CN115316960B - Brain nerve activity regulation and control and brain information synchronous reading system - Google Patents

Abstract

The invention discloses a brain nerve activity regulation and control and brain information synchronous reading system. The recording light source module emits imaging light beams through the imaging light source and passes through the stimulation module; the stimulation module emits a stimulation light beam through a stimulation light source; the signal acquisition module receives the imaging light beam and the stimulating light beam, inputs the imaging light beam and the stimulating light beam to neurons of a brain region of an organism by using the implanted multimode optical fiber, regulates and controls the brain nerve activity of the organism and excites the neurons to generate signals, acquires fluorescence and returns the fluorescence to be sent to the signal recording module; the signal recording module receives the fluorescence and converts the fluorescence into an electric signal. The invention can realize single recording function, single stimulation function and recording stimulation synchronization function, realize different experimental paradigms, ensure the stability of the system, effectively avoid the influence of high-power stimulation light on imaging light and measuring signal light, and has the advantages of compatibility of experimental organisms, magnetic compatibility, synchronous stimulation and recording and adjustable stimulation parameters.

Description

A brain neural activity regulation and brain information synchronous reading system

technical field

The invention relates to a synchronous detection system for brain nerve activity of a living body, in particular to a system for regulating brain nerve activity and synchronously reading brain information.

Background technique

The mammalian brain is divided into many different brain regions according to their functional similarities. Each brain region corresponds to different types of neural information. Understanding the functions of each brain region requires certain manipulation and recording techniques. Through the precise regulation of specific brain regions of freely moving animals and the recording of corresponding neural activities, it can help to better understand the functions of each brain region, provide functional information of relevant brain regions in specific behaviors, and provide important information for further understanding and treatment of related diseases. data and theoretical support.

Magnetic resonance imaging (MRI) and positron emission tomography (PET) can observe the neuronal activity caused by various external stimuli at the whole brain level. However, this whole-brain-based observation method lacks precise spatial and temporal resolution, making it difficult to apply to the study of real-time brain functions of freely moving organisms, and it is also impossible to distinguish brain regions. Electrophysiological technology uses energy such as electricity and sound to stimulate organisms, and records the electrical phenomena that occur in organisms through metal or glass electrodes implanted in the body, with single-cell scale and high time precision. However, electrode recording is susceptible to electromagnetic and motion interference, which makes it impossible to record for a long time, and the synchronization of stimulation and recording sites cannot be achieved. Neuronal activity releases large amounts of calcium ions, so calcium imaging can be used to track neuronal action potentials and understand the activity of neuronal clusters. Due to the highly scattering and highly absorbing properties of biological tissue, traditional optical stimulation and calcium imaging methods (such as confocal and two-photon) cannot cover deep (>1mm) regions below the cerebral cortex. Optical fiber recording technology utilizes the excellent light guiding ability of optical fiber to provide a solution for reading and recording the neural activity of freely behaving organisms. The optical fiber probe is small in size, good in flexibility and high in sensitivity, and can read and record the neural activity of relevant brain regions for a long time.

At present, the existing optical fiber recording system is not compatible with the optical stimulation regulation module, and an additional optical fiber needs to be introduced to realize the optical stimulation regulation function, resulting in the difference between the regulation site and the signal reading site.

Contents of the invention

In order to solve the problems existing in the background technology, the present invention proposes a brain nerve activity regulation and brain information synchronous reading system, which can introduce light stimulation regulation on the basis of ensuring the accuracy of signal reading, and realize the light stimulation of a single optical fiber Regulation and synchronous reading of brain information. Moreover, the invention has the advantages of being compatible with experimental organisms, synchronously regulating light stimulation and reading and recording signals, and having adjustable stimulation parameters.

The technical scheme that the present invention adopts is as follows:

Including the recording light source module, the imaging light beam emitted by the imaging light source passes through the stimulation module and then reaches the signal acquisition module;

Including a stimulating module, which sends a stimulating light beam to the signal acquisition module through a stimulating light source;

Including the signal acquisition module, which receives the imaging beam from the recording light source module and the stimulation beam from the stimulation module, and uses the implanted multimode optical fiber to input to the neurons in the brain area of the organism, regulates the neural activity of the brain of the organism and excites the neurons Signals are generated to realize real-time collection of neural signals in the biological brain area and monitoring of light sources; at the same time, multi-mode optical fibers are used to collect the fluorescence generated after the imaging beam is irradiated on neurons and sent back to the signal recording module; the imaging beam and the stimulating beam are coupled regulation.

It includes a signal recording module, which receives fluorescence from the signal acquisition module and converts it into an electrical signal.

The imaging light source emits an imaging light beam with a central wavelength of 473 nanometers, and the stimulating light source emits a stimulating light beam with a central wavelength of 589 nanometers.

The recording light source module includes an imaging light source, a second half-wave plate, a second scanning galvanometer, a third doublet lens, a first reflection mirror, a fourth doublet lens and a second reflection mirror; the imaging light source, the second half-wave The wave plate and the second scanning galvanometer are arranged sequentially along the same linear optical axis, the second scanning galvanometer, the third doublet lens, and the first reflector are arranged sequentially along the same linear optical axis, the first reflector, the fourth doublet lens and the second mirror are arranged in sequence along the same straight line optical axis; the imaging light source emits an imaging beam, which is sequentially rotated by the second half-wave plate, reflected by the second scanning galvanometer, then transmitted by the third doublet lens, reflected by the first After being reflected by the mirror, transmitted by the fourth doublet lens, and reflected by the second mirror, it enters the polarization beam splitter prism, and is incident into the signal acquisition module after being reflected by the polarization beam splitter prism.

