WO2021003782A1 - 一种无标记全光学神经调控与成像的方法与装置 - Google Patents

一种无标记全光学神经调控与成像的方法与装置 Download PDF

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WO2021003782A1
WO2021003782A1 PCT/CN2019/098679 CN2019098679W WO2021003782A1 WO 2021003782 A1 WO2021003782 A1 WO 2021003782A1 CN 2019098679 W CN2019098679 W CN 2019098679W WO 2021003782 A1 WO2021003782 A1 WO 2021003782A1
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oct
imaging
optical
signal
stimulation
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French (fr)
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李鹏
姚霖
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0062—Arrangements for scanning
    • A61B5/0066—Optical coherence imaging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/102—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for optical coherence tomography [OCT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/02—Detecting, measuring or recording pulse, heart rate, blood pressure or blood flow; Combined pulse/heart-rate/blood pressure determination; Evaluating a cardiovascular condition not otherwise provided for, e.g. using combinations of techniques provided for in this group with electrocardiography or electroauscultation; Heart catheters for measuring blood pressure
    • A61B5/026—Measuring blood flow
    • A61B5/0261—Measuring blood flow using optical means, e.g. infrared light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4058—Detecting, measuring or recording for evaluating the nervous system for evaluating the central nervous system
    • A61B5/4064—Evaluating the brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0622—Optical stimulation for exciting neural tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B9/00—Measuring instruments characterised by the use of optical techniques
    • G01B9/02—Interferometers
    • G01B9/0209—Low-coherence interferometers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0626—Monitoring, verifying, controlling systems and methods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0659—Radiation therapy using light characterised by the wavelength of light used infrared
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection

Definitions

  • the present invention belongs to the field of biomedical imaging, and relates to optical coherence tomography (Optical Coherence Tomography, OCT) and neuromodulation technology, in particular to a label-free all-optical neuromodulation and imaging method and device based on the principle of low coherence interference, It can be used in basic and applied neuroscience research to study the response and mapping of biological cortical neurons under near-infrared pulse stimulation.
  • OCT optical Coherence Tomography
  • neuromodulation technology in particular to a label-free all-optical neuromodulation and imaging method and device based on the principle of low coherence interference
  • the brain is the core of the life movement of organisms and the focus of research by scientists. When the brain is subjected to external stimulation, it will produce functional signal changes, which are closely related to biological functions. Therefore, the ability to realize nerve regulation and imaging, and accurately analyze the changes of nerve function signals, is of great significance to the pathological research of human diseases.
  • unit physiological recording is a common method for monitoring neural function.
  • its sampling is limited by the geometry of the electrode recording site, and the electrode needs to be inserted into the brain.
  • imaging methods that can achieve neural imaging, each with its own advantages and disadvantages.
  • two-photon imaging can perform dense sampling in the x, y, and z axis directions, and can achieve cell-level resolution, but is limited by the sampling field of view, and requires virus injection or gene transfection to label cells.
  • Multiphoton imaging depth can reach 1mm or more, but it is not practical for larger animal models.
  • the optical imaging method using Voltage Sensitive Dye (VSD) staining can achieve large-scale, high-time resolution (1-10ms) imaging, but in large animals, VSD tissue staining and light
  • VSD Voltage Sensitive Dye
  • Optical Intrinsic Signal Imaging (OISI) based on hemodynamic signals is often used for larger-scale imaging, which eliminates the need to add exogenous substances to the brain.
  • OISI signals are highly correlated with neuronal population responses and can be used to draw cortical columns, but OISI cannot achieve deep signal detection.
  • the purpose of the present invention is to address the shortcomings of the prior art, and propose a method and device for label-free all-optical nerve regulation and imaging, which combines near-infrared laser pulse stimulation and OCT technology.
  • the invention is a label-free all-optical method, which is used to regulate and map brain nerve functions in a non-contact, large-scale, and deeply distinguishable manner in the cerebral cortex, and the depth can reach 1 mm.
  • a label-free all-optical neuromodulation and imaging method including:
  • the described use of infrared laser pulses to excite neural activity includes: a single stimulation process consists of three stages: the baseline section t0 has no laser energy, the stimulation section t1 has laser energy, and the recovery section t2 has no laser energy; and the stimulation section t1 is near 1870nm.
  • Infrared pulsed laser the pulse parameters of the laser use 250us pulse width, 200Hz pulse frequency, a total of 100 pulse sequences; the above laser stimulation process (t0+t1+t2) repeats the experiment many times to improve signal stability.
  • infrared laser pulses to stimulate neural activity includes: using a chopper method or a current and voltage trigger control method to achieve a specific laser pulse width and frequency.
  • optical coherence tomography OCT to synchronously collect optical scattering signals caused by neural activity of biological tissues includes: adopting external trigger control, clock signal and other methods to realize synchronous collection of the two processes of infrared laser pulse stimulation and OCT recording.
  • OCT optical coherence tomography
  • the use of OCT to synchronously collect optical scattering signals caused by neural activity includes: using OCT to perform two-dimensional or three-dimensional repeated scanning and imaging of the scattered signal samples of biological tissues, and the repeated scanning and imaging at the same spatial position includes complete laser stimulation process time t0+t1+t2, and OCT imaging adopts one of the following methods: time-domain OCT imaging method that changes the optical path of the reference arm by scanning; or spectral-domain OCT imaging method that uses a spectrometer to record spectral interference signals; uses a swept light source to record spectral interference Sweep frequency OCT imaging method of signal.
  • the biological tissue may be cerebral cortex and the like.
  • the extraction of brain function signals using OCT scattering signals specifically includes: using OCT scattering signals to extract neural response signals in non-vascular areas, and the steps include: processing OCT scattering signals to obtain OCT blood flow contrast images, OCT blood flow contrast images Remove the position of the blood vessel in the space where the acquisition location is located in the middle; compare the scattering signal of the baseline segment (t0) to calculate the relative change of the OCT scattering signal; use the scattering signal of the baseline segment (t0) to filter out the continuous significant change signal points of the OCT scattering signal ; Use the screened significant change signal points as a mask, and use the mask to process the relative changes of the OCT scattering signal to obtain a functional OCT (fOCT) signal; average the functional OCT (fOCT) signals of all test processes to reduce noise.
