A photostimulation recording system

Abstract

The present invention provides a light stimulation recording system, which includes a light source, a collimated light path, a light path screening device, a coupling lens, a single-mode optical fiber, a detector, a fiber optic probe and an acquisition card; the light source includes a light for emitting detection light, a control light and a light for suppressing fluorescence; the light path screening device includes a plurality of dichroic mirrors, and the plurality of dichroic mirrors are used to reflect or transmit the detection light, the control light and the light for suppressing fluorescence to the coupling lens; and the fiber optic probe transmits the fluorescence reflected or transmitted from the coupling lens to the detector. In the above technical scheme, by using a first light emitting light source, a second light emitting light source and an external light source to emit lasers with different functions, the state of the object under different neural signals can be detected, and different detection effects can be achieved, so that it can simultaneously perform activation and inhibition experiments of G‑CAMP6 and NPHR3.0 / JAWS.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescence detection in living organisms, and in particular to a light stimulation recording system. Background Art

[0002] The mammalian brain has an extremely complex structure and function, and the understanding and analysis of the functions of various brain nuclei requires certain observation and recording methods. Real-time observation and recording of neuronal activity in the relevant brain regions of the subject under a specific behavioral paradigm can provide important data references and theoretical support for our understanding of the functional mechanism of the nucleus and further understanding and treatment of related neurological diseases.

[0003] For example, the known patents CN208864289U, entitled “Dual-color fluorescence excited neural signal optical fiber recording system” and CN208822748U, entitled “A device for detecting fluorescence in living organisms” disclose technical solutions that use laser to stimulate the G-camp6 sodium ion indicator in the brain of the organism to produce fluorescence to observe the brain nerve activity of the organism. However, in the above two patents, only the G-camp6 mouse fluorescence experiment can be performed, and the functions are relatively simple. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a light stimulation recording system.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides a light stimulation recording system, which includes a light source, a collimated light path, a light path screening device, a coupling lens, a single-mode optical fiber, a detector, an optical fiber probe and an acquisition card; the detector is connected to the acquisition card; the optical fiber probe is connected to the coupling lens through the single-mode optical fiber;

[0007] The light source comprises a first light source for emitting detection light; a second light source for emitting control light; and an external light source for emitting light for suppressing fluorescence;

[0008] The optical path screening device includes a plurality of dichroic mirrors, and the plurality of dichroic mirrors are used to reflect or transmit the detection light, the control light and the light for suppressing fluorescence to the coupling lens; and reflect or transmit the feedback light transmitted by the optical fiber probe to the coupling lens to the detector.

[0009] In a specific implementation scheme, the collimating optical path includes: a first collimating objective lens, a second collimating objective lens, and a third collimating objective lens; wherein,

[0010] The first collimating objective lens is used to collimate the detection light emitted by the first light source;

[0011] The second collimating objective lens is used to collimate the reference light emitted by the second light source;

[0012] The third collimating lens is used to collimate the fluorescence-suppressing light emitted by the external light source. The collimating effect of the light emitted by the three light sources can be improved by setting three collimating lenses.

[0013] In a specific embodiment, the plurality of dichroic mirrors include: a first dichroic mirror, a second dichroic mirror, and a third dichroic mirror; wherein,

[0014] The first dichroic mirror is used to transmit the detection light and reflect the control light;

[0015] The second dichroic mirror is used to transmit the fluorescence-suppressing light and the fluorescence, and reflect the detection light and the control light transmitted or reflected by the first dichroic mirror;

[0016] The third dichroic mirror is used to transmit the light that suppresses fluorescence and reflect the fluorescence transmitted by the second dichroic mirror. The combination of light paths is achieved by setting a plurality of dichroic mirrors, thereby simplifying the structure of the entire system.

[0017] In a specific implementation scheme, the optical path screening device further includes: a first optical filter, a second optical filter and a third optical filter;

[0018] The first filter, the first collimating lens, and the first light source are arranged along a first axis;

[0019] The second filter, the second collimating lens, and the second light source are arranged along a second axis;

[0020] The third filter and the detector are arranged along a fourth axis;

[0021] The external light source and the third collimating lens are arranged along the third axis; wherein,

[0022] The first axis is parallel to the fourth axis; the second axis is parallel to the third axis, and the first axis is perpendicular to the third axis. The effect of stray light on the experiment can be reduced by setting the filter.

