Non-invasive subcranial fluorescence imaging monitoring method and device for freely moving small animals

Abstract

The present application provides a non-invasive subcranial fluorescence imaging monitoring method and device for freely moving small animals, wherein the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals comprises: infiltrating a liquid chelating agent and a transparent filling liquid of the skull into the non-invasive complete skull of the small animal; fixing a base on the skull of the small animal, carrying a miniature microscope on the base, and additionally setting a long-pass filter matching the fluorescence to be detected at the outlet end of the emission light module of the miniature microscope; combining an environmental camera and a miniature microscope, imaging the subcranial fluorescence signal of the freely moving small animal in real time, and monitoring the fluorescence changes of the subcranial fluorescence signal in real time. This method realizes the long-term stable, clear, and infection-free synchronous and real-time monitoring of the subcranial fluorescence signal of the small animal at the full cortical scale under free movement and the free behavior of the animal.

Description

Technical Field

[0001] The present invention relates to the field of medical imaging technology, and in particular to a non-invasive subcranial fluorescence imaging monitoring method and device for freely moving small animals. Background Art

[0002] At present, the main methods of recording large-scale neural activity include high-density electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) in a state where the movement of small animals is restricted. Among them, EEG is used to collect electrical signals transmitted to the scalp by group neuronal activity by fixing electrodes on the biological scalp. Such electrical signals are weak, easily interfered with, and have low spatial resolution; functional magnetic resonance imaging (fMRI) uses magnetic resonance imaging to measure changes in brain blood flow caused by neuronal activity to indirectly reflect neuronal activity. Its sampling rate and time resolution of the obtained data are very low. Other neuronal imaging techniques also include restricting the organism under a fixed microscope so that it cannot move freely, using large-scale craniotomy to replace the skull with transparent glass, grinding the skull thin or chemically transparent, and other surgical methods to image the entire surface of the cerebral cortex. The aforementioned method of recording neuronal activity requires restricting the free movement of the organism, and the surgical procedure is complicated and has an extremely high risk of intracranial infection. There is also the possibility of thickening of the skull or loss of chemical transparent reagents, which makes it impossible to guarantee long-term and stable monitoring of neurons. Summary of the invention

[0003] The embodiments of the present invention provide a method and device for non-invasive subcranial fluorescence imaging monitoring of freely moving small animals, so as to solve the problem that the method of recording neuronal activity requires restricting the free movement of the organism, the surgical procedure is complicated and has the risk of infection, and there is also the problem that long-term and stable monitoring of neurons cannot be guaranteed due to the thickening of the skull or the loss of chemical transparent reagents.

[0004] A non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals, comprising:

[0005] A non-invasive skull window is constructed on the skull of the small animal, and a liquid chelating agent and a skull transparent filling liquid are infiltrated into the non-invasive intact skull of the small animal;

[0006] A base is fixed on the skull of the small animal, and a miniature microscope is carried on the base. The miniature microscope includes an excitation light module for emitting blue light and an image sensor chip for imaging. A long-pass filter matching the fluorescence to be detected is additionally arranged at the outlet end of the emission light module of the miniature microscope;

[0007] Combined with an environmental camera and a miniature microscope, the fluorescence signal under the skull of freely moving small animals can be imaged in real time, forming a real-time and synchronous monitoring of animal behavior and weak fluorescence signals at the scale of the entire cortex;

[0008] The image sensor chip captures the changes in the fluorescence signal under the skull, converts it into an electrical signal, and then transmits the electrical signal to the monitoring end through a micro cable to monitor the fluorescence changes of the fluorescence signal under the skull in real time.

[0009] Furthermore, a non-invasive skull window is constructed on the skull of the small animal, including:

[0010] The miniature microscope is set at the midline of the skull of the small animal so as to form a viewing window under the complete skull of the small animal on the image sensor chip.

