Multi-core fiber photometry recording system and method

Abstract

Disclosed are a multi-core fiber photometry recording system and method, belonging to the technical field of neural signal recording. The system introduces a multi-core fiber to record a calcium signal in existing fiber photometry recording systems. Compared with traditional single-core fiber recording, the multi-core fiber can divide the recorded brain region into dozens or even more sub-regions, and record calcium signals of respective corresponding regions by means of individual fiber cores in the fiber, thereby obtaining a higher spatial resolution while maintaining the advantages of a fiber photometry recording method, and supporting multi-brain-region recording. Compared with an optical fiber bundle, the multi-core fiber has less loss in a signal transmission process, and can obtain higher-quality data.

Description

A multi-core optical fiber photometry recording system and method TECHNICAL FIELD

[0001] The present application belongs to the technical field of neural signal recording, and relates to a multi-core optical fiber photometry recording system and method. BACKGROUND

[0002] In recent years, calcium indicator proteins (such as GCamp proteins) are usually expressed in neurons by means of genetic methods. When neurons are stimulated and generate action potentials, the concentration of calcium ions changes, which in turn causes the fluorescence intensity of the calcium indicator protein excited by light to change in real time with the change in the concentration of calcium ions. By collecting the fluorescence of the calcium indicator protein excited in real time, the activation and inhibition of neurons can be deduced. The change in the fluorescence of the calcium indicator protein caused by the change in the concentration of calcium ions in neurons is called the calcium response of neurons.

[0003] To obtain high-quality calcium signals, it is necessary to minimize brain damage to avoid the influence of bleeding and inflammatory reactions on signal quality, and to minimize the relative displacement between the brain and the lens to reduce the interference caused by movement. In the research field of neuroscience, it is often necessary to record the calcium response of deep brain regions of freely moving mice, and therefore implantable recording methods have been developed, and the optical path has been simplified and miniaturized to the maximum extent.

[0004] One method is to implant a miniature lens (Grin lens, usually 500-1000 um in diameter) into the brain of a mouse, and to fix a miniaturized fluorescence microscope or a miniaturized two-photon microscope on the surface of the skull of the mouse, so as to realize the recording of the calcium response of single cells in the deep brain region of the freely moving mouse. However, due to the damage caused by the implantation of the lens, the limited range of adjustment of the focusing of the lens, and the interference caused by the relative movement of the lens and the brain, when this method is used to study brain regions that are relatively deep and have less dense neurons, the number of successfully recorded neurons will be greatly reduced compared to the recording of the cerebral cortex and the hippocampus.

[0005] Another method is to further simplify the optical path, use a single single-core optical fiber to conduct excitation light and record calcium signals, that is, fiber photometry (as shown in FIG. 1). Compared with micro-microscope recording, fiber photometry has the following advantages: first, the implanted optical fiber is thinner (usually 200 μm in diameter) than the micro-lens, and causes less damage to the brain. Second, a ceramic head is used to fix the optical fiber implanted in the brain, and the ceramic head is only 1.25 mm in diameter and has negligible weight, which almost does not affect the behavior of the mouse. Third, due to the thinness of the optical fiber and the simplicity of the optical path, fiber photometry can simultaneously record up to a dozen brain regions to study the interaction between different brain regions, while micro-microscope recording can only support single or a small number (at most 4) of site recording. Fourth, due to the simplicity of the optical path, the cost of fiber photometry is greatly reduced. However, fiber photometry can only record the neurons expressing calcium indicator protein in a brain region as a whole, and cannot distinguish the calcium signals of individual neurons. TECHNICAL PROBLEM

[0006] The purpose of the present application is to overcome the above-mentioned shortcomings of the prior art, and to provide a multi-core fiber photometry system and method to solve the problem that the fiber photometry method in the prior art can only record the overall response of the neural nucleus, and has low spatial resolution. Although micro-microscope imaging can obtain calcium response information at the neuron scale, it has the disadvantages of causing greater damage to the brain, greater influence on the spontaneous behavior of the animal, being not conducive to simultaneous recording of multiple brain regions, and being high in cost. TECHNICAL SOLUTION

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A multi-core fiber photometry system comprises a light source, a digital micromirror device and a light splitting module are sequentially arranged on the output light path of the light source, an objective lens is arranged on the reflected light path of the light splitting module, a multi-core optical fiber is coupled to the light outlet of the objective lens, and the other end of the multi-core optical fiber is used to collect excited calcium indicator protein fluorescence.