[0023] In a specific embodiment, the first filter has a transmission bandwidth of 40 nm, an od value of at least 4, and a wavelength range of 430 nm to 510 nm;

[0024] The second filter has a transmission bandwidth of 40nm, an od value of at least 4, and a wavelength range of 390nm-430nm;

[0025] The third filter has a transmission bandwidth of 40 nm, an OD value of at least 5, and a wavelength range of 505 nm-545 nm.

[0026] In a specific embodiment, the first dichroic mirror is arranged at 45° to the first axis and the second axis;

[0027] The second dichroic mirror is disposed at 45° with the third axis and the second axis;

[0028] The third dichroic mirror is disposed at 45 degrees to the third axis and the fourth axis.

[0029] In a specific embodiment, the method further comprises a focusing lens disposed between the third filter and the detector to improve the receiving effect of fluorescence.

[0030] In a specific embodiment, the detection light is blue light.

[0031] In a specific embodiment, the control light is violet light.

[0032] In a specific embodiment, the light that inhibits fluorescence is yellow light.

[0033] It can be seen from the above description that in the above technical scheme, by adopting the first light source, the second light source, and the external light source to emit lasers with different functions, the state of the object under different neural signals can be detected, and different detection effects can be achieved, so that it is possible to simultaneously carry out activation and inhibition experiments of G-CAMP6 and NPHR3.0 / JAWS. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a structural block diagram of a light stimulation recording system provided by an embodiment of the present invention;

[0035] Figure 2 Schematic diagram of the structure of the light stimulation recording system provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] To facilitate understanding of the light stimulation recording system provided by the embodiment of the present invention, G-CAMP6 is first explained. G-CAMP6 is a new type of calcium ion indicator that is sensitive to calcium ions in cells or organelles. Its fluorescence intensity is proportional to the calcium ion concentration and can be used to observe calcium ion-mediated signal transmission processes. Based on the advantages of G-CAMP6 such as high signal-to-noise ratio, good light resistance, and the ability to observe changes in intracellular calcium ions in vivo for a long time, it has been widely used in the field of life science research, such as physiology, cell biology, and clinical research. Through genetic technology, a vector virus containing the G-CAMP6 gene is injected into the target organ and expressed in the target organ, thereby producing G-CAMP6 in the target organ.

[0038] The light stimulation recording system provided in the embodiment of the present invention is used to use excitation light to generate fluorescence in G-CAMP6 in specific target cells of the object to be detected, and simultaneously detect the intensity of the fluorescence, thereby corresponding to the change in intracellular calcium ion concentration, so as to observe and record the neuronal activity of the brain area related to the object to be detected in real time, providing important data reference and theoretical support for understanding the functional mechanism of the nucleus and further understanding and treating related neurological diseases.

[0039] The light stimulation recording system provided in the embodiment of the present invention mainly includes a light source, a collimated optical path, an optical path screening device, a coupling lens, a single-mode optical fiber, a detector, an optical fiber probe and an acquisition card; wherein the light source is used to provide light to illuminate the object to be detected. In the embodiment of the present invention, a mouse is used as the object to be detected. The collimated optical path is used to collimate the light emitted by the light source to ensure the reliability of light propagation. The optical path screening device is used to combine reflection or transmission of the light emitted by the light source to simplify the entire device. The coupling lens is used to couple the above-mentioned light, and the optical fiber probe is connected to the coupling lens through a single-mode optical fiber, and the optical fiber probe is used to detect the neuronal activity of the mouse, and the detector feeds back the fluorescence of the mouse to the acquisition card. The acquisition card is used to record the neural responses corresponding to the specific behavioral actions of the mouse. To facilitate the understanding of the light stimulation recording system provided in the embodiment of the present invention, the following is combined with the attached Figure 1 A detailed description is given.

[0040] like Figure 1 As shown in Figure 1 It is a structural block diagram of the light stimulation recording system provided by the present invention. The light source of the light stimulation recording system provided by the embodiment of the present invention includes three light sources, specifically a first light source 1, a second light source 2 and an external light source LD, wherein the first light source 1 is an LED light source, which is used to emit detection light, and the detection light can be selected as blue light. The second light source 2 is also an LED light source, which is used to emit control light, and the control light can be selected as purple light. The external light source LD is an LD light source, which is used to emit light that inhibits fluorescence, and the light that inhibits fluorescence can be selected as yellow light.