[0011] Furthermore, before the liquid chelating agent and the skull transparent filling liquid are infiltrated into the non-invasive intact skull of the small animal, the method further comprises:

[0012] The skull surface skin is partially exposed and cleaned to create an infiltration site for the infiltration of liquid chelating agents and skull transparent filling fluid.

[0013] Furthermore, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals also includes:

[0014] The environmental camera monitors the free behavior of small animals in real time in correspondence with the real-time fluorescence changes, forming a free-moving image;

[0015] Based on free-moving images and real-time fluorescence changes, the neurobehavioral signals of small animals are synchronously recorded and analyzed.

[0016] Furthermore, the image sensor chip captures the changes of the fluorescence signal under the skull, converts it into an electrical signal, and transmits the electrical signal to the monitoring end through a micro cable to monitor the fluorescence changes of the fluorescence signal under the skull in real time, including:

[0017] The image sensor chip converts the fluorescence signal under the skull into electrical signals and sends the electrical signals to the monitoring end through a micro cable, which is used to realize real-time online monitoring of the fluorescence signals of the cortex of small animals at the mesoscopic scale and the free behavior of animals based on electrical signals.

[0018] Furthermore, the liquid chelating agent is ethylene glycol ditetraacetic acid or tetraacetic acid with a concentration of 8% to 11%.

[0019] Furthermore, before a long-pass filter matching the fluorescence to be detected is additionally provided at the outlet end of the emission light module, the method further includes:

[0020] According to the fluorescence wavelength of the fluorescence signal under the skull, determine the long-pass filters with different central wavelengths.

[0021] Furthermore, the image sensor chip captures changes in fluorescence signals under the skull, including:

[0022] Continuously extract subcranial fluorescence signals, including weak fluorescence signals at the whole cortex scale and activity correlations between various cortical brain regions;

[0023] Based on the changes in the fluorescence signal under the skull, a time series curve of the intensity of the fluorescence signal in a single brain region and cortical mesoscopic scale is formed.

[0024] Furthermore, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals also includes:

[0025] At preset time intervals, the brightness of time-series fluorescence signals corresponding to multiple brain regions in the skull is collected through a micro-microscope;

[0026] Real-time monitoring of the brightness of time-series fluorescence signals in different brain regions and the free behavior of animals was performed to generate a cortical connection relationship map and draw a distribution map of fluorescence changes in different brain regions when the animals were in free behavior.

[0027] A non-invasive subcranial fluorescence imaging monitoring device for freely moving small animals, comprising: a miniature microscope, a monitoring end, and a miniature cable for connecting the miniature microscope and the monitoring end for transmitting subcranial fluorescence signals; wherein the outlet end of the excitation light of the miniature microscope is provided with a long-pass filter for separating the excitation light and the emission light, and the non-invasive subcranial fluorescence imaging real-time monitoring device for freely moving small animals realizes the aforementioned non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals.

[0028] The above-mentioned non-invasive subcranial fluorescence imaging monitoring method and device for free-moving small animals avoids the traditional method of monitoring fluorescence by opening the skull by infiltrating liquid chelating agents into the non-invasive intact skull. A miniature microscope with a long-pass filter is placed at the outlet of the emission light module on the top of the skull of the small animal. This can ensure the free movement of the small animal without the need for green light correction. During fluorescence imaging, impurity reflected light is filtered out to improve the signal-to-noise ratio, and the noise caused by the system light source or a large amount of reflected light is filtered out, which significantly improves the signal specificity. It avoids the excessive reflected light in the traditional craniotomy detection scheme, which causes a large number of brain areas including the visual cortex, and the fluorescence signal changes cannot be directly monitored in real time, and cannot be effectively extracted in offline analysis, resulting in the risk of cortical signal loss; it also avoids the infection risks and surgical challenges of skull removal at the full cortical scale. For a long time, the application of weak fluorescence signals under intact skulls has been limited to experimental schemes and settings for restricted or fixed animals. The operation of the present application is simple, and the instrument imaging does not need to damage the skull of freely moving small animals, thereby achieving long-term stable, clear, infection-free, synchronous and real-time monitoring of the subcranial fluorescence signal at the full cortical scale of the small animals in free movement and the free behavior of the animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0030] Figure 1 A schematic diagram of the monitoring environment of a non-invasive subcranial fluorescence imaging monitoring method for a freely moving small animal in one embodiment of the present invention is shown;