[0009] A plurality of cores are arranged in the multi-core optical fiber.

[0010] The light splitting module is simultaneously arranged on the return light path of the multi-core optical fiber, a photon detector is arranged on the return light path after the light reflection module, and the electrical signal output by the photon detector is recorded.

[0011] The present application is further improved in that:

[0012] Preferably, the light source is an LED lamp, and the wavelength of the laser emitted by the LED lamp is 470 nm and 410 nm.

[0013] Preferably, the light splitting module is a two-way light splitting mirror.

[0014] Preferably, a band-pass filter and a lens are arranged between the light splitting module and the photon detector.

[0015] Preferably, an electrical signal output end of the photon detector is connected with a data acquisition module.

[0016] Preferably, a phase-locked amplifier is arranged between the photon detector and the data acquisition module.

[0017] Preferably, an optical fiber connector is arranged at an end of the multi-core optical fiber for collecting calcium indicator fluorescence, and a ferrule needle is connected to the optical fiber connector and arranged in a detected object.

[0018] A multi-core optical fiber photometric recording method based on the above system, and the specific process is as follows: a light source emits laser of a specific wavelength, the laser is processed by a micro-mirror in a digital micro-mirror device, is reflected by a light splitting module, reaches a set fiber core in a multi-core optical fiber, the fiber core transmits the laser to a specific neuron group to excite calcium indicator fluorescence, the calcium indicator fluorescence passes through the light splitting module through the same fiber core and reaches a photon detector, and the photon detector converts the light signal into an electrical signal.

[0019] Preferably, the light source emits one laser at one time, and the laser is transmitted to one set fiber core.

[0020] Preferably, the electrical signal is converted into a point spread function, and the point spread function is processed by deconvolution. Advantages

[0021] Compared with the prior art, the present application has the following advantages:

[0022] The application discloses a multi-core optical fiber optometry recording system, which introduces a multi-core optical fiber recording calcium signal on the basis of an existing optical fiber optometry recording system. Compared with traditional single-core optical fiber recording, the multi-core optical fiber can divide the recorded brain region into dozens of sub-regions for recording respectively, has higher spatial resolution on the basis of maintaining the advantages of the optical fiber optometry recording method, and supports multi-brain region recording. Compared with an optical fiber bundle, the multi-core optical fiber has smaller loss in the signal transmission process, and can theoretically obtain higher quality data. The system retains the optical fiber optometry recording method on the basis of the existing optical fiber optometry recording method, has the advantages of small damage to the brain, no influence on the spontaneous behavior of animals, support for synchronous recording of multiple brain regions, simple operation, high success rate and the like, and replaces the traditional single-core optical fiber with the multi-core optical fiber. In the free-moving animals, the recorded brain region is divided into dozens of sub-regions for recording respectively, calcium response signal recording is performed on several neuron sub-populations in a brain region nucleus, the spatial resolution is improved, and the calcium response signal recording of the neuron sub-populations in multiple brain regions can be performed simultaneously (with a very short time interval) by using the multi-core optical fiber.

[0023] The application further discloses a multi-core optical fiber optometry recording method, which improves the spatial resolution of the optical fiber optometry recording under the premise of considering the cost, not increasing the damage to the brain, experimental difficulty and retaining the recording of the living body which can move freely, and first proposes calcium recording of the neuron group scale between the brain region nucleus scale and the cell scale. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a principle diagram of the optical fiber optometry recording method of the prior art;

[0025] Fig. 2 is a structure schematic diagram of the multi-core optical fiber optometry recording system of the application;

[0026] Fig. 3 is a structure schematic diagram of the multi-core optical fiber;

[0027] Fig. 4 is a physical diagram of the multi-core optical fiber.