The stimulation module includes a stimulation light source, a first half-wave plate, a first doublet lens, a second doublet lens, a first scanning galvanometer, a scanning mirror, an infinity correction sleeve lens and a polarization beam splitter prism; a laser source, The first half-wave plate, the first doublet lens, the second doublet lens, and the first scanning galvanometer are sequentially arranged along the same linear optical axis, and the laser source emits a stimulating beam, which is sequentially polarized and rotated by the first half-wave plate. The doublet lens assembly composed of the first doublet lens and the second doublet lens is expanded and collimated, reflected by the first scanning galvanometer, then transmitted through the scanning mirror in turn, transmitted by the infinity corrected sleeve lens, and then incident on the polarization beam splitter prism , it is incident into the signal acquisition module after being transmitted through the polarization beam splitter prism.

The signal acquisition module includes a dichroic mirror, an objective lens, a first multimode fiber, a self-calibration module and a photodetector; the dichroic mirror, an objective lens, the first multimode fiber and the self-calibration module are sequentially along the same straight optical axis Arrangement, the stimulating light beam and the imaging light beam are all incident on the dichroic mirror through the polarization beam splitter prism of the stimulating module to be transmitted, and then transmitted and converged by the objective lens and then incident on one end of the first multimode optical fiber, the other end of the first multimode optical fiber and the self The input terminal of the calibration module is connected, and there are two output terminals of the self-calibration module. The two output terminals are respectively connected to the photodetector and the neurons in the brain area of the organism. The transmitted stimulating beam and imaging beam are respectively incident on the self-calibration module. photodetectors and neurons in brain regions of living organisms;

The self-calibration assembly includes a 1x2 fused fiber coupler, a second multimode fiber and a third multimode fiber; a bundled end of the 1x2 fused fiber coupler is used as the input end of the self-calibration assembly and is connected to the other end of the first multimode fiber , the two branch ends of the 1x2 fused fiber coupler are respectively connected to the photodetector and neurons in the brain area of the living body through the second multimode fiber and the third multimode fiber.

The signal recording module includes an optical filter, the fifth doublet lens and a camera; the optical filter, the fifth doublet lens and the camera are sequentially arranged along the same straight line optical axis, and the fluorescence coming from the signal acquisition module passes through the optical filter successively After the light is filtered and converged by the fifth doublet lens, it is incident into the camera, and is detected and collected by the camera.

The optical fiber end faces of the first multimode optical fiber, the second multimode optical fiber and the third multimode optical fiber are designed with an 8-degree bevel and an optical coating is added.

It also includes a vibrating mirror controller, which is electrically connected to the second scanning vibrating mirror of the recording light source module, the first scanning vibrating mirror of the stimulation module, and the photodetector of the signal acquisition module respectively, and the vibrating mirror controller collects the photodetector detected by the photodetector in real time. The electrical signal is used to feedback and adjust the work of the second scanning galvanometer of the recording light source module and the first scanning galvanometer of the stimulation module. Finally, the coupling control of the imaging beam and the stimulating beam is realized.

Make the detection surface perpendicular to the optical axis of the objective lens of the signal acquisition module, and establish the x direction and y direction perpendicular to each other on the detection surface:

The first scanning galvanometer adjusts the x direction, and is used to deflect the stimulation beam in the x direction, so as to realize light stimulation with different frequencies and times;

The second scanning galvanometer adjusts the x direction and the y direction, and is used for deflecting the stimulating beam in the x direction and the y direction, so as to realize optical imaging.

Combining the on and off switching of two different states of the first scanning galvanometer with the on and off switching of the second scanning galvanometer, three different modes of the system are constructed:

Mode 1, single record mode

Control the first scanning galvanometer in the state off and the second scanning galvanometer in the state on, control the photodetector to sample at intervals to obtain a light intensity signal of a lower intensity of the imaging beam, and the camera keeps continuously collecting images in real time;

Mode 2, single stimulation mode

Control the first scanning galvanometer in the state of on and the second scanning galvanometer in the state of off, control the photodetector to sample at intervals to obtain a higher intensity light intensity signal of the stimulating beam, and the camera does not work and does not collect images;

Mode 3. Recording stimulus synchronization mode

Control the first scanning galvanometer in the state on and the second scanning galvanometer in the state on, control the photodetector to sample at intervals, alternately obtain the light intensity signal of the lower intensity of the imaging beam and the light intensity signal of the higher intensity of the stimulation beam, The camera keeps capturing images continuously in real time.

In the present invention, the chromatic aberration introduced by the imaging beam and the stimulating beam due to the difference in wavelength, and the position difference of the focus point after the introduction of the objective lens due to incomplete registration of the beam, by applying a deflection voltage to the scanning galvanometer, ensure the strict registration of the initial xy direction of the stimulating beam and the imaging beam , to ensure accuracy.