[0031] Figure 2 A first flow chart of the non-invasive subcranial fluorescence imaging monitoring method for free-moving small animals in the first embodiment of the present invention is shown;

[0032] Figure 3 A schematic diagram of the structure of a miniature microscope in a non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals in a first embodiment of the present invention is depicted;

[0033] Figure 4 A second flow chart of the non-invasive subcranial fluorescence imaging monitoring method for free-moving small animals in the second embodiment of the present invention is shown;

[0034] Figure 5 A diagram showing the whole-skin-scale fluorescence signal collection environment of a small animal in free movement in the non-invasive subcranial fluorescence imaging monitoring method for a small animal in free movement according to the first embodiment of the present invention;

[0035] Figure 6 A curve diagram showing the change in fluorescence signal intensity of the brain region related to the right nose of a mouse when the mouse is moving freely in the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals in the first embodiment of the present invention;

[0036] Figure 7 A comparison diagram of subcranial fluorescence signals captured by the real-time fluorescence imaging monitoring device provided by the present application in the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals in the first embodiment of the present invention is shown;

[0037] Figure 8 A graph showing the stable acquisition of fluorescence changes of intracranial fluorescence signals over a six-month period in the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals in the first embodiment of the present invention is shown.

[0038] Description of Figure Numbers:

[0039] 1. Freely moving small animals; 2. Miniature microscope; 3. Miniature cable with a diameter of <1mm and 50 ohms; 4. Monitoring terminal; 4. Conical mirror body; 5. Blue bandpass filter; 6. Green or red 525nm bandpass filter; 7. Image sensor chip; 8. Longpass filter matching the fluorescence to be detected; 9. Complete skull of small animals; 10. Plano-convex lens. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In the course of animal evolution, the more evolved the species, the stronger its ability to adapt to the surrounding environment. Higher animals can respond quickly to changes in the surrounding environment thanks to their developed nervous systems. The type of nerve cells in the nervous system that are responsible for signal transmission and processing are called neurons. The intelligence level of higher animals depends on the number of neurons in their bodies and the complexity of the interconnections between neurons. Lower animals such as hydras have only hundreds of neurons in their bodies, which are interconnected to form a simple neural network.

[0042] Consciousness is the thought content that animals perceive that causes their brains to think. Different species of animals have significantly different numbers of neurons, and the number of neurons in the same species of animals also varies to a certain extent. It can be said that almost every animal has a different number of neurons. Therefore, the number of consciousnesses of an individual animal is related to the number of neurons in its brain, which is why there are quantitative differences in the thought activities and corresponding behaviors of different animals.

[0043] The mouse brain has about 71 million neurons. With the development of brain science research, more and more studies have shown that a single behavior is often encoded by many neurons in multiple brain regions. Therefore, new tools and methods of large-scale optical imaging technology are urgently needed to analyze the relationship between the activity state of neurons at the cortical scale and behavior in small animals under free behavior.

[0044] The existing method of recording neuronal activity requires fixing small animals to restrict the free movement of the organism, and the surgical process is complicated and has the risk of infection. In addition, there is the risk of thickening of the skull or loss of chemical transparent reagents, which makes it impossible to ensure long-term and stable monitoring of neurons. Based on this situation, the present application proposes an embodiment. Figure 1 Under the experimental environment shown, a non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals is provided. Figure 2 As shown, the specific steps include:

[0045] S10. Construct a stable, durable and non-invasive skull window on the skull of a small animal, and infiltrate a liquid chelating agent and a skull transparent filling liquid into the non-invasive intact skull of the small animal to form a durable and stable skull window.