[0028] In the figure, 1 is a light source, 2 is a digital micromirror device, 3 is a light splitting module, 4 is an objective lens, 5 is a multi-core optical fiber, 6 is a core insertion needle, 7 is an optical fiber joint, 8 is a band-pass filter, 9 is a lens, 10 is a photon detector, 11 is a phase-locked amplification module, 12 is a data acquisition module, and 13 is a fiber core. Embodiment of the application

[0029] The application will be further described in detail below with reference to the drawings:

[0030] In the description of the application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the application; the terms "first", "second", "third" are only for the purpose of description, and cannot be understood as indicating or implying relative importance; in addition, unless otherwise expressly specified and limited, the terms "mounting", "connecting", "connecting" should be understood in a broad sense, for example, it can be fixed connection, but also can be detachable connection; can be directly connected, but also indirectly connected through an intermediate medium, can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the application can be understood according to the specific circumstances.

[0031] When neurons are stimulated, the concentration of calcium ions inside the cell changes. This change plays a crucial role in the function of neurons. In the resting state, the concentration of calcium ions inside most neurons is maintained at a low level, about 50-100 nM. However, when neurons are stimulated and generate action potentials, the concentration of calcium ions inside them increases rapidly. This is because the propagation of action potentials causes voltage-gated calcium channels on the cell membrane to open, allowing high concentrations of calcium ions outside the cell to enter the cell. This rapid change in calcium ion concentration is crucial for many functions of neurons, such as the release of neurotransmitters in synaptic vesicles, the regulation of gene expression, and the metabolic processes of cells. By recording calcium signals in neurons, researchers can understand the excitatory state of neurons, information transmission, and their response to various stimuli. In neuroscience research, calcium indicators are often used to record neuronal calcium responses. Calcium indicators are compounds or proteins that can specifically bind to calcium ions and emit fluorescence under the action of excitation light, and the fluorescence intensity can change in real time with the concentration of calcium ions in the cell. To test the change in fluorescence, a miniature lens can usually be installed in the mouse brain, but this method is difficult to achieve synchronous recording of multiple brain regions; as shown in Figure 1, excitation light (such as blue laser (473nm laser), which can also be replaced by LED) is transmitted by a single-core optical fiber to conduct excitation light and record calcium signals. After being reflected by a dichroic mirror 3, the excitation light is transmitted into the brain through the optical fiber, causing the calcium indicator protein (such as GCaMP5g shown in the figure) to emit green fluorescence. The real-time light intensity change of green fluorescence reflects the neural activity of the specific nucleus of the brain. The green fluorescence is transmitted through the optical fiber and passes through the dichroic mirror to the photon detector 10 (such as CCD, CMOS, etc.), and the bandpass filter 8 is used to filter out stray light to prevent signal interference. The fluorescence signal detected by the photon detector 10 is processed by the lock-in amplification module 11 and the data acquisition module 12 to form continuous changes in neural calcium activity data over time, but this method can only record neurons expressing calcium indicator proteins in one brain region.

[0032] To solve the above problems, referring to Figure 2, the first aspect of the present application provides a multi-core optical fiber photometric recording system, which comprises a light source 1, a digital micromirror device 2, a light splitting module 3, an objective lens 4, a multi-core optical fiber 5, a pin 6, a multi-core optical fiber joint 7, a bandpass filter 8, a lens 9, a photon detector 10, a lock-in amplification module 11 and a data acquisition module 12.