By controlling the opening and closing of two scanning galvanometers, the present invention can realize single recording function, single stimulation function, and recording and stimulation synchronous function, and cooperate with each other to realize different experimental paradigms.

The intensity of the signal is closely related to the power of the imaging beam. In order to ensure the accuracy and stability of the signal during the recording process, a 1x2 fused fiber coupler is used to realize the 1-point and 2-way of the multimode fiber outlet, and one of them is used as the monitoring path. The self-calibration component is introduced to monitor the imaging beam and stimulation beam in real time, and the beam that deviates from the optical axis is corrected by fine-tuning the voltage value of the scanning galvanometer to ensure the stability of the system.

It has been verified by experiments that the combination of long-wavelength stimulating light and short-wavelength imaging light in the present invention can effectively reduce spectral interference, match the notch dichroic mirror of the corresponding wavelength, and can effectively avoid the impact of high-power stimulating light on imaging light and measurement signal light. Real-time recording of signals during stimulation.

The present invention matches the corresponding control scheme and self-calibration system through the structure of double-scanning galvanometers, and combines specially processed multi-mode optical fibers to simultaneously perform optical fiber recording and light stimulation, and realizes the regulation of biological brain nerve activity and real-time signal monitor.

The beneficial effects of the present invention are:

The present invention increases the setting of optical stimulation on the basis of optical fiber recording, and selects single recording, single stimulation or stimulation recording synchronization mode according to the situation, and correspondingly realizes brain region information reading, brain nerve activity regulation, and brain nerve regulation and information synchronous reading Two functions.

Moreover, the present invention is designed with an 8-degree slope on the end face of the multimode optical fiber, and adds an optical coating to reduce the end face reflection of high-power stimulating beams and improve the transmission and collection efficiency of nerve signals.

In the present invention, the scanning galvanometer is used to control the stimulation beam. Through the galvanometer controller and the control program, the system can achieve the fastest stimulation frequency of 1 kHz, and the stimulation frequency and stimulation time can be adjusted. The use of the scanning galvanometer ensures that the stimulation beam and imaging Beams and tight registration for guaranteed accuracy.

At the same time, self-calibration components are introduced to monitor the imaging beam and stimulation beam in real time to ensure the stability of the system. Experimental results verify that it can realize the regulation of optical stimulation of a single optical fiber and the synchronous recording of nerve signals, with low signal noise and high sensitivity.

The invention has the advantages of compatibility with experimental organisms, magnetic compatibility, synchronous stimulation and recording, and adjustable stimulation parameters.

Description of drawings

In order to explain the present invention in more detail, it is illustrated in conjunction with the following figures. The accompanying drawings show a system diagram of the method of the present invention, listing specific elements and explaining them accordingly.

Fig. 1 is a block diagram of the system structure of the present invention.

Fig. 2 is a schematic diagram of the system of the present invention.

Figure 3 is a timing diagram of single recording, single stimulation mode and stimulus recording synchronization mode.

Figure 4 is the signal recorded by the camera in the sample-free simultaneous stimulation recording mode.

Figure 5 is the signal recording of experimental animals: (I) is the signal recorded by the camera in the synchronous stimulation recording mode of the experimental animal, and (II) is the signal recorded by the camera in the single recording mode of the experimental animal.

Fig. 6 is a situation diagram of an experimental paradigm and experimental results combining three modes in an embodiment.

In the figure: stimulus light source 1, first half-wave plate 2, first doublet lens 3, second doublet lens 4, first scanning galvanometer 5, scanning mirror 6, infinity correction sleeve lens 7, imaging light source 8 , the second half-wave plate 9, the second scanning mirror 10, the third doublet lens 11, the first mirror 12, the fourth doublet lens 13, the second mirror 14, the polarization beam splitter prism 15, the dichroic mirror 16, objective lens 17, first multimode fiber 18, 1x2 fused fiber coupler 19, second multimode fiber 20, third multimode fiber 21, photodetector 22, optical filter 23, fifth doublet lens 24, Camera 25, vibrating mirror controller 26.

Detailed ways

In order to make the purpose and technical solution of the present invention clearer, the present invention will be further described below in conjunction with the accompanying drawings and specific implementation.

As shown in Figure 1, it includes a recording light source module, a stimulation module, a signal acquisition module and a signal recording module.

Including the recording light source module, the imaging light beam is sent by the imaging light source to the signal acquisition module after passing through the stimulation module, the power of the imaging light source is adjustable, and the power is 0-100 microwatts;

Including the stimulation module, the stimulation light beam is sent to the signal acquisition module through the stimulation light source, the power and frequency of the stimulation light source are adjustable, the power is 0-20 mW, and the frequency is 0.01-1kHz;

Including the signal acquisition module, which receives the imaging beam from the recording light source module and the stimulation beam from the stimulation module, and uses the implanted multimode optical fiber to input to the neurons in the brain area of the organism, regulates the neural activity of the brain of the organism and excites the neurons Generate signals to realize real-time acquisition of neural signals in the biological brain area and monitoring of light sources; at the same time, multi-mode optical fibers are used to collect the fluorescence generated after the imaging beam irradiates neurons and send them back to the signal recording module;

Including the signal recording module, which receives the fluorescence from the signal acquisition module and converts it into an electrical signal, and realizes the real-time recording of the signal of 0-100 frames.