[0046] Specifically, liquid chelating agents are used to chelate calcium ions in the skull without opening the skull. Chelation refers to the effect that metal ions are linked to two or more coordinating atoms (non-metals) in the same molecule by coordination bonds to form a heterocyclic structure (chelate ring) containing metal ions. The formation of the chelate ring makes the chelate more stable and permeable than non-chelate complexes with similar composition and structure.

[0047] Calcium ions play an important role in many physiological activities. In addition to playing an important role in muscle cell contraction, calcium ions are also one of the important "barometers" of neuronal activity: when the neuronal membrane potential depolarizes and the generated action potential is transmitted to the axon terminal of the neuron, the voltage-gated calcium ion channel on the cell membrane opens, a large number of calcium ions flow in, and vesicles containing neurotransmitters are released from the presynaptic membrane to the postsynaptic membrane, so that downstream neurons can receive upstream signals. Therefore, calcium ion imaging can track neuronal action potentials, thereby helping to understand the activities of neuronal clusters, and is used in a variety of studies such as perception, learning and memory, and social behavior.

[0048] Furthermore, after the scalp is removed during surgery, a transparent skull filling liquid, such as 502 glue or dental cement, is used to fill the skull pores, prevent the evaporation of skull water, and improve the optical transparency of the skull after the scalp is removed. There are cavities on the skull, which usually contain physiological fluids. After the scalp is opened, they will evaporate and make the skull opaque. The use of a transparent skull filling liquid to fill the cavities can make the skull transparent again, and it can also prevent water evaporation and make the skull transparent again. For example, if 502 glue is added, it is necessary to wait until the skull is dry before adding it. After fixation, a thin mirror surface is formed, which can form a stable window for imaging. The liquid chelating agent and the transparent skull filling liquid make the skull transparent again, and at the same time, prevent the evaporation of skull water, and build a long-term stable window above the intact skull.

[0049] This embodiment does not pose a risk of intracranial infection to small animals, while increasing the transparency and stability of the skull and maintaining the transmittance of the skull for a long time through dental cement or 502, thereby maintaining the long-term monitorability of the skull.

[0050] S20. A base is fixed on the skull of the small animal, and a miniature microscope is carried on the base. The miniature microscope includes an excitation light module for emitting blue light and an image sensor chip for imaging. A long-pass filter matching the fluorescence to be detected is also arranged at the outlet end of the emission light module.

[0051] Specifically, a base is fixed on the skin of the skull of a small animal, and a miniature microscope is used to image the weak fluorescence signal under the skull of the small animal when it moves freely. The miniature microscope includes an excitation light module for sending blue light and an image sensor chip for imaging. A long-pass filter matching the fluorescence to be detected is also arranged at the outlet end of the emission light module to filter out the excessive impurity reflected light from the skull of the small animal, thereby improving the signal-to-noise ratio of the weak fluorescence signal under the skull and realizing real-time monitoring of the cortical mesoscopic level neural activity when the animal moves freely.

[0052] In this embodiment, a small and light base plate is fixed on the skull of the small animal, and the micro microscope is clamped on the base plate during monitoring. After the experiment, the micro microscope can be removed from the base plate to reduce the moving weight of the small animal.

[0053] Existing imaging equipment is not equipped with a long-pass filter, resulting in high overall nonspecific fluorescence brightness. The reflection of the excitation light in the imaging results is extremely strong. When the small animals behave freely, the real-time fluorescence signal in the brain area is difficult to directly observe.

