An all-optical closed-loop multi-color fluorescence modulation system for conscious animals

Abstract

The application provides a full-optical closed-loop multi-color fluorescence regulation system for a conscious animal, comprising: a light source module providing a plurality of fluorescence excitation lights and optogenetic excitation lights; an excitation light beam combining module realizing beam combining of the plurality of fluorescence excitation lights and the optogenetic excitation lights in space; a fiber coupling module transmitting the combined excitation lights to a brain region of the conscious animal; a fluorescence signal filtering module for realizing beam splitting and frequency domain filtering of fluorescence signals reversely transmitted by the fiber coupling module in space; a signal acquisition module composed of a plurality of detectors, which respectively converts the fluorescence signals subjected to the beam splitting and the frequency domain filtering into electric signals and records the electric signals by an acquisition card; and a signal feedback modulation module dynamically adjusting parameters of the optogenetic excitation lights according to the acquired fluorescence signals. The application can realize real-time quantitative recording of physiological state changes of tissues while performing optogenetic regulation of the conscious animal, can self-adaptively regulate activities of the tissues, and realizes more precise and efficient individualized regulation effect.

Description

Technical Field

[0001] This invention relates to the field of closed-loop light regulation, and in particular to a fully optical closed-loop multicolor fluorescence regulation system for conscious animals. Background Technology

[0002] The mammalian brain is a highly complex biological system containing a vast number of nerve cells. Neurons are interconnected by intricate nerve fiber connections, forming neural networks and neural circuits that govern various brain functions. Understanding the relationship between brain activity and experimental animal behavior primarily involves real-time dynamic observation and regulation of neuronal activity in specific brain regions of behavioral animals. Traditional methods for recording neural activity in freely moving animals include multichannel electrophysiological recording; however, this method cannot distinguish cell types and the acquired signals are susceptible to electromagnetic interference.

[0003] In recent years, with the development of chemistry and molecular biology, fluorescent probe technology has seen tremendous advancements. Various fluorescent probes, such as calcium ion fluorescent probes and voltage-sensitive fluorescent probes, can be used to monitor changes in neuronal activity in real time. Fiber optic recording systems, as a relatively new method for recording neural activity, utilize calcium ion / voltage-sensitive fluorescent probes to optically monitor the activity of neurons of specific cell types in freely moving small animals. While this method offers advantages such as cell specificity and resistance to electromagnetic interference, the fluorescence signals acquired are easily affected by photobleaching, movement, and the concentration of the fluorescent probe, making it impossible to obtain accurate physiological information about changes in neuronal activity. Furthermore, standalone fiber optic recording systems cannot simultaneously record and further modulate the nervous system, hindering a deeper understanding of the functional mechanisms of neural nuclei / circuits.

[0004] Application number 202021586705.3, patent titled "An Optical Fiber Recording System with Optogenetic Function," discloses an optical fiber recording system with optogenetic function, enabling simultaneous use of optogenetics and optical fiber recording. However, this invention lacks a closed-loop control unit, making it unable to adjust external interventions in real time based on the organism's own neural activity signals. Summary of the Invention

[0005] The main objective of this invention is to overcome the aforementioned deficiencies in the prior art and propose a fully optical closed-loop multicolor fluorescence modulation system for conscious animals. This system enables multicolor fiber optic recording, effectively eliminating the influence of photobleaching, animal movement, and fluorescent probe concentration on the relative quantification of biological information. Simultaneously, this system is organically combined with optogenetic optical modulation technology to achieve closed-loop control, adaptively regulating neurons in different individuals and under different states, achieving more precise and efficient personalized modulation, and providing an effective research tool for further understanding and treating related neurological diseases.

[0006] The present invention adopts the following technical solution:

[0007] A fully optical closed-loop multicolor fluorescence modulation system for conscious animals, the system comprising: a light source module, an excitation light beam combining module, an optical fiber coupling module, a fluorescence signal filtering module, a signal acquisition module, and a signal feedback modulation module; the light source module is connected to the excitation light beam combining module, the combined excitation light is connected to the optical fiber coupling module, the fluorescence signal generated by the excitation light is then received by the signal acquisition module through the fluorescence signal filtering module via the optical fiber coupling module, and the signal feedback modulation module adjusts the stimulation parameters of the excitation light in real time according to the acquired signal, together forming a closed-loop optical modulation system.