  • OCT scattering signals remove the position of the blood vessel in the space where the acquisition location is located in the middle
  • compare the scattering signal of the baseline segment (t0) to calculate the relative change of the OCT scattering signal
  • OCT scattering signals to extract brain function signals specifically includes: using OCT blood flow contrast (OCTA) technology to extract blood flow signal changes in the vascular region during stimulation.
  • OCT blood flow contrast OCTA
  • a set of near-infrared laser stimulation device to stimulate the neural activity of the target brain tissue
  • a set of synchronization control unit respectively connected to the near-infrared laser stimulation device and OCT optical coherence detection device, used to synchronize the two processes of laser stimulation and OCT recording;
  • One or more processors are respectively connected to the near-infrared laser stimulation device and the OCT optical coherent detection device for analyzing, processing and detecting the OCT scattering signal.
  • the near-infrared laser stimulation device has a certain difference between its working wavelength and the center wavelength of the OCT system.
  • the near-infrared laser generated by the near-infrared laser stimulation device uses the 1870nm band, and the imaging detection of the OCT optical coherent detection device uses the 1300nm band.
  • the OCT optical coherent detection device uses one of the following:
  • the one or more processors include: calculating changes in nerve scattering signals in non-vascular areas and changes in blood flow signals in vascular areas before and after laser stimulation to obtain brain function information.
  • the present invention has the following beneficial effects and advantages:
  • infrared laser pulses are used to excite neural activity
  • OCT is used to simultaneously collect optical scattering signals caused by neural activities
  • OCT scattering signals are used to extract brain function signals.
  • the spectral overlap of the optogenetic probe and calcium indicator leads to crosstalk between the stimulation and imaging channels.
  • the working wavelength of the near-infrared laser stimulation of the present invention and the central wavelength of the OCT system, which ensures that the two optical channels of stimulation and imaging do not interfere with each other, wherein the stimulation adopts the 1870nm band and the imaging adopts the 1300nm band.
  • the stimulation and imaging channels of the present invention work in the infrared waveband, and the penetration depth is deeper than that of visible light, which can reach 1mm.
  • the invention does not require injection of contrast agents or viral transfection, making it compatible with non-human primate studies, and possibly with human studies in a clinical environment.
  • Figure 1 is a schematic diagram of the method of the present invention
  • Figure 2 is a schematic diagram of the device of the present invention.
  • Figure 3 is a schematic diagram of an embodiment of the present invention.
  • FIG. 4 is a timing diagram of near-infrared laser stimulation according to an exemplary embodiment of the present invention.
  • Fig. 5 is an OCT structure diagram of an exemplary embodiment of the present invention.
  • Fig. 6 is an OCT blood flow contrast diagram according to an exemplary embodiment of the present invention.
  • Fig. 7 is a graph of electrophysiological signal results according to an exemplary embodiment of the present invention.
  • FIG. 8 is a diagram of the spatial distribution result of fOCT signals according to an exemplary embodiment of the present invention.
  • FIG. 9 is a graph of the time distribution result of fOCT signals according to an exemplary embodiment of the present invention.
  • Fig. 10 is a graph of the correlation result of fOCT signal stimulation intensity according to an exemplary embodiment of the present invention.
  • Fig. 11 is a graph showing the results of blood flow velocity changes in an exemplary embodiment of the present invention.
  • 1-Infrared laser pulse excites neural activity; 2-OCT collects optical scattering signal caused by neural activity; 3-OCT scattering signal relative change calculation; 11-light source; 12-beam splitter; 13-reference arm collimator 14-Plane high reflection mirror; 15-Sample arm collimator; 16-Scanning galvanometer; 17-Objective lens; 18-Sample to be tested; 19-Interference signal detection device; 20-Signal processor; 21-Polarization controller 31-low-coherence broadband light source; 32-optical circulator; 33-fiber coupler; 34-first fiber collimating device; 35-focusing lens; 36-plane high reflector; 37-second fiber collimating device; 38-scanning galvanometer; 39-focus lens; 40-sample dispersion device; 41-third fiber collimator device; 42-grating; 43-Fourier transform lens; 44-high-speed line scan camera; 45-signal processor Module and calculation unit; 46-first polarization controller; 47-second polar
  • each operation is described as a plurality of discrete operations, but the described order does not represent the order of implementing the operations.
  • the depth z direction is the direction along the incident optical axis
  • the x-y plane is the plane perpendicular to the optical axis, where x and y are orthogonal, and x represents the OCT transverse fast scanning direction, and y represents the slow scanning direction.
  • the method of the present invention is shown in Figure 1. Firstly, near-infrared pulse stimulation is used to stimulate the nerve activity of the target brain, then the OCT system is used to collect the changes in the scatter signal caused by the nerve activity, and finally the OCT scatter signal is used to extract the brain function signal.
  • the stimulation process includes 3 time periods: baseline section t0 has no laser energy, stimulation section t1 has laser energy, and recovery section t2 has no laser energy; stimulation section t1 uses 1870nm near-infrared laser with 250us pulse width , 200Hz pulse frequency, a total of 100 pulse sequences; the above laser stimulation process (t0+t1+t2) can be repeated several times to improve signal stability.
  • the OCT system uses two-dimensional or three-dimensional OCT scanning imaging of the scatter signal samples, repeat the scanning and imaging at the same spatial position for a certain period of time (total time t0+t1+t2), and record the spectrum with a spectrometer
  • the spectral domain OCT imaging method of interference signals (or the time domain OCT imaging method of changing the optical path of the reference arm by scanning and the swept frequency OCT imaging method of using a swept light source to record the spectral interference signal).
  • the OCT scattering signal is used to extract the brain function signal, and the changes of the nerve scattering signal in the non-vascular area and the blood flow signal change in the vascular area before and after laser stimulation are calculated to obtain the brain function information.
  • First combine the OCT blood flow angiogram to eliminate the position of blood vessels in the space and eliminate the influence of blood flow.
  • the specific steps are: determine the base value of the OCT scattered signal, I(z,x,t) Indicates the OCT intensity signal, the OCT intensity signal is the absolute value of the OCT scattering signal, z is the depth direction, x is the lateral direction, and t is the time dimension.
  • the blank time before stimulation is t0.