[0054] This embodiment emits blue light through a micro microscope, such as Figure 3 As shown, the fluorescent protein or molecule in the superficial layer of the cortex of a small animal that is freely moving is excited through the window under the intact skull, and an additional long-pass filter, such as ET500lp or JB510, is added in front of the traditional long-pass filter, such as ET525 / 50nm, to filter out the excessively strong reflected excitation light in the field of view, thereby improving the signal-to-noise ratio of the fluorescence signal to be detected under the skull and improving the specificity of the weak fluorescence signal passing through the intact skull. The blue light or other wavelength excitation light corresponding to the fluorescence to be detected is filtered with a long-pass filter that matches the fluorescence to be detected, such as a green long-pass filter. This embodiment is also suitable for fluorescence detection of other colors, and only the excitation light and the filter need to be replaced.

[0055] S30. Combined with an environmental camera and a miniature microscope, the fluorescence signals under the skull of freely moving small animals are imaged in real time, forming real-time and synchronous monitoring of animal behavior and weak fluorescence signals at the whole cortex scale.

[0056] S40. The image sensor chip captures the changes in the fluorescence signal under the skull, converts it into an electrical signal, and transmits the electrical signal to the monitoring end through a micro cable to monitor the fluorescence changes of the fluorescence signal under the skull in real time.

[0057] Specifically, the whole-cortical window is visualized stably and long-term, combined with a small-mass, approximately 3-gram miniature microscope that is fixed above the skull during the experiment, to enable long-term and stable real-time imaging of centimeter-scale fluorescence signals throughout the cortex.

[0058] The excitation light and the emission light are separated by a filter to filter out the laser reflected light, and then the calcium fluorescence signal of the small animal in a moving state is imaged on the surface of the image sensor chip, such as the calcium signal, to form a fluorescence signal under the skull.

[0059] The non-invasive subcranial fluorescence imaging monitoring method for free-moving small animals provided in this embodiment avoids the traditional method of monitoring fluorescence by opening the skull by infiltrating a liquid chelating agent into the non-invasive intact skull. A miniature microscope with a long-pass filter is placed at the outlet of the emission light module on the top of the skull of the small animal. This ensures the free movement of the small animal without the need for green light correction. When performing fluorescence imaging, it filters out impurity reflected light, improves the signal-to-noise ratio, and filters the noise caused by the system light source or a large amount of reflected light. The signal specificity is significantly improved, and the excessive reflected light in the traditional craniotomy detection scheme is avoided. The fluorescence signal changes in a large number of cortical brain areas, such as the visual cortex, cannot be directly monitored in real time, and cannot be effectively extracted in offline analysis, resulting in the risk of cortical signal loss. Avoid the infection risks and surgical challenges of skull removal at the full cortical scale. For a long time, the application of weak fluorescence signals under the intact skull has been limited to experimental schemes and settings for restricted or fixed animals. The operation of the present application is simple, and the instrument imaging does not need to damage the skull of freely moving small animals, thereby achieving long-term stable, clear, infection-free, synchronous and real-time monitoring of the subcranial fluorescence signal at the full cortical scale of the small animals in free movement and the free behavior of the animals.

[0060] In a specific embodiment, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals further comprises the following steps:

[0061] The miniature microscope is set at the midline of the skull of the small animal so as to form a viewing window under the complete skull of the small animal on the image sensor chip.

[0062] Specifically, at the midline of the skull, that is, above the intact skull, while the animal is moving freely, a miniature microscope is used to monitor the animal's behavior and changes in clear fluorescent signals in each cortical brain region in real time, with an imaging resolution of 50-150 microns.

[0063] In a specific embodiment, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals further specifically includes the following steps before step S10, that is, before the liquid chelating agent and the skull transparent filling liquid are infiltrated into the non-invasive intact skull of the small animal:

[0064] Part of the skull surface skin is exposed and cleaned to leave a penetration site for the infiltration of liquid chelating agents and skull transparent filling liquid. A long-term stable transparent window is formed on the intact skull to image the real-time changes of weak fluorescence under the skull through the skull.

[0065] Specifically, the liquid chelating agent and the transparent skull filling liquid can be injected into the penetration site through a syringe or the like, and penetrate into the entire cortex through the skull surface skin, thereby achieving non-invasive enhancement of the fluorescence reaction.