The imaging light source emits a blue imaging light beam with a central wavelength of 473 nm, and the stimulating light source emits a yellow stimulating light beam with a central wavelength of 589 nm.

As shown in Figure 2, the recording light source module includes an imaging light source 8, a second half-wave plate 9, a second scanning galvanometer 10, a third doublet lens 11, a first reflection mirror 12, a fourth doublet lens 13 and a second Mirror 14; imaging light source 8, the second half-wave plate 9, and the second scanning vibrating mirror 10 are arranged sequentially along the optical axis of the same straight line, and the second scanning vibrating mirror 10, the third doublet lens 11, and the first reflecting mirror 12 are arranged along the same line. The linear optical axes are arranged sequentially, and the first reflector 12, the fourth doublet lens 13 and the second reflector 14 are arranged sequentially along the same linear optical axis; the imaging light source 8 emits an imaging beam, which is polarized and rotated by the second half-wave plate 9 in turn, After being reflected by the second scanning galvanometer 10, it is transmitted through the third doublet lens 11, reflected by the first reflector 12, transmitted by the fourth doublet lens 13, reflected by the second reflector 14, and then incident into the polarization beam splitter prism 15. After being reflected by the polarization beam splitter prism 15, it enters the dichroic mirror 16 of the signal acquisition module.

In the above process, the imaging beam is expanded through the doublet lens assembly composed of the third doublet lens 11 and the fourth doublet lens 13 . The direction deflection scanning process is performed on the stimulation beam through the first scanning galvanometer 5 .

As shown in Figure 2, the stimulation module includes a stimulation light source 1, a first half-wave plate 2, a first doublet lens 3, a second doublet lens 4, a first scanning galvanometer 5, a scanning mirror 6, and an infinity correction sleeve Lens 7 and polarization splitter prism 15; laser source 1, first half-wave plate 2, first doublet lens 3, second doublet lens 4, first scanning vibrating mirror 5 are arranged in sequence along the same straight line optical axis, laser source 1 Stimulating light beams are emitted, which are sequentially polarized and rotated by the first half-wave plate 2, collimated and expanded by the doublet lens assembly composed of the first doublet lens 3 and the second doublet lens 4, and reflected by the first scanning galvanometer 5 , and then transmitted through the scanning mirror 6, the infinity correction sleeve lens 7 and then incident into the polarization beam splitter prism 15, and then incident into the dichroic mirror 16 of the signal acquisition module after being transmitted through the polarization beam splitter prism 15.

In the above process, the stimulus beam is collimated and expanded by the doublet lens assembly composed of the first doublet lens 3 and the second doublet lens 4 . The direction deflection scanning process is performed on the stimulation beam through the first scanning galvanometer 5 .

The scanning mirrors 6 are all laser scanning lenses. When the incident light angle changes relative to the optical axis of the lens, the scanning lens can also produce a flat imaging surface.

The combination of the infinity correction sleeve lens 7 and the scanning mirror 6 forms a telecentric system.

The polarizing beam splitter 15 is used to transmit the stimulating beam and reflect the imaging beam. The polarizing beam splitter 15 is designed on the intersection optical path of the imaging beam and the stimulating beam, passes through the stimulating beam and reflects the imaging beam, the inclined plane of the polarizing beam splitting prism 15 is placed at an angle of 45° to the imaging beam, and the imaging beam passes through the polarizing beam splitting prism 15 and meets the stimulating beam completely coincident.

The first doublet lens 3 and the second doublet lens 4 constitute the first group of 4F systems, the scanning mirror 6 and the infinity correction sleeve lens 7 constitute the second group of 4F systems, the third doublet lens 11 and the fourth doublet lens 13 constitute the third group of 4F systems.

As shown in Figure 2, the signal acquisition module comprises dichroic mirror 16, objective lens 17, the first multimode optical fiber 18, self-calibration module and photodetector 22; Dichroic mirror 16, objective lens 17, the first multimode optical fiber 18 Arranged sequentially along the same optical axis as the self-calibration module, the stimulating beam and the imaging beam are both incident on the dichroic mirror 16 through the polarization beam splitter prism 15 of the stimulating module for transmission, and then transmitted and converged by the objective lens 17 and then incident on the first multi-mode One end of the optical fiber 18, the other end of the first multimode optical fiber 18 is connected to the input end of the self-calibration module, there are two output ends of the self-calibration module, and the two output ends are respectively connected to the photodetector 22 and neurons in the brain area of the living body , the transmitted stimulating light beam and imaging light beam are respectively incident on the photodetector 22 and neurons in the brain area of the organism through the self-calibration module;

When the stimulating light beam is irradiated to neurons, it will excite the light-sensitive protein expressed in the neuron cells, which is selective for the passage of cations or anions, causing the membrane potential on both sides of the cell membrane to change, so as to achieve the purpose of selectively exciting or inhibiting the cells;

The excitatory or inhibitory activity of the cells will cause rapid changes in the concentration of free calcium ions in the cells. Imaging beams irradiate the neurons, which will excite the calcium indicator expressed in the nerve cells, and show the calcium ion concentration in the neurons through fluorescent signals. . The fluorescence returns to the dichroic mirror 16 in reverse according to the original path of the imaging beam, and after being reflected, it is detected and received by the signal recording module.