  • the OCT scattering signal during this period is averaged to obtain the OCT scattering signal base value I Baseline before stimulation:
  • N is the number of frames collected in the corresponding t0 time period.
  • 3 ⁇ (z,x) represents the standard deviation of all pixels at position (z,x);
  • this pixel is defined as a positive signal pixel:
  • the positive effective signal pixels and the negative effective signal pixels are screened out, a mask is generated, and the fOCT signal is obtained.
  • the response value of the negative effective signal pixel is reversed, and then together with the response value of the positive effective signal pixel, the final fOCT signal is generated through averaging processing to reduce noise and improve the signal-to-noise ratio.
  • the removed blood vessel area uses OCT angiography (OCTA) technology to extract hemodynamic response signals during stimulation, and synthesize neural responses to obtain brain function signals.
  • OCT angiography OCTA
  • Figure 2 shows a schematic diagram of the label-free all-optical nerve regulation and imaging device of the present invention.
  • the main structure of the low-coherence interferometry part of the device is an interferometer, which is composed of 11-17, 19, and 21.
  • the light emitted by the light source 11 is divided into two parts by the beam splitter 12: one of the light enters the interference
  • the reference arm of the instrument is irradiated on the plane high reflector 14 through the reference arm collimator 13; another beam of light enters the sample arm and is focused on the sample to be tested after collimation 15 and light path reflection; the sample 18 is placed on the sample The focal plane of the arm objective lens 17.
  • the reflected back by the reference arm and the sample arm interferes and is received by the interference signal detection device 19.
  • the polarization controller 21 is used to adjust the polarization state of the light beam to maximize the signal interference effect.
  • the label-free all-optical neuromodulation and imaging device shown in Fig. 2 specifically includes:
  • the light source 11 uses broadband low-coherence light
  • the plane mirror 14 can move along the optical axis
  • the interference signal detection device 19 is a one-point detector.
  • the interference signals of the two arms are detected by the point detector 19, and the low-coherence interference detection of the scattered signals in the z direction of a certain spatial depth, so as to obtain the depth spatial dimension sample volume .
  • the light source 11 uses broadband low-coherence light
  • the plane mirror 14 is fixed
  • the interference signal detection device 19 uses a spectrometer.
  • the interference signal passes through the line camera in the spectrometer while recording the interference spectrum.
  • the Fourier analysis method is used to analyze the interference spectrum signal, and the scattering information in the depth z direction is obtained in parallel, so as to obtain the sample volume in the depth dimension space.
  • the light source 11 is a swept frequency light source
  • the plane mirror 14 is fixed
  • the interference signal detection device 19 is a point detector.
  • the point detector records the low-coherence interference spectrum of the swept-frequency light source in time sharing. Sampling Fourier analysis of the interference spectrum signal, parallel acquisition of the depth z direction scattering information, so as to obtain the depth dimension space sampling volume.
  • the OCT scanning imaging methods involved in the description of FIG. 1 can be combined to analyze the relative movement of blood flow and surrounding tissues to generate OCTA blood flow movement contrast, and to enhance the spatial correspondence.
  • Figure 3 shows an exemplary embodiment using the present invention disclosed herein.
  • Mark all-optical neuromodulation and imaging device including broadband low-coherence light source 31, optical circulator 32, fiber coupler 33 with a split ratio of 50:50, first polarization controller 46, first fiber collimator 34, focusing lens 35.
  • the signal controller 52 uses Cygnus Technology, PG4000A digital controller; the laser 53 uses a fiber-coupled semiconductor laser with a working band of 1870nm; the broadband low-coherence light source 31 uses a super-luminescent diode light source with a center wavelength of 1325nm and a bandwidth of 100nm.
  • the line scan camera 44 is a line scan camera composed of 2048 pixel units, and the scan lens 51 in the sample arm is a lens with a focal length of 36 mm.
  • the laser 53 emits near-infrared laser light and irradiates the test sample through the laser stimulation fiber 54.
  • the light emitted by the low-coherence broadband light source 31 used in the device of the present invention enters the optical fiber coupler 33 with a splitting ratio of 50:50 after passing through the optical circulator 32, and the light emitted from the optical fiber coupler 33 is divided into two sub-beams :
  • One beam of light is connected to the first fiber collimating device 34 in the reference arm through the first polarization controller 46 through the fiber, and irradiated to the plane high reflector after being focused by the collimation, dispersion compensator 48 and dispersion compensation and focusing lens 35 36;
  • Another beam of light is connected to the second fiber collimation device 37 of the sample arm part through the second polarization controller 47 through the optical fiber, after collimating, the scanning galvanometer 38 light path reflection and the focusing lens 39, the focusing lens 49 are focused and illuminated To the sample to be tested.
  • the dichroic mirror 50 is used to realize the 90° turning of the OCT detection light path of the light emitted by the focusing lens 49.
  • the scanning galvanometer 38 in the sample arm is fixed, so that the low-coherence interferometer can parallelly detect and obtain the depth direction scattering signals at the same position in the sample space at different times.
  • the light path in the sample arm conducts the light beam through the single-mode fiber, and the light scattered back from the sample to be tested plays a role of spatial filtering, that is, effectively reducing the multiple scattering components in the scattered signal.
  • the light reflected by the plane high reflector 36 in the reference arm interferes with the backscattered light of the measured sample in the sample arm at the fiber coupler 33.
  • the interference light is detected and recorded by the spectrometer (including devices 41 to 44), and then The signal controller 52 and the calculation unit 45 collect and analyze the signal.
  • the light stimulation unit and the OCT signal acquisition unit are synchronized by means of external trigger control, clock signals, etc.
  • the present invention can obtain neural response and hemodynamic response before and after stimulation and their characteristics.
  • the near-infrared laser stimulation sequence is shown in Figure 4. There is no laser energy in the t0 period, the laser energy starts to work in the t1 period, and the t2 period is the recovery period without laser energy.
  • the pulse width of each pulse in the output laser pulse sequence Adjustable, pulse period is adjustable. In the specific implementation here, the fixed pulse width of each pulse is 250 ⁇ s, and the pulse period is 5 ms.
  • Near-infrared laser stimulation is a 0.5s pulse train composed of 100 such pulses. The laser is output through a special optical fiber and irradiated to the target area of the rat optical observation window.