[0066] In a specific embodiment, if Figure 4 As shown, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals further specifically includes the following steps:

[0067] S40. The free behavior of the small animals corresponding to the real-time fluorescence changes is monitored by the environmental camera to form a free-moving image.

[0068] S50. Synchronously record and analyze the neurobehavioral signals of small animals based on free-moving images and real-time fluorescence changes.

[0069] Specifically, the fluorescence signal is projected onto the imaging chip of the micro-microscope to form a fluorescence signal under the skull. When the animal is able to move freely and the skull is intact, the animal behavior can be monitored through the micro-microscope device and the common environmental camera during the experiment, realizing the free behavior of the small animal and the real-time online synchronous monitoring of the fluorescence signal under the skull. The fluorescence signal monitored by the device is highly specific, so there is no need for online correction (such as green light correction) or offline calculation, and the fluorescence signal under the intact skull can be visualized in real time.

[0070] In a specific embodiment, the non-invasive subcranial fluorescence imaging monitoring method for free-moving small animals, in step S40, that is, the image sensor chip captures the subcranial fluorescence signal and transmits the subcranial fluorescence signal to the monitoring end through a micro cable, specifically includes the following steps:

[0071] The image sensor chip converts the fluorescence signal under the skull into electrical signals and sends the electrical signals to the monitoring end through a micro cable, which is used to realize real-time online monitoring of the fluorescence signals of the cortex of small animals at the mesoscopic scale and the free behavior of animals based on electrical signals.

[0072] In a specific embodiment, in the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals, the liquid chelating agent is ethylene glycol ditetraacetic acid or tetraacetic acid with a concentration of 8% to 11%.

[0073] Specifically, after the skull skin is cut and cleaned, the liquid chelating agent forms a stable long-term fluorescence transmittance window on the intact skull. Combined with the fluorescence imaging device, the fluorescence signal under the skull is monitored in real time while the animal moves freely. The liquid chelating agent can be 8% to 11% ethylene glycol ditetraacetic acid (EGTA) or tetraacetic acid (BAPTA) to enhance the permeability of the skull.

[0074] In a specific embodiment, before step S20, that is, before a long-pass filter matching the fluorescence to be detected is additionally provided at the outlet end of the emission light module, the following steps are further specifically included:

[0075] According to the fluorescence wavelength of the fluorescence signal under the skull, determine the long-pass filters with different central wavelengths.

[0076] Specifically, the conventional green filter has a central wavelength of 500nm to 550nm and a bandwidth of 45nm to 60nm.

[0077] In this embodiment, according to the wavelength of the fluorescence to be detected, for example, green, a green long-pass filter is selected to pass wavelengths above 500nm or 510nm and cut wavelengths below 500nm or 510nm. For red, a red long-pass filter is selected to pass wavelengths above 540nm and cut wavelengths below 540nm.

[0078] The bioluminescence that passes through the skull is quite weak. The excitation light, for example, is 470nm. The two coexist in the test sample, and the wavelength interval between the excitation spectrum and the emission spectrum of the fluorescence is very small, such as around 15nm. Through the use of additional bandpass filters, the isolation between the excitation light and the emission light can be improved, and the specificity of trace fluorescence can be improved. When the skull is intact, the fluorescence signal of the skull cortex can be monitored in real time, which is used to synchronously evaluate the real-time monitoring of fluorescence changes at the scale of the entire cortex and the free behavior of animals.

[0079] In a specific embodiment, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals, in step S40, that is, the image sensor chip captures the change of the subcranial fluorescence signal, specifically includes the following steps:

[0080] S41. Continuously extract subcranial fluorescence signals, including weak fluorescence signals at the whole cortex scale and activity correlations between cortical brain regions.