The dichroic mirror 16 is used to transmit the stimulating light beam and the imaging light beam, and reflect the fluorescent signal back in reverse. The dichroic mirror 16 is a notch dichroic mirror, the transmission band 1 has a wavelength range of 350-500 nm, and the transmission band 2 has a wavelength range of 530-1600 nm. Used to reflect retrograde fluorescent signals through the stimulating and imaging beams.

The photodetector 22 adopts a photodiode, and when receiving a light intensity signal, a corresponding photocurrent value will be displayed at the receiving end.

The self-calibration assembly includes a 1x2 fused fiber coupler 19, a second multimode fiber 20 and a third multimode fiber 21; a bundled end of the 1x2 fused fiber coupler 19 is used as an input end of the self-calibration assembly and the first multimode fiber 18 is another One end is connected, and the two branch ends of the 1×2 fused fiber coupler 19 are respectively connected to the photodetector 22 and neurons in the brain region of the living body through the second multimode fiber 20 and the third multimode fiber 21 .

Both the stimulating light beam and the imaging light beam are divided into two light beams by the 1×2 fused optical fiber coupler 19 , and then divided into two light beams, which are respectively incident on the photodetector 22 and the neuron connection in the brain area of the living body.

When the stimulation beam and the imaging beam are due to errors such as mechanical component drift, the rear focus position of the objective lens 17 is shifted, thereby affecting the coupling efficiency of the stimulation beam and the imaging beam into the first multimode optical fiber 18 . At this moment, the photodetector 22 recognizes that the output end power of the third multimode optical fiber 21 has dropped, and the photodetector 22 converts the light intensity signal into an electrical signal, and the oscillating mirror controller 26 controls to change the first scanning oscillator according to the electrical signal. mirror 5, or the mirror angle in the second scanning galvanometer 10. When the light intensity signal recognized by the photodetector 22 reaches the target value, the focal position of the light beam behind the objective lens 17 is at the center of the end face of the first multimode optical fiber 18, and the coupling efficiency reaches the highest.

Or when the hardware clock is unstable, there is a deviation between the actual stimulation frequency and the set frequency. The photodetector 22 feeds back the frequency signal to the vibrating mirror controller 26 to change the applied signal frequency of the vibrating mirror until the frequency of the stimulating beam collected by the photodetector 22 in real time reaches the target value.

The use of 1x2 fused fiber coupler 19 divides the light beam emitted from the first multimode fiber 18 into two and enters the second multimode fiber 20 and the third multimode fiber 21. The second multimode fiber 20 is connected to the biological brain The neurons in the area are connected, and the third multimode optical fiber 21 is connected to the photodetector 22, which ensures real-time monitoring and feedback of signals during the experiment.

The intensity of the signal is closely related to the power of the imaging beam, and the accuracy and stability of the signal can be guaranteed during the recording process through self-calibration components.

As shown in Figure 2, the signal recording module includes optical filter 23, the fifth doublet lens 24 and camera 25; Fluorescence coming from the module is filtered by the filter 23 in turn, converged by the fifth doublet lens 24, and then incident into the camera 25, where it is detected and collected by the camera 25.

The first multimode fiber 18 , the second multimode fiber 20 , and the third multimode fiber 21 are all multimode fibers with a diameter of 200 microns and a numerical aperture of 0.37.

The filter 19 has a transmission bandwidth of 20 nanometers, an OD value of 4, and a central wavelength of 515 nanometers.

The fiber end faces of the first multimode fiber 18, the second multimode fiber 20, and the third multimode fiber 21 are designed with an 8-degree slope and an optical coating is added to reduce the end face reflection of the high-power stimulating beam and improve signal transmission and collection efficiency.

Also comprise vibrating mirror controller 26, respectively with the second scanning vibrating mirror 10 of recording light source module, the first scanning vibrating mirror 5 of stimulating module, the photodetector 22 of signal acquisition module electrically connected, like this photodetector 22 passes vibrating mirror The controller 26 is connected to the first scanning vibrating mirror 5 and the second scanning vibrating mirror 10 to send real-time feedback signals. The work of mirror 10 and the first scanning galvanometer 5 of the stimulation module is adjusted by feedback.

When the power and frequency of the stimulating beam changed, the light intensity of the stimulating beam was collected in real time by the photodetector 22 and converted into an electrical signal, and the vibrating mirror controller 26 controlled to change the lens angle in the first scanning vibrating mirror 5 according to the electrical signal, Until the light intensity of the stimulating light beam collected by the photodetector 22 in real time reaches the target value;

When the power and frequency of the imaging beam change, the light intensity of the imaging beam is collected in real time by the photodetector 22 and converted into an electrical signal, and the vibrating mirror controller 26 changes the mirror angle in the second scanning vibrating mirror 10 according to the electrical signal control, Until the light intensity of the imaging beam collected by the photodetector 22 in real time reaches the target value.

The vibrating mirror controller uses a signal acquisition card to receive the light signal from the photodetector and apply the calibrated vibrating mirror voltage value to the scanning vibrating mirror.