[0033] The light source output end of the light source 1 is communicated with the light source entrance of the digital micromirror device 2, the light source exit of the digital micromirror device 2 is provided with a light splitting module 3, the light splitting module 3 reflects the light output from the digital micromirror device 2 and changes the direction to the input into the objective lens 4, the light direction of the objective lens 4 and the light direction of the digital micromirror device 2 are perpendicular to each other, the light exit of the objective lens 4 and the optical fiber of the multi-core optical fiber 5 are coupled, the light emitted by the light source 1 enters the specific fiber core 13 in the multi-core optical fiber 5 after being focused by the objective lens 4, the other end of the multi-core optical fiber 5 is provided with the optical fiber joint 7, the optical fiber joint 7 is connected with the plug pin 6, and part of the plug pin 6 is arranged in the detected object. The light splitting module 3 is provided with a band-pass filter 8 away from the objective lens 4, a lens 9 is arranged behind the band-pass filter 8, the light exit of the lens 9 is connected with a photon detector 10, the signal exit of the photon detector 10 is connected with a lock-in amplifier module 11, the signal exit of the lock-in amplifier module 11 is connected with a data acquisition module 12, and the converted signal is displayed at the corresponding position by the data acquisition module 12.

[0034] Referring to Fig. 3, the multi-core optical fiber 5 of the present application is an optical fiber with multiple fiber cores 13 in a common cladding, common multi-core optical fiber structures include four-core, seven-core, etc., the actual number and distribution of the fiber cores can be designed according to actual needs, and the multi-core optical fiber of the present application does not produce optical coupling between the fiber cores, and the signals do not interfere with each other during propagation. Each fiber core 13 can excite different regions in the detected object, and the recorded brain regions are divided into dozens or even more sub-regions for recording respectively, and the use of multi-core optical fiber for photometric recording of neuronal calcium response can obtain higher spatial resolution.

[0035] The light source 1 is a laser light source, which can be replaced by other specific wavelength light sources meeting the requirements, such as LED, etc.

[0036] As a preferred scheme, the light source of the present application can emit LED lamps with wavelengths of 470 nm and 410 nm.

[0037] It should be noted that the laser emitted by the light source 1 in the present application can activate the region corresponding to one fiber core, but by adjusting the time interval of the laser emitted by the light source 1, different regions can be activated at a very short time interval, and the specific activation region and the time interval between region activations can be determined according to experimental needs, so that multiple regions can be excited almost at the same time.

[0038] Digital mirror device (DMD) is a kind of light switching element, which is a reflective spatial light modulator, and its basic structure is composed of thousands of aluminum square micro-mirrors, which are orderly integrated on a CMOS substrate. Each micro-mirror unit is a pixel unit, which includes a micro-mirror, a hinge, an addressing electrode and the like. Through software, the loaded image digital signal is converted into a voltage deflection signal, and is matched with the addressing electrode, so that each micro-mirror unit can be independently driven by the hinge rotating device below, to realize the modulation of light signals.

[0039] The digital micro-mirror device 2 is used for encoding and processing the excitation light, so that the excitation light emitted from the light source 1 can reach different cores 13 through different micro-mirrors in the digital micro-mirror device 2, and then activate different set regions. The digital micro-mirror device 2 is connected with a computer, so that the reflection of laser through which micro-mirror is controllable, and the intensity of the reflected excitation light is also controllable. Thus, the excitation light of each optical fiber core is individually tuned, and the intensity of the excitation light output by each optical fiber core is adjusted; the DMD is further used to realize the time-sharing light supply of each optical fiber core, to avoid the mutual interference of the fluorescent signals of adjacent regions, and to improve the signal quality.

[0040] The light splitting module 3 is a two-way beam splitter, which is a device capable of splitting an incident light beam into two different spectral bands according to wavelength differences. In the present application, the two-way beam splitter can split the light sources with wavelengths of 470nm and 410nm into different spectral bands and input them into corresponding optical fiber cores. The laser with a wavelength of 410nm serves as reference laser, and calibrates the stability of the light source in the fiber-optic recording system. By comparing the output of the standard laser with the output of the actual laser source, the fluctuation or drift of the light source can be detected and corrected, to ensure the accuracy and reliability of the measurement results.

[0041] In the present application, the dual-color fiber-optic recording system is taken as an example, and the application can also be used in a multi-color (adding violet light (410nm) as reference light and adding green excitation light, etc.) fiber-optic recording system.

[0042] The photon detector 10 mentioned in the present application can adopt a charge coupled device (CCD), a complementary metal oxide semiconductor (CMOS) or other light signal detection devices meeting the requirements.

