 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.

Images