[0041] Continue to refer Figure 1 , the collimating optical path provided by the embodiment of the present invention includes three collimating objective lenses, and the three collimating objective lenses correspond to the three light sources one by one. The three collimating objective lenses are respectively: a first collimating objective lens, a second collimating objective lens, and a third collimating objective lens. For the convenience of description, the first collimating objective lens is named as collimating objective lens F1, the second collimating objective lens is named as collimating objective lens F2, and the third collimating objective lens is named as collimating objective lens F3; wherein, the collimating objective lens F1 is used to collimate the detection light emitted by the first luminous light source 1; the collimating objective lens F2 is used to collimate the control light emitted by the second luminous light source 2; and the collimating objective lens F3 is used to collimate the light that suppresses fluorescence emitted by the external light source LD. When setting, the first luminous light source 1 and the collimating objective lens F1 are coaxially arranged along the first axis, the second luminous light source 2 and the collimating objective lens F2 are coaxially arranged along the second axis, and the external light source LD and the collimating objective lens F3 are coaxially arranged along the third axis. wherein, the first axis is perpendicular to the second axis and the third axis respectively.

[0042] Continue to refer Figure 1 The optical path screening device provided by the embodiment of the present invention includes multiple dichroic mirrors and multiple filters, wherein the filters can be selectively set, and the optical path screening device can also only include multiple dichroic mirrors.

[0043] Continue to refer Figure 1 , multiple filters include a first filter D1, a second filter D2 and a third filter D3; wherein the first filter D1 is arranged along the first axis with the collimating lens F1 and the first light source 1; the second filter D2 is arranged along the second axis with the collimating lens F2 and the second light source 2; the third filter D3 is arranged along the fourth axis with the detector C1; wherein the second axis is perpendicular to the fourth axis. When three filters are specifically arranged, the first filter D1 has a transmission bandwidth of 40nm, an od value of at least 4 (optimal 5), and a wavelength range of 430nm-510nm (optimal 450nm-490nm); the second filter D2 has a transmission bandwidth of 40nm, an od value of at least 4 (optimal 5), and a wavelength range of 390nm-430nm; the third filter D3 has a transmission bandwidth of 40nm, an od value of at least 5 (optimal 6), and a wavelength range of 505nm-545nm.

[0044] First of all, the characteristic of the dichroic reflector is that it reflects the excitation light incident at an angle of 45° and transmits the fluorescence incident at an angle of 45°. When in use, multiple dichroic mirrors are used to reflect or transmit the detection light, the control light and the light for suppressing fluorescence to the coupling lens 3; and transmit the fluorescence reflected or transmitted by the optical fiber probe 5 to the coupling lens 3 to the detector C1. Figure 1As shown in , the plurality of dichroic mirrors include a first dichroic mirror B1, a second dichroic mirror B2 and a third dichroic mirror B3. The first dichroic mirror B1 is used to transmit the detection light and reflect the control light, that is, the first dichroic mirror B1 can transmit the light of the first filter D1 and reflect the light of the second filter D2 at the same time; Figure 1 As shown in , when the first dichroic mirror B1 is set, it is set at 45° to the first axis and the second axis. The light transmitted by the first filter D1 can directly pass through the first dichroic mirror B1, and the light transmitted by the second filter D2 is reflected by the first dichroic mirror B1. The second dichroic mirror B2 transmits the light that suppresses fluorescence and the fluorescence, and reflects the detection light and the comparison light transmitted or reflected by the first dichroic mirror B1, that is, the second dichroic mirror B2 can reflect the detection light and the comparison light, and can transmit the light that suppresses fluorescence and the signal light returned by the optical fiber probe 5. When the second dichroic mirror B2 is set, the second dichroic mirror B2 is set at 45° to the third axis and the second axis. The third dichroic mirror B3 is used to transmit the light that suppresses fluorescence, and reflect the fluorescence transmitted by the second dichroic mirror B2, that is, the third dichroic mirror B3 can transmit the light of the collimating objective lens F3, and can reflect the fluorescence returned by the second dichroic mirror B2. When the third dichroic mirror B3 is set, the third dichroic mirror B3 is set at 45° to the third axis. As shown in Figure 1 As shown in FIG. 1 , the first dichroic mirror B1 , the second dichroic mirror B2 and the third dichroic mirror B3 are all arranged in parallel.