Make the detection surface perpendicular to the optical axis of the objective lens 17 of the signal acquisition module, and establish mutually perpendicular and orthogonal x directions and y directions on the detection surface:

The first scanning galvanometer 5 adopts a one-dimensional galvanometer system to adjust the x-direction, and is used to deflect the stimulation beam in the x-direction, so as to realize light stimulation of different frequencies and times;

The second scanning galvanometer 10 adopts a two-dimensional galvanometer system to adjust the x direction and the y direction, and is used to deflect the imaging beam in the x direction and the y direction to realize optical imaging.

Due to the chromatic aberration caused by the difference in wavelength between the imaging beam and the stimulating beam, and the difference in the position of the focal point due to the incomplete registration of the beams, the imaging beam should be corrected so that it completely coincides with the stimulating beam. When calibrating the system, the calibration of the stimulating beam is performed first. Apply voltage to the first scanning galvanometer 5 until the light intensity recognized by the photodetector 22 reaches the maximum value. At this time, the coupling efficiency of the stimulation beam to the first multimode fiber 18 after passing through the objective lens 17 reaches the maximum. Let the voltage applied to the first scanning galvanometer 5 at this time be the deflection voltage value in its on state. When the imaging beam is calibrated, the first scanning galvanometer 5 is applied with the above-mentioned calibrated deflection voltage to make it in the on state, and by fine-tuning the lens angle of the second scanning galvanometer 10, after the imaging beam passes through the polarizing beam splitter prism 15, and the stimulating beam in the Strict registration in the xy direction realizes strict alignment of the focus positions of the two beams exiting the objective lens, and the voltage value applied by the second scanning galvanometer 10 at this time is used as the deflection voltage value when the second scanning galvanometer 10 is in the on state. In subsequent experiments, by applying calibrated deflection voltages to the first scanning galvanometer 5 and the second scanning galvanometer 10 respectively or simultaneously, the stimulation, imaging, Or simultaneous stimulus imaging.

Combining the on and off switching of two different states of the first scanning galvanometer 5 with the on and off switching of the second scanning galvanometer 10, three different modes of the system are constructed:

Mode 1, single record mode

Control the first scanning galvanometer 5 in the state off and the second scanning galvanometer 10 in the state on, control the photodetector 22 to sample at intervals to obtain a light intensity signal of a lower intensity of the imaging beam, and the camera 25 keeps continuously collecting images in real time; It only realizes the signal reading and recording functions.

Mode 2, single stimulation mode

Control the first scanning galvanometer 5 in the state on and the second scanning galvanometer 10 in the state off, control the photodetector 22 to sample at intervals to obtain a higher intensity light intensity signal of the stimulating beam, and the camera 25 does not work and does not collect images; It only realizes the brain information light stimulation regulation function.

Mode 3. Recording stimulus synchronization mode

Control the first scanning vibrating mirror 5 in the state on and the second scanning vibrating mirror 10 in the state on, control the photodetector 22 to alternately sample at intervals, obtain the light intensity signal of the lower intensity of the imaging beam, and the higher intensity signal of the stimulating beam Intensity of the light intensity signal, the camera 25 keeps continuously collecting images in real time, realizing the brain information light stimulation regulation function and the brain information synchronous recording function.

The first scanning galvanometer 5 has the following two switching states:

When the first scanning galvanometer 5 applies an additional voltage, it is in the state on. The first scanning galvanometer 5 controls the stimulation beam to propagate along the optical axis, and is coupled into the first multimode optical fiber 18 through the objective lens 17. From the second multimode The optical fiber 20 exits to neurons for stimulation;

When the first scanning galvanometer 5 does not apply an additional voltage, the state is off, and the first scanning galvanometer 5 controls the stimulation beam to deviate from the optical axis, so that the focus of the beam passing through the objective lens 17 deviates from the input end of the first multimode optical fiber 18. The end face cannot be emitted from the second multimode optical fiber 20 to neurons for stimulation;

The first scanning galvanometer 5 is controlled to switch between the state on and the state off to form a stimulating beam of frequency.

The second scanning galvanometer 10 has the following two switching states:

When the second scanning galvanometer 10 does not apply an additional voltage, it is in the state on. The second scanning galvanometer 10 controls the imaging light beam to propagate along the optical axis, and is coupled into the first multimode optical fiber 18 through the objective lens 17. From the second multimode optical fiber The mode fiber 20 exits to the neuron to excite and generate fluorescence for imaging;

When the second scanning galvanometer 10 applies an additional voltage, the state is off, and the second scanning galvanometer 10 controls the imaging beam to deviate from the optical axis, so that the focus of the beam passing through the objective lens 17 deviates from the end face of the input end of the first multimode optical fiber 18 , the neurons cannot be emitted from the second multimode optical fiber 20 for excitation imaging.

Embodiments of the present invention are as follows:

In this embodiment, the experimental animal mouse is used to stimulate the light-sensitive protein expressed by neurons in a specific brain region of the mouse and read the fluorescent signal generated in the neural activity. Neurons express the light-sensitive protein halophilic rhodopsin (NpHR), which inhibits neuronal activity when stimulated by yellow light. Neurons express calcium indicator GCaMP6, which can be excited by blue light to produce fluorescence, and can observe and record neuron activities in real time.