  • FIG. 5 shows the structure diagram of OCT acquisition.
  • OCT can realize three-dimensional structure imaging
  • FIG. 5A is a projection diagram of the OCT structure
  • FIG. 5B is a cross-sectional view of the dotted line in FIG. 5A.
  • FIG. 6 shows an OCT blood flow radiography (OCTA) image.
  • the three-dimensional structure signal collected by the OCT is processed by the OCTA technology to obtain the three-dimensional blood flow signal.
  • FIG. 6A is an OCTA projection view
  • FIG. 6B is a cross-sectional view at the position of the dotted line in FIG. 6A.
  • Figure 7 shows the results of electrophysiological signals under different stimulation intensities (0.3J, 0.5J, 0.7J, 1.0J/cm 2 /pulse). As the intensity of near-infrared laser stimulation increases, the spike rate increases. The results show that the near-infrared laser stimulation of the rat trunk motor sensory cerebral cortex can cause the excitatory activity of neurons. Therefore, the changes in the fOCT response signal observed later may be potentially related to the activity of neurons caused by external stimuli.
  • Figures 8-11 show graphs of the results of changes in the fOCT functional signal of rat brain cortex obtained in this example.
  • the present invention verifies the temporal and spatial distribution of the neural response in the non-vascular area, as well as its relationship with the stimulation intensity, as well as the hemodynamic response in the vascular area.
  • the OCT system collects signals from the center and surrounding areas of the near-infrared laser stimulation area to observe the fOCT function response (ROI1: center, ROI2: edge, ROI3: external). It can be observed in the result graph that the near-infrared laser stimulation caused a relative change in the OCT scattering signal in the rat brain cortex (the signal change in Fig. 8A is quantitatively analyzed in Fig. 8B).
  • ROI1 contains the largest number of effective pixels ( ⁇ 449 pixels); ROI2 contains less effective pixels ( ⁇ 354 pixels); ROI3 does not show a significant change in scattering.
  • the fOCT signal reflects the spatial positioning of the neural signal as shown in Figure 8A, and the average amplitude of all effective pixels and the time-varying process of the induced fOCT signal is shown in Figure 8B.
  • the time correspondence of fOCT changes can be maintained to the depth range of the cortex 600um.
  • the fOCT signal time is also closely related to the near-infrared laser stimulation time.
  • the changes in the OCT scattering signal are synchronized with the near-infrared laser stimulation. All pixels with significant changes in the experiment were averaged for the population response. There is a similar time coincidence in the scattering changes in Figure 9A. If the stimulus start time is different, the response start time delay and the response peak time delay are quantified in Figure 9B. The results show that the fOCT response is synchronized in time with the laser stimulation, and the time delay is about 30ms.