[0045] Continue to refer Figure 1 A focusing lens E1 is arranged between the third filter D3 and the detector C1. The fluorescence reflected by the third dichroic lens passes through the third filter D3, is focused by the focusing lens E1 and then transmitted to the detector C1. The detector C1 may be a photomultiplier tube, which receives the light signal and converts it into an electrical signal.

[0046] Continue to refer Figure 1 The optical coupling circuit includes a coupling lens 3, which can couple light and collimate light. The coupling lens 3 can be made of transparent glass or plastic. The coupling lens 3 is connected to the optical fiber probe 5 through a single-mode optical fiber 4.

[0047] like Figure 1In the optical path shown in the figure, the first light source 1 emits a blue light with a wavelength range of 450nm-490nm, which is collimated by the collimating lens F1, passes through the first filter D1 and the first dichroic mirror B1, and is reflected by the second dichroic mirror B2 to the coupling lens 3; the second light source 2 emits a purple light with a wavelength range of 390nm-430nm, which is collimated by the collimating lens F2, passes through the second filter D2, is reflected by the first dichroic mirror B1 to the second dichroic mirror B2, and is reflected by the second dichroic mirror B2 to the coupling lens 3; the external light source LD emits a yellow light with a wavelength range of 587nm-591nm, which passes through the collimating lens F3, passes through the third dichroic mirror B3, the second dichroic mirror B2 to the coupling lens 3.

[0048] The fluorescent proteins of the neurons under test are marked by calcium-sensitive fluorescent probes and common fluorescent probes respectively. The two different light beams are coupled to the single-mode optical fiber 4 through the coupling lens 3 and then enter the optical fiber probe 5. The optical fiber probe 5 irradiates the mouse brain. After the mouse brain is irradiated, the signal light emitting G-camp fluorescence is activated. The signal light is received by the optical fiber probe 5 and enters the single-mode optical fiber 4. After the single-mode optical fiber 4 is collimated by the coupling lens 3, it enters the second dichroic mirror B2 and reaches the third dichroic mirror B3, is reflected to the third filter D3, and then enters the detector C1 after being focused by the focusing lens E1. The optical signal is converted into an electrical signal and sent to the acquisition card 6. The acquisition card 6 is connected to the computer to record and observe the information. At this time, if the external light source LD is turned on, the yellow light emitted by the external light source LD inhibits the fluorescence expression of the mouse, and the fluorescence emitted to the detector C1 will decrease rapidly. The external performance of the mouse is: from suddenly becoming lively to suddenly curling up in place, and when the external light source LD is turned off, the mouse returns to normal.

[0049] It can be seen from the above description that the light stimulation recording system provided in the embodiment of the present invention can detect the state of different neural signals of the object under test and achieve different detection effects by adopting the first light source 1, the second light source 2, and the external light source LD to emit lasers with different functions, thereby realizing activation and inhibition experiments of G-CAMP6 and NPHR3.0 / JAWS at the same time.

[0050] It should be understood that Figure 1 The placement positions of the first light source 1, the second light source 2 and the external light source LD shown in the figure are not limited to Figure 1 In the manner shown, the positions of the first light source 1, the second light source 2 and the external light source LD may also be swapped. In the embodiment of the present invention, the arrangement positions of the three light sources are not specifically limited.