Referring to Figure 3, the system has three modes, mode one is single recording mode, mode two is single stimulation mode, and mode three is recording and stimulation synchronous mode. By setting the alternating frequency and duration of the on and off states of the first and second scanning galvanometers, the switching of modes 1, 2, and 3 can be realized, thereby forming different stimulation paradigms. The frequency is 20Hz, the total stimulation time is 5 seconds, and the stimulation paradigm is 15 seconds when the stimulation is stopped. In each stimulation period of 5 seconds, the first scanning galvanometer scans the stimulation beam into the multimode fiber 18 and stays there for 5 milliseconds, then scans out the multimode fiber 18 and stays there for 45 milliseconds to form a stimulation beam with a frequency of 20 Hz.

Referring to FIG. 4 , in the no-sample synchronous stimulation recording mode, the signal recorded by the camera 25 is smooth, indicating that there is no crosstalk of the stimulation beam to the signal.

Referring to FIG. 5 , this embodiment adopts a stimulation paradigm in which the single stimulation time is 5 milliseconds, the stimulation frequency is 20 Hz, the total stimulation duration is 5 seconds, and the stimulation stop duration is 15 seconds. The synchronous stimulation recording signal is shown in (I) of Fig. 5. Turn off the stimulation module, and record the signal of the experimental animal as shown in (II) of Figure 5. Therefore, the present invention can realize the synchronous stimulation and recording of a single optical fiber, the regulation of brain nerve activity and the synchronous reading of brain information, with good signal quality and adjustable stimulation parameters.

Referring to FIG. 6 , this embodiment presents an experimental paradigm combining three modes and corresponding experimental results. In this example, the purpose of the experiment is to manipulate the motor behavior of the mouse and record the corresponding signals, so as to study the function of the motor brain area of the mouse.

During the experiment, first, use mode 1: single recording mode to monitor the quality of the fluorescent signal of the mouse, and judge whether the expression of the calcium indicator is completed in the neuron cells. Use mode 3: Stimulus recording synchronization mode, monitor the quality of mouse stimulation signals, and judge whether the light-sensitive protein is expressed in neuron cells. When the light-sensitive protein is expressed, the subsequent experiments can be started.

In the formal experiment, use mode 2: single stimulation mode, and analyze the movement behavior of mice during the stimulation process by shooting video, and judge the specific movement function controlled by the optical fiber implanted in the brain area.

The calcium ion indicator is sensitive to light, and continuous light will cause it to photobleach, resulting in the inability of the calcium ion indicator to emit fluorescence or weakening of the fluorescence, which will affect the accuracy of the experimental results.

During the experiment, a brief mode 1: single recording mode was performed at an interval of 5 minutes, in order to determine whether photobleaching had affected neuronal signals in mice and affected the experimental results. Stop the experiment when the signal value is lower than expected. Simultaneously conduct a brief mode three: Stimulation-recording synchronous mode, the purpose is to judge whether the light-sensitive protein loses its activity or its activity weakens, and whether the cells are effectively activated by the stimulating beam. Also, stop the experiment when the signal value is lower than expected.

This operation mode effectively avoids the influence of the continuous imaging beam on the biological signal, and also avoids the damage of the biological tissue caused by the heat generated by the long-term beam.

Claims (6)