[0043] Photon detector 10 is a photoelectric device for converting light energy into electrical signals, and the photon detector works by external photoelectric effect or internal photoelectric effect. In the external photoelectric effect, photons are absorbed by the photoelectric cathode, and the electrons with sufficient energy escape to become free photoelectrons, and then form a photocurrent signal. The internal photoelectric effect mainly involves semiconductor materials, and the photons directly excite the bound state electrons of the materials into conduction electrons, participate in conduction, and realize photoelectric conversion. These electrical signals are proportional to the number of received photons, thereby realizing the detection and measurement of optical radiation. Photodiodes, phototubes, photoresistors, photomultipliers, photovoltaic cells or four-quadrant detectors can be used. In the present application, the photon detector 10 can convert the optical signal transmitted back by the optical fiber into an electrical signal.

[0044] The phase-locked amplifier 11 is an amplifier for separating a specific carrier frequency signal from a large interference environment, and works on the basis of synchronous detection principle. In the present application, the phase-locked amplifier is used to obtain the target electrical signal from the electrical signal transmitted back by the photon detector.

[0045] The data acquisition module 12, DAQ, captures the analog or digital signal from the phase-locked amplifier 11, and at the same time, the captured analog signal is converted into a digital signal for processing and analysis by a computer. The converted digital signal is transmitted to a computer or other processing unit for further data processing, storage and display.

[0046] One end of the multi-core optical fiber 5 is provided with a fiber joint 7, and the fiber joint 7 is connected with the ferrule needle 6. The fiber joint 7 is a detachable joint, one end of which is fixedly connected with the multi-core optical fiber 5, and the other end is fixedly connected with the ferrule needle 6. The ferrule needle 6 is inserted into the object to be detected. The ferrule needle 6 and the fiber joint 7 are LC connectors, and the LC connector is a Lucent connector. On the one hand, the LC connector is combined with the multi-core optical fiber to form an LC ferrule needle implanted in the brain, and on the other hand, the LC connector is combined with the multi-core optical fiber jumper to ensure that the jumper is perfectly aligned with the multi-core optical fiber implanted in the brain. When recording multiple brain regions, a prefabricated high-precision multi-core optical fiber MPO connector will be used.

[0047] The second aspect of the present application discloses a multi-core optical fiber recording method. The multi-core optical fiber conducts excitation light and collects calcium indicator protein emitted fluorescent signals in real time. A photon detector (such as CMOS or CCD) is used to record the fluorescent signals in real time, and each fiber core 13 is an independent recording unit. The specific process includes the following steps:

[0048] The light source 1 emits laser of specific wavelength, the laser is processed by the micro-mirror set by the digital micro-mirror device 2, and then reaches the light splitting module 3. The laser passes through the objective lens 4 and reaches the corresponding fiber core 13. The opening and closing of the laser in each fiber core 13 can be adjusted at the millisecond level. The laser is transmitted to the specific neuron group in the nucleus through the fiber core 13 to excite the calcium indicator fluorescence. The calcium indicator fluorescence is transmitted back through the same fiber core 13. The calcium indicator fluorescence passes through the objective lens 4 and reaches the photon detector 10 through the light splitting module 3. The signal of the photon detector 10 is recorded through the phase-locked amplification module 11 and the data acquisition module 12.

[0049] As a preferred solution, after the laser is transmitted from the digital micro-mirror device 2, the excitation light of the adjacent optical fiber core and the collected fluorescence may interfere with each other. Therefore, time-sharing excitation and deconvolution means are used to distinguish the signals of the neuron groups corresponding to different optical fiber cores.

[0050] Specifically, the light source 1 can only transmit the laser signal through one fiber core 13 to reach the designated area to excite the fluorescence each time the laser is emitted. Therefore, the light source 1 excites different areas by different excitation times.

[0051] Ideally, the fluorescence signal feedback from a certain site is only received by the fiber core 13 directly above the emission signal, and the fluorescence signals below different fiber cores 13 do not interfere with each other. However, in actual situations, the fluorescence signal of a certain site is received by different optical fibers, but the fluorescence signal received by the fiber core 13 directly above is stronger, and the fluorescence signals received by other fiber cores 13 are weaker. Therefore, after the signal is recorded by the data acquisition module 12, deconvolution is used to remove noise to distinguish the calcium responses of the neuron groups corresponding to different optical fiber cores.