  • Figures 10A-10B show the functional response at different stimulus intensities (0.3, 0.5, 0.7, 1.0 J/cm 2 /pulse).
  • the increase in the intensity of the near-infrared laser stimulation leads to an increase in the amplitude of the change of the fOCT signal.
  • the result is shown in FIG. 10A.
  • the peak value of the change in the fOCT signal has a linear relationship with the stimulation intensity, and the result is shown in FIG. Due to the high sensitivity of interferometric detection, the amplitude of the fOCT signal change is relatively large (the slope of linear fitting: 2.1).
  • Figure 11 shows the hemodynamic response of increasing the OCT acquisition frequency and different stimulation intensities (0.5, 0.7, 1.0 J/cm 2 /pulse).
  • the increase in the intensity of the near-infrared laser stimulation also leads to an increase in the velocity of the blood vessel area.
  • the time delay is about 1s.
  • the above experimental results fully illustrate that the present invention can simultaneously carry out neural regulation and imaging, there is no crosstalk phenomenon of regulation/imaging channels, no contrast agent injection and virus transfection are needed, and the near-infrared band can realize regulation and imaging of deep tissues.
  • the invention can realize non-marked all-optical nerve regulation and imaging, and can simultaneously perform non-contact precise stimulation and brain function signal detection on the target area.

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Abstract

本发明公开了一种无标记全光学神经调控与成像的方法与装置。利用红外激光脉冲激发神经活动，利用OCT采集神经活动引发的光学散射信号，实现无标记全光学神经调控与成像，具体包括：将红外激光瞄准脑部目标区域，射入一定时间序列的激光脉冲，利用OCT对目标区域进行同步扫描成像，分析OCT散射信号相比于激光刺激前的相对变化，得到基于OCT的脑功能信号，同步实现神经调控与成像。本发明可以同步开展神经调控和成像，不存在调控/成像通道的串扰现象，无需注射造影剂和病毒转染，近红外波段可实现组织深部区域的调控与成像。

Description

一种无标记全光学神经调控与成像的方法与装置 技术领域
本发明属于生物医学成像领域，涉及光学相干层析成像技术(Optical Coherence Tomography,OCT)和神经调控技术，尤其涉及一种基于低相干干涉原理的无标记全光学神经调控与成像的方法与装置，可用于基础与应用神经科学研究中，研究近红外脉冲刺激下生物皮质神经元的响应和映射情况。
背景技术
大脑是生物体生命运动的核心，也是科学家研究的焦点。大脑在经受外部刺激时，会产生功能信号变化，这种变化与生物机能密切相关。因此，能够实现神经调控与成像，准确分析神经功能信号变化，对人类疾病病理研究具有重大意义。
对于许多旨在神经调控和功能区追踪的脑功能研究，需要给予脑部神经刺激，研究其功能响应。其中，电刺激是神经刺激的常用工具，但它受到电流的扩散作用影响，经常导致激活额外的脑电路，容易产生副作用。此外，刺激伪影使得同时进行电生理刺激和信号记录存在困难。与此相比，光学刺激技术的发展有明显的优势。其中，光遗传学是一种特异性细胞刺激技术，其与麻醉和清醒行为动物中的电记录相容。然而，在诸如灵长类动物中，它需要注射病毒，病毒表达的时间较长，通常为4-6周，刺激位点仅限于病毒表达的位点。其他更大规模的神经刺激方法也有(如经颅磁刺激、超声等)，但空间分辨率低。
对于神经信号记录，单位生理学记录是监测神经功能的常用方法。然而，其取样受到电极记录部位几何形状的限制，并且需要将电极***脑中。神经信号成像方面，目前已有许多成像方式可以实现神经成像，各有其优缺点。其中，双光子成像可以在x，y和z轴方向进行密集采样，而且能够达到细胞级分辨率，但受采样视场的限制，并且需要通过注射病毒或基因转染来标记细胞。多光子成像深度可以达到1mm或更大，但对于较大的动物模型不实用。为了监测种群水平的神经活动，利用电压敏感染料(Voltage Sensitive Dye,VSD)染色的光学成像方法可实现大规模、高时间分辨率(1～10ms)成像，但是在大型动物中，VSD组织染色和光动力学损伤的相关性限制了其推广。基于血液动力学信号的内源信号光成像(Optical Intrinsic Signal Imaging,OISI)常用于更大规模成像，可以无需向脑中加入外源性物质。OISI信号与神经元群体响应相关性高，可用 于绘制皮质柱，但是OISI无法实现深度信号探测。
以上技术已有不同的组合，实现了神经刺激和信号记录。然而，目前的方法无法同时具有非接触式(无需***或应用任何材料)、大规模(mm到cm的比例)、深度可分辨(可以区分不同的深度)，并且易于应用于大型动物模型等特征。所以目前迫切需要一种全光学手段同时进行神经调控与成像的方法与装置，可以同时进行无接触精准刺激和信号检测。
发明内容
本发明的目的是针对现有技术的不足，提出了一种无标记全光学神经调控与成像的方法与装置，结合了近红外激光脉冲刺激和OCT技术。
本发明是无标记的全光学方法，用于在大脑皮层中以无接触，大规模，深度可分辨的方式调控和映射脑神经功能，深度可达到1mm。