[0081] S42. Based on the changes in the fluorescence signal under the skull, a curve diagram of the time series changes in the intensity of the fluorescence signal in a single brain region and cortical mesoscopic scale is formed.

[0082] Specifically, the superficial layer of the cortex is the subcutaneous layer at a depth of about 100 micrometers. The subcranial fluorescent signal includes calcium fluorescence and / or neurotransmitter fluorescent probes and the like.

[0083] like Figure 5 The whole-skin scale fluorescence signal collection environment diagram of the small animal in free movement is shown in the figure. For example, the R-NO in the right nose-related brain area of ​​the mouse in the figure is the target observation position. By continuously extracting the subcranial fluorescence signal reflected by calcium ions at the target observation position, the fluorescence signal at R-NO can be obtained. Figure 6 The time-series-based fluorescence signal intensity change curve shown is the change in fluorescence signal intensity in the brain area related to the right nose of the mouse when it moves freely.

[0084] In a specific embodiment, the non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals further specifically comprises the following steps:

[0085] S40. At preset time intervals, the brightness of the time-series fluorescence signals corresponding to multiple brain regions in the skull is collected through a micro microscope.

[0086] S50. Monitor the brightness of the time-series fluorescence signals in different brain regions and the free behavior of animals in real time, generate a cortical connection relationship map, and draw a distribution map of fluorescence changes in different brain regions when the animals are in free behavior.

[0087] This embodiment also provides a non-invasive subcranial fluorescence imaging monitoring device for free-moving small animals, comprising: a miniature microscope, a monitoring terminal, and a miniature cable for connecting the miniature microscope and the monitoring terminal for transmitting subcranial fluorescence signals. The outlet end of the excitation light of the miniature microscope is provided with a long-pass filter for separating the excitation light and the emission light, and the non-invasive subcranial fluorescence imaging monitoring device for free-moving small animals implements the non-invasive subcranial fluorescence imaging monitoring method for free-moving small animals as described above. Figure 7 As shown, the left side of the figure is a fluorescence signal image under the skull taken without the fluorescence imaging real-time monitoring device provided by the present application, and the right side is a fluorescence signal image under the skull taken with the fluorescence imaging real-time monitoring device provided by the present application (including a cutoff filter in the excitation light band to improve the signal-to-noise ratio of the system).

[0088] The non-invasive subcranial fluorescence imaging monitoring device for free-moving small animals provided in this embodiment avoids the traditional method of monitoring fluorescence by opening the skull by infiltrating a liquid chelating agent into the non-invasive intact skull. A miniature microscope with a long-pass filter is placed at the outlet of the emission light module on the top of the skull of the small animal. This ensures the free movement of the small animal without the need for green light correction. When performing fluorescence imaging, it filters out impurity reflected light, improves the signal-to-noise ratio, and filters the noise caused by the system light source or a large amount of reflected light. The signal specificity is significantly improved, and the excessive reflected light in the traditional craniotomy detection scheme is avoided. The fluorescence signal changes in a large number of cortical brain areas, such as the visual cortex, cannot be directly monitored in real time, and cannot be effectively extracted in offline analysis, resulting in the risk of cortical signal loss. Avoid the infection risks and surgical challenges of skull removal at the full cortical scale. For a long time, the application of weak fluorescence signals under the intact skull has been limited to experimental schemes and settings for restricted or fixed animals. The operation of the present application is simple, and the instrument imaging does not need to damage the skull of freely moving small animals, thereby achieving long-term stable, clear, infection-free, synchronous and real-time monitoring of the subcranial fluorescence signal at the full cortical scale of the small animals in free movement and the free behavior of the animals.

[0089] Specifically, Figure 8 The fluorescence changes of the intracranial fluorescence signal are shown in a stable acquisition graph for up to six months.

[0090] It should be understood that the order of execution of the steps in the above embodiment does not necessarily mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiment of the present invention.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention.