[0008] Specifically, the light source module consists of multiple LED light sources or laser light sources, which are used to excite the fluorescent probe to generate fluorescent signals that reflect biological tissue information and to perform photoregulation on cells expressing photosensitive proteins.

[0009] Specifically, the excitation light beam combining module consists of several fiber collimators, filters, and dichroic mirrors; the fiber collimators are used to collimate and transmit the light output from the light source, the filters are used to filter out light in bands other than the required wavelength, and the dichroic mirrors are used to spatially combine the light from each light source.

[0010] Specifically, the fiber coupling module consists of a multi-band dichroic mirror, a fiber collimator, two multimode step-index fibers, and a fiber slip ring. The two multimode step-index fibers are connected by a fiber slip ring to reduce the entanglement of the fibers caused by the movement of small animals, which affects signal acquisition. The excitation light is reflected by the multi-band dichroic mirror and coupled into the multimode step-index fiber by the fiber collimator. It is then transmitted through the fiber slip ring to the brain tissue of the small animal expressing fluorescent probes and photosensitive proteins. The fluorescence signal generated in the region of interest is then transmitted in reverse to the multimode step-index fiber.

[0011] Specifically, the fluorescence signal filtering module consists of several filters, a dichroic mirror, and an optical fiber collimator; the dichroic mirror is used to spatially split the collected fluorescence signal according to the wavelength range, the filters are used to filter out light other than the fluorescence signal wavelength, and the optical fiber collimator couples the fluorescence into the optical fiber.

[0012] Specifically, the signal acquisition module consists of an avalanche diode photodetector and a data acquisition card; the avalanche diode photodetector converts the fluorescence signal coupled into the optical fiber into an electrical signal, which is then recorded by the data acquisition card.

[0013] Specifically, the signal feedback adjustment module consists of a signal generator; the signal generator compares and analyzes the collected fluorescence signal with the user-defined trigger threshold, and generates a corresponding trigger signal based on the analysis result to adjust the parameters of the light source.

[0014] As can be seen from the above description of the present invention, compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) This invention proposes a fully optical closed-loop multicolor fluorescence modulation system for conscious animals. This system can achieve multicolor fiber optic recording, effectively eliminating the influence of photobleaching, animal movement, and fluorescent probe concentration on the relative quantification of biological information. At the same time, this system is organically combined with optogenetic optical modulation technology to achieve closed-loop control. It can adaptively regulate neurons in different individuals and in different states, achieving more precise and efficient personalized regulation, and providing an effective research tool for further understanding and treatment of related neurological diseases.

[0016] (2) By setting up multi-channel multicolor fluorescence excitation, the present invention can accurately quantify the physiological information of the neural tissue to be detected by fluorescence ratio detection method, eliminating the influence of photobleaching, motion and fluorescence probe concentration on fluorescence signal; by using signal feedback modulation module, optogenetics and fiber optic recording system can be organically combined to achieve more precise and efficient personalized closed-loop control. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the framework of the fully optical closed-loop multicolor fluorescence regulation system for conscious animals as described in an embodiment of the present invention.

[0018] Figure 2 This is a structural diagram of the fully optical closed-loop multicolor fluorescence regulation system for conscious animals as described in an embodiment of the present invention.

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative effort are within the scope of protection of the present application.

[0021] This invention proposes a fully optical closed-loop multicolor fluorescence modulation system for conscious animals. This system enables multicolor fiber optic recording, effectively eliminating the influence of photobleaching, animal movement, and fluorescent probe concentration on the relative quantification of biological information. Furthermore, this system is organically combined with optogenetic optical modulation technology to achieve closed-loop control, adaptively modulating neurons in different individuals and under different states, achieving more precise and efficient personalized modulation, and providing an effective research tool for further understanding and treating related neurological diseases.