本发明的目的是通过下述技术方案实现的：
一、一种无标记全光学神经调控与成像的方法，包括：
S1、利用红外激光脉冲激发神经活动；
S2、利用OCT同步采集神经活动引发的光学散射信号；
S3、利用OCT散射信号提取脑功能信号。
所述的利用红外激光脉冲激发神经活动，包括：单次刺激过程由3个阶段组成：基线段t0没有激光能量，刺激段t1有激光能量，恢复段t2没有激光能量；刺激段t1采用1870nm近红外波段的脉冲激光；激光的脉冲参数采用250us脉冲宽度，200Hz脉冲频率，总计100个脉冲序列；上述激光刺激过程(t0+t1+t2)重复试验多次，提高信号稳定性。
所述的利用红外激光脉冲激发神经活动，包括：采用斩波器方式、或者电流、电压触发控制的方式实现特定的激光脉冲宽度和频率。
所述的利用光学相干层析成像OCT同步采集生物组织神经活动引发的光学散射信号，包括：采用外部触发控制、时钟信号等方式实现红外激光脉冲刺激与OCT记录两个过程的同步采集。
所述的利用OCT同步采集神经活动引发的光学散射信号，包括：利用OCT对生物组织的散射信号样本进行二维或三维空间的重复扫描成像，在同一空间位置重复扫描成像包含完整激光刺激过程时间t0+t1+t2，并且OCT成像采用以下方式之一：通过扫描改变参考臂光程的时间域OCT成像方法；或者利用光谱仪记录光谱干涉信号的光谱域OCT成像方法；利用扫频光源记录光谱干涉信号的扫频OCT成像方法。
所述的生物组织可以为大脑皮层等。
所述的利用OCT散射信号提取脑功能信号，具体包括：利用OCT散射信号，提取非血管区域的神经响应信号，其步骤包括：用OCT散射信号处理获得OCT血流造影图，OCT血流造影图中剔除采集位置所在空间中血管的位置；对比基线段(t0)的散射信号，计算OCT散射信号的相对变化；利用基线段(t0)的散射信号，筛选出OCT散射信号连续的显著变化信号点；以筛选出的显著变化信号点作为掩模，用掩模对OCT散射信号的相对变化处理得到功能OCT(fOCT)信号；对所有试验过程的功能OCT(fOCT)信号进行平均，减小噪声。
所述的利用OCT散射信号提取脑功能信号，具体包括：利用OCT血流造影(OCTA)技术，提取刺激过程中血管区域的血流信号变化。
近红外激光刺激的工作波长和OCT***中心波长存在一定的差异，保证刺激与成像两个光学通道互不干扰，包括：近红外激光刺激采用1870nm波段，OCT成像采用1300nm波段。
二、无标记全光学神经调控与成像装置：
一套近红外激光刺激装置，用于激发目标脑部组织的神经活动；
一套OCT光学相干探测装置，用于对二维或者三维空间内的光学散射信号进行OCT采集；
一套同步控制单元，分别连接近红外激光刺激装置和OCT光学相干探测装置，用于同步实现激光刺激与OCT记录两个过程；
一个或多个处理器，分别连接近红外激光刺激装置和OCT光学相干探测装置，用于分析处理探测得到OCT散射信号。
所述的近红外激光刺激装置，其工作波长和OCT***中心波长存在一定的差异，近红外激光刺激装置产生的近红外激光采用1870nm波段，OCT光学相干探测装置的成像探测采用1300nm波段。
所述的一OCT光学相干探测装置是采用以下的一种：
包括低相干光源、干涉仪和探测器；
或者包括低相干光源、干涉仪和光谱仪；
或者包括扫频宽光谱光源、干涉仪和探测器。
所述的一个或多个处理器包括：计算在激光刺激前后，非血管区域的神经散射信号变化和血管区域的血流信号变化，得到脑功能信息。
本发明相比现有技术具有以下有益效果和优势：
结合近红外激光脉冲刺激和OCT技术，利用红外激光脉冲激发神经活动，并利用OCT同时采集神经活动引发的光学散射信号，利用OCT散射信号提取 脑功能信号。
相比于已有的光遗传学和光学钙成像的组合，其由于光遗传探针和钙指示剂光谱重叠，导致刺激和成像通道之间存在串扰。本发明近红外激光刺激的工作波长和OCT***中心波长存在一定的差异，保证刺激与成像两个光学通道互不干扰，其中刺激采用1870nm波段，成像采用1300nm波段。
相比于已有的光遗传学和光学钙成像的组合，其由于组织内强烈可见光散射，导致刺激和成像深度有限。本发明刺激和成像通道皆工作在红外波段，相比于可见光，穿透深度更深，可达到1mm。
相比于已有的光遗传学和光学钙成像的组合，其需要对实验动物进行病毒转染，不适合用于非人类灵长类动物研究。本发明不需要注射造影剂或进行病毒转染，使其与非人类灵长类动物研究兼容，并且可能与临床环境中的人体研究相容。
附图说明
图1为本发明方法的示意图；
图2为本发明装置的示意图；
图3为本发明实施例的示意图；
图4为本发明示例性实施例的近红外激光刺激时序图；
图5为本发明示例性实施例的OCT结构图；
图6为本发明示例性实施例的OCT血流造影图；
图7为本发明示例性实施例的电生理信号结果图；
图8为本发明示例性实施例的fOCT信号空间分布结果图；
图9为本发明示例性实施例的fOCT信号时间分布结果图；
图10为本发明示例性实施例的fOCT信号刺激强度相关性结果图；
图11为本发明示例性实施例的血流速度变化结果图。
其中：1-红外激光脉冲激发神经活动；2-OCT采集神经活动引发的光学散射信号；3-OCT散射信号的相对变化计算；11-光源；12-分束器；13-参考臂准直镜；14-平面高反射镜；15-样品臂准直镜；16-扫描振镜；17-物镜；18-待测样品；19-干涉信号探测装置；20-信号处理器；21-偏振控制器；31-低相干宽带光源；32-光环形器；33-光纤耦合器；34-第一光纤准直器件；35-聚焦透镜；36-平面高反射镜；37-第二光纤准直器件；38-扫描振镜；39-聚焦透镜；40-样品分散装置；41-第三光纤准直器件；42-光栅；43-傅里叶变换透镜；44-高速线阵相机；45-信号处理器模块与计算单元；46-第一偏振控制器；47-第二偏振控制器；48：色 散补偿器；49：聚焦透镜；50：二向色镜；51：扫描透镜；52：信号控制器；53：激光器；54：激光刺激光纤。
具体实施方式
下面将结合附图对本发明的具体实施方式作详细说明，附图形成本文的一部分。需要注意的是，这些说明及示例仅仅为示例性的，不能被理解为限制了本发明的范围，本发明的保护范围由随附的权利要求书限定，任何在本发明权利要求基础上的改动都是本发明的保护范围。
为了便于理解本发明的实施例，将各操作描述成多个离散的操作，但是，描述的顺序不代表实施操作的顺序。
本描述中针对样品测量空间采用基于空间方向的x-y-z三维坐标表示。这种描述仅仅用于促进讨论，而不意欲限制本发明的实施例的应用。其中：深度z方向为沿入射光轴的方向；x-y平面为垂直于光轴的平面，其中x与y正交，且x表示OCT横向快扫描方向，y表示慢扫描方向。
上述i，I，t等表示变量，仅仅用于促进讨论，而不意欲限制本发明的实施例的应用，可以是1,2,3等任一数值。
本发明方法如图1所示，首先利用近红外脉冲刺激，激发目标脑部神经活动，然后利用OCT***采集神经活动引起的散射信号变化，最后利用OCT散射信号提取脑功能信号。
利用近红外脉冲刺激，刺激过程包括3个时间段：基线段t0没有激光能量，刺激段t1有激光能量，恢复段t2没有激光能量；刺激段t1采用1870nm近红外波段的激光，250us的脉冲宽度，200Hz的脉冲频率，总计100个脉冲序列；上述激光刺激过程(t0+t1+t2)可以重复试验多次以提高信号稳定性。