[0052] The specific process is that after the signal is recorded by the data acquisition module 12, the light intensity received by different optical fiber cores is recorded by a single point light source at different positions under the optical fiber. For a region, the corresponding point spread function (PSF) is obtained. After recording the calcium response of the neuron group in the experiment, the point spread function is processed by the deconvolution algorithm to remove noise, and the calcium response of the neuron group corresponding to different optical fiber cores is obtained. The relationship between the measured image and the collected image is shown in the following two formulas. Exemplarily, the denoiser can be a Wiener filter or a Richardson-Lucy algorithm, and the degree of denoising can be adjusted according to the actual situation.

[0053] [Measured image] = [PSF] * [ideal image]

[0054] [ideal image] = [measured image] * [PSF] -1

[0055] It should be noted that the single brain region recording is taken as an example in the embodiments of the application, and the application can also be used for multi-brain region recording.

[0056] The above merely provides the preferred embodiments of the application, and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A multi-core optical fiber optical logging system, characterized by, The application relates to a calcium indicator protein fluorescence acquisition device, which comprises a light source (1), a digital micromirror device (2) and a light splitting module (3) arranged in sequence on an output light path of the light source (1), an objective lens (4) arranged on a reflected light path of the light splitting module (3), a multi-core optical fiber (5) coupled with a light outlet of the objective lens (4), and the other end of the multi-core optical fiber (5) being used for collecting excited calcium indicator protein fluorescence. A plurality of fiber cores (13) are arranged in the multi-core optical fiber (5). The light splitting module (3) is arranged on a return light path of the multi-core optical fiber (5), a photon detector (10) is arranged on the return light path after the light splitting module (3), and an electrical signal output by the photon detector (10) is recorded.

2. A multi-core fiber optical logging system according to claim 1, characterized in that, The light source (1) is an LED lamp, and the laser emitted by the LED lamp has a wavelength of 470nm and 410nm.

3. A multi-core fiber-optic photometric recording system according to claim 1, characterized in that, The light splitting module (3) is a two-way light splitting mirror.

4. A multi-core fiber-optic photometric recording system according to claim 1, characterized in that, A band-pass filter (8) and a lens (9) are arranged between the light splitting module (3) and the photon detector (10).

5. A multi-core fiber-optic photometric recording system according to claim 1, characterized in that, A data acquisition module (12) is connected to an electrical signal output end of the photon detector (1).

6. A multi-core fiber-optic photometric recording system according to claim 5, characterized in that A phase-locked amplifier (11) is arranged between the photon detector (1) and the data acquisition module (12).

7. A multi-core fiber-optic photometric recording system according to claim 1, characterized in that, An optical fiber joint (7) is arranged at an end of the multi-core optical fiber (5) used for collecting calcium indicator protein fluorescence, an insertion core needle (6) is connected to the optical fiber joint (7), and the insertion core needle (6) is arranged in a detected object.

8. A method of recording the intensity of light based on the system of claim 1, characterized by, Specifically, the light source (1) emits laser with a specific wavelength, the laser is processed by micro-mirrors in the digital micromirror device (2), is reflected by the light splitting module (3), reaches the set fiber core (13) in the multi-core optical fiber (5), the fiber core (13) transmits the laser to a specific neuron group to excite calcium indicator protein fluorescence, the calcium indicator protein fluorescence passes through the light splitting module (3) through the same fiber core (13) and reaches the photon detector (10), and the photon detector (10) converts the optical signal into an electrical signal.

9. The multi-core optical fiber optical recording method according to claim 8, characterized by, The light source (1) emits one laser at one time, and the laser is transmitted to one set fiber core (13).

10. The multi-core optical fiber optical recording method according to claim 8, characterized by, The electrical signal is converted into a point spread function, and the point spread function is processed through deconvolution.

Images