[0022] like Figure 1 As shown, this embodiment discloses a fully optical closed-loop multicolor fluorescence modulation system for conscious animals. It is characterized by comprising a light source module 1, an excitation light beam combining module 2, an optical fiber coupling module 3, a fluorescence signal filtering module 4, a signal acquisition module 5, and a signal feedback modulation module 6. The light source module 1 is connected to the excitation light beam combining module 2. The combined excitation light is connected to the optical fiber coupling module 3. The fluorescence signal generated by the excitation light is then received by the signal acquisition module 5 via the fluorescence signal filtering module 4 and the optical fiber coupling module 4. The signal feedback modulation module 6 adjusts the stimulation parameters of the excitation light in real time according to the acquired signal. All modules together constitute a closed-loop optical modulation system.

[0023] like Figure 2 As shown, in this embodiment, the light source module consists of an LED light source 1 with a center wavelength of 430nm, a laser light source 2 with a center wavelength of 561nm, and an LED light source 3 with a center wavelength of 620nm. LED light source 1 and LED light source 3 together serve as the ATP fluorescent probes AT1.03 and Ca. 2+ The excitation source of the fluorescent probe REX-GECO1 enables the targeting of ATP and Ca in neuronal cells. 2+ Fluorescence ratio detection allows for precise determination of changes in relative content. Laser source 2 serves as the excitation source for the photosensitive protein eArchT3.0, enabling photomodulation of nerve cells.

[0024] In this embodiment, the excitation beam combining module consists of fiber collimators 4, 5, and 6, filters 7, 8, and 9 (430 / 24; 561 / 10x; 620 / 20m), and dichroic mirrors 10 and 11. The fiber collimators are used to collimate and transmit the light output from the light source. Filters 7, 8, and 9 have parameters of 430 / 24, 561 / 10, and 620 / 20, respectively, and are used to filter out light in wavelengths other than the desired wavelength. Dichroic mirrors 10 and 11 are 605nm and 490nm long-pass dichroic mirrors, respectively, used to spatially combine the light from the various light sources.

[0025] In this embodiment, the fiber coupling module consists of a multi-band dichroic mirror 12, a fiber collimator 13, multimode step-index fibers 14 and 16, and a fiber slip ring 15. The multimode step-index fibers 14 and 16 are connected by the fiber slip ring 15 to reduce the entanglement effect of small animal movement on the fiber optic signal acquisition. Excitation light, after being reflected by the multi-band dichroic mirror 12 with parameters ZT440 / 514 / 561 / 640rpc, is coupled by the fiber collimator 13 into the multimode step-index fiber 14. It is then transmitted through the fiber slip ring 15 and the multimode step-index fiber 16 to the brain tissue of the small animal expressing the fluorescent probe and photosensitive protein. The fluorescence signal generated in the region of interest is then transmitted back to the multimode step-index fiber.

[0026] In this embodiment, the fluorescence signal filtering module consists of filters 18, 21, 24, and 26; dichroic mirrors 17, 20, and 23; and fiber collimators 19, 22, 25, and 27. Long-pass dichroic mirrors 17, 20, and 23, with parameters of 505nm, 567nm, and 638nm respectively, are used to spatially split the acquired fluorescence signal according to the wavelength range. Filters 18, 21, 24, and 26, with parameters of 480 / 40, 535 / 30, 585 / 20, and 700 / 75 respectively, are used to filter out light outside the fluorescence signal wavelength. Fiber collimators 19, 22, and 27 are used to couple the fluorescence into the optical fiber.

[0027] In this embodiment, the signal acquisition module consists of avalanche diode photodetectors 28, 29, 30, and 31 and an acquisition card 32. The avalanche diode photodetectors 28, 29, 30, and 31 convert the fluorescence signal coupled into the optical fiber into an electrical signal, which is then recorded by the acquisition card 32.

[0028] In this embodiment, the signal feedback adjustment module consists of a signal generator 33. The signal generator 33 compares and analyzes the fluorescence signal acquired by the acquisition card 32 with the user-defined trigger threshold, and generates a corresponding trigger signal based on the analysis result to adjust the parameters of the light source.