利用OCT***采集神经活动引起的散射信号变化，对散射信号样本进行二维或三维空间的OCT扫描成像，在同一空间位置重复扫描成像一定时间(总时间t0+t1+t2)，利用光谱仪记录光谱干涉信号的光谱域OCT成像方法(或者通过扫描改变参考臂光程的时间域OCT成像方法和利用扫频光源记录光谱干涉信号的扫频OCT成像方法)。
利用OCT散射信号提取脑功能信号，计算在激光刺激前后，非血管区域的神经散射信号变化和血管区域的血流信号变化，得到脑功能信息。首先结合OCT血流造影图，剔除空间中血管位置，消除血流的影响。数据处理部分，先对比刺激前(t0)的散射信号，计算OCT散射信号在激光刺激中(t1)的相对变化， 其具体步骤是：确定OCT散射信号基值，I(z,x,t)表示OCT强度信号，OCT强度信号为OCT散射信号的绝对值，z为深度方向，x为横向方向，t为时间维度。
刺激之前的空白时间为t0段，将此段时间的OCT散射信号平均得到刺激之前的OCT散射信号基值I Baseline：
Figure PCTCN2019098679-appb-000001
其中，N为对应t0时间段采集的帧数。
用dR/R表示实时采集的OCT散射信号相比于OCT散射信号基值的相对变化：
Figure PCTCN2019098679-appb-000002
为了提高计算效率，并不是所有时刻的信号点都用于计算，需要按照一定原则筛选。基本步骤如下：如果(z,x,t i)位置像素点起始的连续5帧的OCT强度信号强度值小于I Baseline减去3σ(z,x)之后的值，则此像素点定义为负有效信号像素，表示为：
I(z,x,t i:t i+4)<I Baseline-3σ(z,x)  (3)
其中，3σ(z,x)表示(z,x)位置上所有像素点的标准差；
类似地，如果(z,x,t i)位置像素点连续5帧的信号强度值小于I Baseline加上3σ(z,x)之后的值，则此像素点定义为正有效信号像素：
I(z,x,t i:t i+4)>I Baseline+3σ(z,x)   (4)
由此筛选出正有效信号像素和负有效信号像素，生成掩模，得到fOCT信号。在OCT散射信号中，将负有效信号像素的响应值取反，然后和正有效信号像素的响应值一起，通过平均处理产生最终的fOCT信号，以此来减小噪声，提高信噪比。
剔除的血管区域利用OCT血流造影(OCTA)技术，提取刺激过程中血液动力学的响应信号，综合神经响应得到脑功能信号。
图2示出的是本发明的无标记全光学神经调控与成像装置示意图。该装置的低相干干涉测量部分的主体结构为一干涉仪，由11～17、19和21构成，其中光源11发出的光被分束器12分成两部分光束：其中的一束光进入到干涉仪的参考臂，通过参考臂准直镜13照射于平面高反射镜14上；另一束光进入到样品臂，经过准直15和光路反射后聚焦到待测样品上；样品18置于样品臂物镜17的焦平面处。而后参考臂和样品臂各自反射回的光发生干涉后由干涉信号探 测装置19接收。对于光纤型光路，采用偏振控制器21调整光束的偏振态，最大化信号干涉效果。
依据低相干干涉探测信号的不同方式，图2所示的无标记全光学神经调控与成像装置具体包括：
1)时间域测量装置。光源11采用宽带低相干光，平面反射镜14可沿光轴方向移动，干涉信号探测装置19为一点探测器。通过移动平面反射镜14改变参考臂光程，两臂的干涉信号由点探测器19探测到，对某一空间深度的z方向的散射信号的低相干干涉探测，从而得到深度空间维度的采样体。
2)光谱域测量装置。光源11采用宽带低相干光，平面反射镜14固定不动，干涉信号探测装置19采用光谱仪。干涉信号经过光谱仪中的线阵相机同时记录干涉光谱。采用傅里叶分析方法分析干涉光谱信号，并行获取深度z方向的散射信息，从而得到深度维度空间的采样体。
3)扫频测量装置。光源11采用扫频光源，平面反射镜14固定不动，干涉信号探测装置19采用点探测器。点探测器分时记录扫频光源的低相干干涉光谱。采样傅里叶分析干涉光谱信号，并行获取深度z方向的散射信息，从而得到深度维度空间的采样体。
对于上述不同的测量装置，可分别结合图1叙述中所涉及的OCT扫描成像方式，分析血流与周围组织的相对运动生成OCTA血流运动造影，并增强空间对应性。
图3示出的是本文所公开的利用本发明的一个示例性实施例。标记全光学神经调控与成像装置，包括宽带低相干光源31、光环形器32、分光比为50:50的光纤耦合器33、第一偏振控制器46、第一光纤准直器件34、聚焦透镜35、平面高反射镜36、第二偏振控制器47、第二光纤准直器件37、扫描振镜38、物镜39、样品分散装置40、第三光纤准直器件41、光栅42、傅里叶变换透镜43、高速线阵相机44、信号处理器模块与计算单元45、色散补偿器48、聚焦透镜49、二向色镜50、扫描透镜51、信号控制器52、激光器53、激光刺激光纤54。其中信号控制器52采用Cygnus Technology，PG4000A数字控制器；其中激光器53采用工作波段为1870nm的光纤耦合半导体激光器；其中宽带低相干光源31采用中心波长为1325nm、带宽为100nm的超发光二极管光源，高速线阵相机44采用由2048像素单元组成的线阵扫描相机，样品臂中扫描透镜51选用焦距为36mm的透镜。
激光器53发出近红外激光经激光刺激光纤54照射到被测样品上。其中由本发明装置所使用的低相干宽带光源31发出的光，经过光环行器32后进入到 分光比为50:50的光纤耦合器33，从光纤耦合器33出射的光被分成两部分子光束：其中一束光通过光纤经过第一偏振控制器46连接至参考臂中的第一光纤准直器件34，经过准直、色散补偿器48色散补偿和聚焦透镜35聚焦后照射到平面高反射镜36；另一束光通过光纤经过第二偏振控制器47连接至样品臂部分的第二光纤准直器件37，经过准直、扫描振镜38光路反射和聚焦透镜39、聚焦透镜49聚焦后照射到被测样品上。在扫描透镜51之前，采用二向色镜50将聚焦透镜49出射的光线实现OCT探测光路的90°转折。样品臂中的扫描振镜38固定不动，使得低相干干涉仪能够并行探测得到样品空间同一位置在不同时刻的深度方向的散射信号。同时样品臂中的光路通过单模光纤传导光束，对待测样品散射回的光起到空间滤波的作用，即有效地减小散射信号中的多次散射成分。由参考臂中平面高反射镜36反射的光与样品臂中被测样品背向散射的光在光纤耦合器33处干涉，干涉光经过光谱仪(包括器件41～44)探测并被记录，而后由信号控制器52与计算单元45采集并作信号分析处理。光刺激单元和OCT信号采集单元通过采用外部触发控制、时钟信号等方式实现同步。
本发明根据fOCT信号可以得到刺激前后神经响应和血流动力学响应及其特征。近红外激光刺激时序如图4所示，t0时间段无激光能量，t1时间段激光能量开始作用，t2时间段为无激光能量的恢复时期，其中输出的激光脉冲序列中每个脉冲的脉宽可调，脉冲周期可调。这里具体实施中，每个脉冲的固定脉宽为250μs，脉冲周期5ms。近红外激光刺激是由100个这样的脉冲构成0.5s的脉冲链。激光通过特制的光纤输出，照射到大鼠光学观察窗口目标区域。