1. A brain neural activity regulation and brain information synchronous reading system, characterized in that: Including the recording light source module, the imaging light beam emitted by the imaging light source passes through the stimulation module and then reaches the signal acquisition module; The recording light source module includes an imaging light source (8), a second half-wave plate (9), a second scanning galvanometer (10), a third doublet lens (11), a first mirror (12), a fourth The doublet lens (13) and the second reflector (14); the imaging light source (8), the second half-wave plate (9), and the second scanning vibrating mirror (10) are arranged in sequence along the optical axis of the same straight line, and the second scanning vibrating The mirror (10), the third doublet lens (11), and the first reflector (12) are sequentially arranged along the same linear optical axis, and the first reflector (12), the fourth doublet lens (13) and the second reflector (14) Arranged sequentially along the optical axis of the same straight line; the imaging light source (8) emits an imaging beam, which is sequentially rotated by the second half-wave plate (9) and reflected by the second scanning galvanometer (10), and then sequentially passes through the third double After the cemented lens (11) transmits, the first reflector (12) reflects, the fourth doublet lens (13) transmits, and the second reflector (14) reflects, it enters the polarization beam splitter (15), and passes through the polarization beam splitter ( 15) After reflection, it is incident into the signal acquisition module; Including a stimulating module, which sends a stimulating light beam to the signal acquisition module through a stimulating light source; The stimulation module includes a stimulation light source (1), a first half-wave plate (2), a first doublet lens (3), a second doublet lens (4), a first scanning galvanometer (5), a scanning mirror (6), infinity correction sleeve lens (7) and polarization beam splitter prism (15); laser source (1), first half-wave plate (2), first doublet lens (3), second doublet lens (4), the first scanning galvanometer (5) is sequentially arranged along the same straight line optical axis, the laser source (1) emits a stimulating beam, and the stimulating beam is sequentially polarized and rotated by the first half-wave plate (2), and is transmitted by the first doublet lens (3) and the second doublet lens (4) constitute the beam expansion and collimation of the doublet lens assembly, after reflection by the first scanning galvanometer (5), it is transmitted through the scanning mirror (6) in turn, and the infinity correction sleeve lens (7) After being transmitted, it is incident into the polarization beam splitter prism (15), and after being transmitted through the polarization beam splitter prism (15), it is incident into the signal acquisition module; Including the signal acquisition module, which receives the imaging beam from the recording light source module and the stimulation beam from the stimulation module, and uses the implanted multimode optical fiber to input to the neurons in the brain area of the organism, regulates the neural activity of the brain of the organism and excites the neurons Generate signals to realize real-time acquisition of neural signals in the biological brain area and monitoring of light sources; at the same time, multi-mode optical fibers are used to collect the fluorescence generated after the imaging beam irradiates neurons and send them back to the signal recording module; Including a signal recording module, which receives fluorescence from the signal acquisition module and converts it into an electrical signal; The signal acquisition module includes a dichroic mirror (16), an objective lens (17), a first multimode optical fiber (18), a self-calibration module and a photodetector (22); a dichroic mirror (16), an objective lens ( 17), the first multimode optical fiber (18) and the self-calibration module are sequentially arranged along the same straight optical axis, and the stimulation beam and imaging beam are both incident on the dichroic mirror (16) through the polarization beam splitter prism (15) of the stimulation module to generate transmission, and then transmitted through the objective lens (17) and converged, it is incident on one end of the first multimode fiber (18), the other end of the first multimode fiber (18) is connected to the input end of the self-calibration module, and the output end of the self-calibration module has Two, the two output terminals are respectively connected to the photodetector (22) and the neurons in the brain area of the organism, and the transmitted stimulating beam and imaging beam are respectively incident on the photodetector (22) and the brain area of the organism through the self-calibration module of neurons; The self-calibration module includes a 1x2 fused fiber coupler (19), a second multimode fiber (20) and a third multimode fiber (21); a bundled end of the 1x2 fused fiber coupler (19) serves as a self-calibration module The input end of the first multimode fiber (18) is connected to the other end, and the two branch ends of the 1x2 fused fiber coupler (19) respectively pass through the second multimode fiber (20), the third multimode fiber (21) and the photoelectric detectors (22), neuronal connections in brain regions of organisms; Combining the on and off switching of two different states of the first scanning galvanometer (5) with the on and off switching of the second scanning galvanometer (10), three different modes of the system are constructed: Mode 1, single record mode Control the first scanning galvanometer (5) in the state off and the second scanning galvanometer (10) in the state on, control the photodetector (22) to sample at intervals to obtain a light intensity signal of a lower intensity of the imaging beam, the camera ( 25) Keep continuously collecting images in real time; Mode 2, single stimulation mode Control the first scanning galvanometer (5) in the state on and the second scanning galvanometer (10) in the state off, control the photodetector (22) to sample at intervals to obtain a higher intensity light intensity signal of the stimulating beam, and the camera ( 25) Do not collect images when not working; Mode 3. Recording stimulus synchronization mode Control the first scanning galvanometer (5) in the state of on and the second scanning galvanometer (10) in the state of on, control the photodetector (22) to sample at intervals, and alternately obtain the light intensity signal and stimulus of the lower intensity of the imaging beam The camera (25) keeps collecting images continuously in real time for the light intensity signal with a relatively high intensity of the light beam. 2. A system for regulating and synchronously reading brain information according to claim 1, characterized in that: said imaging light source emits an imaging light beam with a central wavelength of 473 nanometers, and said stimulating light source emits a central wavelength Stimulating beam for 589 nm. 3. A brain nerve activity regulation and brain information synchronous reading system according to claim 1, characterized in that: the signal recording module includes a filter (23), a fifth doublet lens (24) and The camera (25); the optical filter (23), the fifth doublet lens (24) and the camera (25) are sequentially arranged along the optical axis of the same straight line, and the fluorescence coming from the signal acquisition module is filtered by the optical filter (23) in sequence , The fifth doublet lens (24) is converged and incident to the camera (25), and is detected and collected by the camera (25). 4. A brain nerve activity regulation and brain information synchronous reading system according to claim 1, characterized in that: the first multimode optical fiber (18), the second multimode optical fiber (20), the third The optical fiber end face of the multimode optical fiber (21) is designed with an 8-degree slope and an optical coating is added. 5. A brain nerve activity regulation and brain information synchronous reading system according to claim 1, characterized in that: it also includes a vibrating mirror controller (26), respectively connected with the second scanning vibrating mirror (10) of the recording light source module ), the first scanning galvanometer (5) of the stimulus module, and the photodetector (22) of the signal acquisition module are electrically connected, and the galvanometer controller (26) collects the electrical signal detected by the photodetector (22) in real time, and records Feedback adjustment is performed on the work of the second scanning galvanometer (10) of the light source module and the first scanning galvanometer (5) of the stimulation module. 6. A brain nerve activity regulation and brain information synchronous reading system according to claim 1, characterized in that: the detection surface is perpendicular to the optical axis of the objective lens (17) of the signal acquisition module, and the mutual detection surface is established on the detection surface. Orthogonal x and y directions: The first scanning galvanometer (5) adjusts the x direction, and is used to deflect the stimulation beam in the x direction, so as to realize light stimulation with different frequencies and times; The second scanning galvanometer (10) adjusts the x direction and the y direction, and is used for deflecting the stimulation beam in the x direction and the y direction to realize optical imaging.

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