Claims

1. A non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals, characterized in that: include: A persistent, stable and non-invasive skull window is constructed on the skull of a small animal, and a portion of the skull surface skin is exposed and cleaned to leave an infiltration position for infiltrating a liquid chelating agent and a skull transparent filling liquid. The liquid chelating agent and the skull transparent filling liquid are injected into the infiltration position through a syringe, infiltrating into the non-invasive complete skull of the small animal, and infiltrating into the entire cortex through the skull surface skin, thereby achieving a non-invasive enhanced fluorescence reaction and forming a persistent and stable window; A base is fixed on the skull of the small animal, and a miniature microscope is carried on the base. The miniature microscope includes an excitation light module for emitting blue light and an image sensor chip for imaging. A long-pass filter matching the fluorescence to be detected is additionally arranged at the outlet end of the emission light module of the miniature microscope; Combining the environmental camera and the miniature microscope, the fluorescence signal under the skull of the freely moving small animal is imaged in real time, so as to form a real-time and synchronous monitoring of the animal behavior and the weak fluorescence signal at the whole cortex scale; The image sensor chip captures the changes of the fluorescence signal under the skull, and after converting it into an electrical signal, transmits the electrical signal to the monitoring end through a micro cable to monitor the fluorescence changes of the fluorescence signal under the skull in real time; based on the fluorescence changes of the fluorescence signal under the skull, a time series change curve of the intensity of the fluorescence signal at the mesoscopic scale of a single brain region and cortex is formed; At preset time intervals, the micro-microscope is used to collect the brightness of time-series fluorescence signals corresponding to multiple brain regions in the skull; The brightness of the time-series fluorescence signals in different brain regions and the free behavior of the animals are monitored in real time, a cortical connection relationship map is generated, and a distribution map of fluorescence changes in different brain regions of the animals when they are in a free behavior state is drawn.

2. The non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals according to claim 1 is characterized in that: The miniature microscope is arranged at the midline position of the skull of the small animal, so as to form a viewing window under the complete skull of the small animal on the image sensor chip.

3. The non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals according to claim 1, characterized in that: Also includes: The free behavior of the small animal corresponding to the real-time fluorescence change is monitored by an environmental camera to form a free-moving image; Based on the free-moving images and the real-time fluorescence changes, the neural behavioral signals of the small animals are synchronously recorded and analyzed.

4. The non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals according to claim 1, characterized in that: The image sensor chip captures the change of the fluorescence signal under the skull, converts it into an electrical signal, and transmits the electrical signal to the monitoring end through a micro cable to monitor the fluorescence change of the fluorescence signal under the skull in real time, including: The image sensor chip converts the fluorescence signal under the skull into an electrical signal, and sends the electrical signal to the monitoring end through the micro cable, so as to realize real-time online monitoring of the fluorescence signal of the cortex of the small animal at a mesoscopic scale and the free behavior of the animal based on the electrical signal.

5. The non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals according to claim 1, characterized in that: The liquid chelating agent is ethylene glycol ditetraacetic acid with a concentration of 8% to 11%.

6. The non-invasive subcranial fluorescence imaging monitoring method for freely moving small animals according to claim 1, characterized in that: Before a long-pass filter matching the fluorescence to be detected is additionally provided at the outlet end of the emission light module, the method further includes: According to the fluorescence wavelength of the fluorescence signal under the skull, long-pass filters with different central wavelengths are determined.

7. A non-invasive subcranial fluorescence imaging monitoring device for freely moving small animals, characterized in that: include: A miniature microscope, a monitoring end, and a miniature cable connecting the miniature microscope and the monitoring end for transmitting fluorescence signals under the skull; wherein the outlet end of the excitation light of the miniature microscope is provided with a long-pass filter for separating the excitation light and the emission light, and the non-invasive fluorescence imaging monitoring device under the skull of a freely moving small animal implements the non-invasive fluorescence imaging monitoring method under the skull of a freely moving small animal as described in any one of claims 1 to 6.

Images