图5示出的是OCT采集的结构图，OCT可实现三维结构成像，图5A为OCT结构投影图，图5B为图5A中虚线位置的截面图。
图6示出的是OCT血流造影(OCTA)图，利用OCTA技术对OCT采集的三维结构信号进行处理，可以得到三维血流信号。图6A为OCTA投影图，图6B为图6A中虚线位置的截面图。
图7示出的是不同刺激强度下(0.3J,0.5J,0.7J,1.0J/cm 2/pulse)电生理信号结果图，随着近红外激光刺激强度增加导致锋电位发放率增加，该结果说明了针对大鼠躯干运动感觉脑皮质的近红外激光刺激，会引起神经元的兴奋活动。因此后续观察到的fOCT响应信号变化，可能与神经元受外界刺激所引起的活动之间存在潜在的关联性。
图8-11示出的是利用本实施例中得到的大鼠脑皮层fOCT功能信号变化结果图。为了表征近红外激光刺激诱发的fOCT信号，本发明验证了非血管区域神经响应的时间和空间分布，以及它与刺激强度的关系，还有血管区域的血流动 力学响应。
如图8A所示，在不移动激光刺激光纤并使用相同的刺激参数情况下，OCT***对近红外激光刺激区域中心及周围区域进行信号采集，观察fOCT功能响应(ROI1：中心，ROI2：边缘，ROI3：外部)。结果图中可观察到，近红外激光刺激引起大鼠脑皮质中OCT散射信号发生相对变化(在图8B中定量分析图8A中的信号变化)。ROI1包含的有效像素数量最大(～449像素)；ROI2包含的有效像素数量减小(～354像素)；ROI3在散射中没有表现出显著变化。综上，fOCT信号反映了神经信号的空间定位如图8A所示，所有有效像素的平均幅度以及诱发的fOCT信号随时间变化过程如图8B所示。此外，如深度分辨序列图8C所示，fOCT变化的时间对应性可以保持至皮层600um的深度范围。
fOCT信号时间上也与近红外激光刺激时间紧密相关。如图9A-9B所示，OCT散射信号的变化与近红外激光刺激同步。试验中所有具有显著变化的像素均针对群体响应进行了平均。在图9A中的散射变化中也有类似的时间重合性。如果刺激开始时间不同，则响应开始时间延迟和响应峰值时间延迟，在图9B中量化。结果显示fOCT响应在时间上与激光刺激同步，时间延迟约为30ms。
图10A-10B显示了不同刺激强度下(0.3,0.5,0.7,1.0J/cm 2/pulse)的功能响应。近红外激光刺激强度增加导致fOCT信号变化幅度增加，结果如图10A所示，fOCT信号变化峰值与刺激强度存在线性关系，结果如图10B所示。由于干涉检测的高灵敏度，fOCT信号变化的幅度较大(线性拟合的斜率：2.1)。
图11显示了提高OCT采集频率，不同刺激强度下(0.5,0.7,1.0J/cm 2/pulse)的血流动力学响应，近红外激光刺激强度增加也导致血管区域流速变化幅度增加，流速变化时间延迟约为1s。
上述实验结果充分说明：本发明可以同步开展神经调控和成像，不存在调控/成像通道的串扰现象，无需注射造影剂和病毒转染，近红外波段可实现组织深部区域的调控与成像。用本发明可以实现无标记全光学神经调控与成像，可以对目标区域同时进行无接触精准刺激和脑功能信号检测。

Claims (10)

  1. 一种无标记全光学神经调控与成像的方法，包括：
    利用红外激光脉冲激发神经活动；
    利用OCT同步采集神经活动引发的光学散射信号；
    利用OCT散射信号提取脑功能信号。
  2. 根据权利要求1所述的无标记全光学神经调控与成像的方法，其特征在于：利用红外激光脉冲激发神经活动，包括：
    单次刺激过程由3个阶段组成：基线段t0没有激光能量，刺激段t1有激光能量，恢复段t2没有激光能量；
    刺激段t1采用1870nm近红外波段的脉冲激光；
    激光的脉冲参数采用250us脉冲宽度，200Hz脉冲频率，总计100个脉冲序列；
    上述激光刺激过程(t0+t1+t2)重复试验多次。
  3. 根据权利要求1所述的无标记全光学神经调控与成像的方法，其特征在于：利用红外激光脉冲激发神经活动，包括：采用斩波器方式、或者电流、电压触发控制的方式实现特定的激光脉冲宽度和频率。
  4. 根据权利要求1所述的无标记全光学神经调控与成像的方法，其特征在于：利用光学相干层析成像OCT同步采集生物组织神经活动引发的光学散射信号，包括：采用外部触发控制、时钟信号等方式实现红外激光脉冲刺激与OCT记录两个过程的同步采集。
  5. 根据权利要求1所述的无标记全光学神经调控与成像的方法，其特征在于：利用OCT同步采集神经活动引发的光学散射信号，包括：利用OCT对生物组织进行二维或三维空间的重复扫描成像，在同一空间位置重复扫描成像包含完整激光刺激过程时间t0+t1+t2，并且OCT成像采用以下方式之一：通过扫描改变参考臂光程的时间域OCT成像方法；或者利用光谱仪记录光谱干涉信号的光谱域OCT成像方法；利用扫频光源记录光谱干涉信号的扫频OCT成像方法。
  6. 根据权利要求1所述的无标记全光学神经调控与成像的方法，其特征在于：利用OCT散射信号提取脑功能信号，具体包括：利用OCT散射信号，提取非血管区域的神经响应信号，其步骤包括：
    用OCT散射信号处理获得OCT血流造影图，OCT血流造影图中剔除采集位置所在空间中血管的位置；
    对比基线段(t0)的散射信号，计算OCT散射信号的相对变化；
    利用基线段(t0)的散射信号，筛选出OCT散射信号连续的显著变化信号点；
    以筛选出的显著变化信号点作为掩模，用掩模对OCT散射信号的相对变化处理得到功能OCT(fOCT)信号；
    对所有试验过程的功能OCT(fOCT)信号进行平均，减小噪声。
  7. 根据权利要求1所述的无标记全光学神经调控与成像的方法，其特征在于：利用OCT散射信号提取脑功能信号，具体包括：利用OCT血流造影(OCTA)技术，提取刺激过程中血管区域的血流信号变化；近红外激光刺激采用1870nm波段，OCT成像采用1300nm波段。
  8. 用于实施权利要求1～7中任一所述方法的无标记全光学神经调控与成像装置，其特征在于包括：
    一套近红外激光刺激装置，用于激发目标脑部组织的神经活动；
    一套OCT光学相干探测装置，用于对二维或者三维空间内的光学散射信号进行OCT采集；
    一套同步控制单元，分别连接近红外激光刺激装置和OCT光学相干探测装置，用于同步实现激光刺激与OCT记录两个过程；
    一个或多个处理器，分别连接近红外激光刺激装置和OCT光学相干探测装置，用于分析处理探测得到OCT散射信号。
  9. 根据权利要求8所述的无标记全光学神经调控与成像装置，其特征在于：
    所述的近红外激光刺激装置，其工作波长和OCT***中心波长存在一定的差异，近红外激光刺激装置产生的近红外激光采用1870nm波段，OCT光学相干探测装置的成像探测采用1300nm波段。
  10. 根据权利要求8所述的无标记全光学神经调控与成像装置，其特征在于：所述的一个或多个处理器包括：计算在激光刺激前后，非血管区域的神经散射信号变化和血管区域的血流信号变化，得到脑功能信息。
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