[0029] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An all-optical closed-loop multi-color fluorescence modulation system for conscious animals, characterized in that, The system comprises a light source module, an excitation light beam combining module, a fiber coupling module, a fluorescence signal filtering module, a signal acquisition module and a signal feedback adjustment module; the light source module is connected with the excitation light beam combining module, the combined excitation light is connected with the fiber coupling module, the fluorescence signal generated by the excitation light is received by the signal acquisition module through the fluorescence signal filtering module, and the signal feedback adjustment module adjusts the stimulation parameters of the excitation light in real time according to the collected signal, thereby forming a closed-loop light regulation system; The light source module is composed of a plurality of LED light sources or laser light sources, which are used for exciting the fluorescence probe to generate the fluorescence signal of the reaction biological tissue information and for light regulation of the cells expressing the light-sensitive protein; the light source module comprises LED light sources with central wavelengths of 430 nm, 620 nm and a laser light source with a central wavelength of 561 nm; the LED light sources with central wavelengths of 430 nm and 620 nm are respectively used for excitation of ATP fluorescence probes and Ca²⁺ fluorescence probes; the laser light source with a central wavelength of 561 nm is used for exciting the light-sensitive protein eArchT3.0; and the signal feedback adjustment module adjusts the stimulation parameters of the excitation light in real time according to the collected signal; The excitation light beam combining module is composed of a plurality of fiber collimators, optical filters and dichroic mirrors; the fiber collimator is used for collimating and transmitting the light output by the light source, the optical filter is used for filtering out light with wavelengths other than the required wavelengths, and the dichroic mirror is used for spatially combining the light of each light source; the central wavelengths of the optical filters corresponding to the LED light sources with central wavelengths of 430 nm, 620 nm and the laser light source with a central wavelength of 561 nm are 430 / 24 nm, 561 / 10 nm and 620 / 20 nm respectively; the light of the LED light source with a central wavelength of 620 nm and the laser light source is combined by the 605 nm long-wave-pass dichroic mirror, and then combined with the light of the LED light source by the 490 nm long-wave-pass dichroic mirror; The fluorescence signal filtering module is composed of a plurality of optical filters, dichroic mirrors and fiber collimators; the dichroic mirror is used for spatially splitting the collected fluorescence signal according to the wavelength range, the optical filter is used for filtering out light with wavelengths other than the fluorescence signal, and the fiber collimator is used for coupling the fluorescence into the optical fiber; the dichroic mirror is used for spatially splitting the collected fluorescence signal according to the wavelength range, and the specific splitting is as follows: the dichroic mirror parameters are 505 nm, 567 nm and 638 nm, one beam of light after splitting by the 505 nm dichroic mirror is filtered by the 480 / 40 optical filter; one beam of light after splitting by the 567 nm dichroic mirror is filtered by the 535 / 30 optical filter; and two beams of light after splitting by the 638 nm dichroic mirror are filtered by the 585 / 20 and 700 / 75 optical filters respectively.

2. The all-optical closed-loop multi-color fluorescence regulation system for conscious animals according to claim 1, wherein, The optical fiber coupling module is composed of a multi-band dichroic mirror, an optical fiber collimator, two multimode step-index optical fibers and an optical fiber slip ring; the two multimode step-index optical fibers are connected by the optical fiber slip ring, which is used to reduce the influence of the movement of small animals on the signal acquisition caused by the winding of the optical fiber; the excitation light is coupled into the multimode step-index optical fiber by the optical fiber collimator after being reflected by the multi-band dichroic mirror, and is transmitted to the small animal brain tissue expressing the fluorescent probe and the light-sensitive protein through the optical fiber slip ring, and the fluorescent signal generated by the region of interest is reversely transmitted to the multimode step-index optical fiber.

3. The all-optical closed-loop multi-color fluorescence modulation system for conscious animals according to claim 1, wherein, The signal acquisition module is composed of an avalanche photodiode photodetector and an acquisition card; the avalanche photodiode photodetector converts the fluorescent signal coupled into the optical fiber into an electric signal and records the electric signal by the acquisition card.

4. The all-optical closed-loop multi-color fluorescence regulation system for conscious animals according to claim 1, wherein, The signal feedback adjustment module is composed of a signal generator; the signal generator compares and analyzes the collected fluorescent signal with the trigger threshold defined by the user, and generates a corresponding trigger signal according to the analysis result to realize the parameter adjustment of the light-regulated light source.

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