[0051] For example, Figure 2 As shown in Figure 2FIG. 2 shows a schematic diagram of the structure of the optical stimulation recording system provided by an embodiment of the present invention. The optical stimulation recording system includes a housing 60, and a first optical path module 30, a second optical path module 20 and a third optical path module 10 arranged in the housing 60, wherein the first optical path module 30 includes Figure 1 The first light source 1, collimating lens F1, first filter D1, first dichroic mirror B1, second light source 1, collimating lens F2 and second filter D2 shown in the figure; the second optical path module 20 includes Figure 1 The second dichroic mirror B2 and the coupling lens 3 are shown in FIG. 1 ; the third optical path module 10 includes Figure 1 The collimating lens F3, the third dichroic mirror B3, the third filter D3 and the focusing lens E1 are shown in FIG. 3 ; the optical signal transmission relationship between the first optical path module 30, the second optical path module 20 and the third optical path module 10 can be referred to Figure 1 As shown in FIG. 6 . An external light source LD is also provided in the housing 60, and the external light source LD is connected to the third optical path module 10 via an optical cable. In addition, a fourth module 40 is also included, and the fourth module 40 includes Figure 1 The third filter D3, focusing lens E1, detector C1, and acquisition card 6 are shown. In addition, a power supply 50 and some control components are provided in the housing and used to power the above-mentioned light sources. It should be understood that Figure 2 The structure of a specific light stimulation recording system is merely exemplified. The light stimulation recording system provided in the embodiment of the present invention may also adopt other structural methods to realize its functions, and the specific implementation form is not limited.

[0052] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A light stimulation recording system, characterized in that: It includes a light source, a collimated optical path, an optical path screening device, a coupling lens, a single-mode optical fiber, a detector, an optical fiber probe and an acquisition card; the detector is connected to the acquisition card; the optical fiber probe is connected to the coupling lens through the single-mode optical fiber; The light source comprises a first light source for emitting detection light; a second light source for emitting control light; and an external light source for emitting light for inhibiting fluorescence, wherein the light for inhibiting fluorescence is yellow light with a wavelength range of 587nm-591nm; The collimating optical path includes a first collimating objective lens, a second collimating objective lens, and a third collimating objective lens; The optical path screening device includes a plurality of dichroic mirrors, and the plurality of dichroic mirrors are used to reflect or transmit the detection light, the control light and the light for suppressing fluorescence to the coupling lens; and reflect or transmit the feedback light transmitted by the optical fiber probe to the coupling lens to the detector; The optical path screening device further includes a first filter, a second filter and a third filter; the first filter, the first collimating lens and the first luminous light source are arranged along a first axis; the second filter, the second collimating lens and the second luminous light source are arranged along a second axis; the third filter and the detector are arranged along a fourth axis; the external light source and the third collimating lens are arranged along a third axis; wherein the first axis is parallel to the fourth axis; the second axis is parallel to the third axis, and the first axis is perpendicular to the third axis; The transmission bandwidth of the first filter is 40nm, the od value is at least 4, and the wavelength range is 430nm-510nm; the transmission bandwidth of the second filter is 40nm, the od value is at least 4, and the wavelength range is 390nm-430nm; the transmission bandwidth of the third filter is 40nm, the od value is at least 5, and the wavelength range is 505nm-545nm.

2. The optical stimulation recording system according to claim 1, characterized in that: in, The first collimating objective lens is used to collimate the detection light emitted by the first light source; The second collimating objective lens is used to collimate the reference light emitted by the second light source; The third collimating objective lens is used to collimate the fluorescence-suppressing light emitted by the external light source.

3. The optical stimulation recording system according to claim 1, characterized in that: The plurality of dichroic mirrors include: a first dichroic mirror, a second dichroic mirror, and a third dichroic mirror; wherein, The first dichroic mirror is used to transmit the detection light and reflect the control light; The second dichroic mirror is used to transmit the fluorescence-suppressing light and the fluorescence, and reflect the detection light and the control light transmitted or reflected by the first dichroic mirror; The third dichroic mirror is used for transmitting the light for suppressing fluorescence and reflecting the fluorescence transmitted by the second dichroic mirror.

4. The optical stimulation recording system according to claim 3, characterized in that: The first dichroic mirror is disposed at 45° to the first axis and the second axis; The second dichroic mirror is disposed at 45° to the third axis and the second axis; The third dichroic mirror is disposed at 45 degrees to the third axis and the fourth axis.

5. The optical stimulation recording system according to claim 1, characterized in that: It also includes a focusing objective lens arranged between the third filter and the detector.

6. The optical stimulation recording system according to any one of claims 1 to 5, characterized in that: The detection light is blue light.

7. The optical stimulation recording system according to claim 6, characterized in that: The contrast light is violet light.

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