CN219105267U

CN219105267U - Head-mounted microscope and microscopic imaging system

Info

Publication number: CN219105267U
Application number: CN202222570681.8U
Authority: CN
Inventors: 张超; 彭晓聿; 李军; 李阳
Original assignee: Hangzhou Bolun Brain Technology Co ltd
Current assignee: Hangzhou Bolun Brain Technology Co ltd
Priority date: 2022-09-26
Filing date: 2022-09-26
Publication date: 2023-05-30
Anticipated expiration: 2032-09-26

Abstract

The present application provides a head-mounted microscope and a microscopic imaging system, the head-mounted microscope including a first dichroic mirror, a second dichroic mirror, and an image sensor; in the optogenetic optical path, the light emitted by the first light source is transmitted by the first dichroic mirror and then reflected to the head of the experimental animal by the second dichroic mirror so as to apply optogenetic stimulation to the experimental animal; in the calcium imaging optical path, the light emitted by the second light source is reflected by the first dichroic mirror and then reflected by the second dichroic mirror to the head of the experimental animal so as to excite the calcium imaging virus of the experimental animal, and the image sensor is utilized to receive the reflected light of the experimental animal so as to collect the neuro-active image of the experimental animal. The head-mounted microscope can be worn on the head of an experimental animal (living animal), is applied to animal behavioural research, and has wide application range.

Description

Head-mounted microscope and microscopic imaging system

Technical Field

The present application relates to the technical field of microscopy, optogenetic and optoelectronic detection, and in particular to head-mounted microscopy and microscopic imaging systems.

Background

Microscopic imaging has important applications in biomedical research, novel material characterization, and other fields. Conventional optical microscopes are based on physical quantities of light absorption, phase gradient, and birefringence of the sample for imaging, and generally suffer from low contrast. Whereas fluorescence microscopy images the spatial-temporal distribution of individual molecular species by exploiting the fluorescent properties of the sample. Wherein, fluorescence is the light radiated in the process that electrons transition from a ground state to an excited state when molecules are excited by light, and return to the ground state after relaxation.

Because fluorescence microscopic imaging has the advantages of being capable of specifically marking, carrying out real-time dynamic imaging on living cells and the like, the fluorescence microscopic imaging becomes a necessary technical means for current biological science research. The fluorescence microscopic imaging can be used for researching the structure and dynamic change of specific cells, and can also be used for researching physiological dynamic process based on calcium ions or voltage sensitive fluorescent molecules. For different cells, fluorescent proteins with different spectral characteristics are respectively used for specific labeling, and dynamic interaction between the fluorescent proteins can be studied.

CN211718616U discloses a simple fluorescent microscope, which belongs to the technical field of microscopic imaging. The light source, the lens and the excitation end optical filter are sequentially arranged to form a first light path, the objective lens, the dichroic mirror, the collection end optical filter, the convex lens, the concave lens and the camera of the smart phone are sequentially arranged to form a second light path, the first light path and the second light path are mutually perpendicular, the first light path and the second light path are intersected at the dichroic mirror, and the biological sample to be observed is arranged below the objective lens. However, the microscope needs to be fixed on a laboratory bench, and can only observe an isolated biological sample, and has limited application range.

Accordingly, there is a need to provide a head-mounted microscope and a microscopic imaging system that address the problems of the prior art.

Disclosure of Invention

The utility model aims at providing a head-mounted microscope and microscopic imaging system, head-mounted microscope can be worn at experimental animal (live animal) head, moves along with experimental animal, is applied to animal behavioural research, and application scope is wide.

The purpose of the application is realized by adopting the following technical scheme:

in a first aspect, the present application provides a head-mounted microscope for wearing on the head of a laboratory animal, the brain region of the laboratory animal being injected with a photo-genetic virus and a calcium imaging virus, the head-mounted microscope for forming a photo-genetic optical path and a calcium imaging optical path;

the head-mounted microscope includes a first dichroic mirror, a second dichroic mirror, and an image sensor;

in the optogenetic optical path, the light emitted by the first light source is transmitted by the first dichroic mirror and then reflected to the head of the experimental animal by the second dichroic mirror so as to apply optogenetic stimulation to the experimental animal;

in the calcium imaging optical path, the light emitted by the second light source is reflected by the first dichroic mirror and then reflected by the second dichroic mirror to the head of the experimental animal so as to excite the calcium imaging virus of the experimental animal, and the image sensor is utilized to receive the reflected light of the experimental animal so as to collect the neuro-active image of the experimental animal.

The beneficial effect of this technical scheme lies in: the method can be used for injecting the photo-genetic virus and the calcium imaging virus into brain regions of experimental animals in advance, wherein the process of injecting the photo-genetic virus is a process of introducing exogenous light-sensitive protein genes into target cells, and the process of injecting the calcium imaging virus is a process of introducing calcium ion indicators into the target cells. As the optogenetic virus is injected into the brain region of the experimental animal, the light-sensitive channel protein is expressed on the cell membrane structure.

In the optogenetic optical path, light emitted by the first light source is transmitted through the first dichroic mirror and then reflected to the head of the experimental animal through the second dichroic mirror, along with the irradiation of the light, the activation and the closing of the photosensitive channel protein on the cell membrane structure can be controlled, and the activation and the closing of the photosensitive protein can control the opening and the closing of the ion channel on the cell membrane, so that the change of the cell membrane voltage, such as the depolarization and the hyperpolarization of the membrane, can be changed. When the membrane voltage depolarizes to a certain level, the neuron is induced to generate a conductive electric signal, namely the neuron is activated; in contrast, when the membrane voltage is hyperpolarized to a certain level, the generation of the action potential of the neuron, namely the inhibition of the neuron, is inhibited, and the activation and inhibition of the neuron is realized, namely the optogenetic stimulation.

In the calcium imaging optical path, light emitted by the second light source is reflected by the first dichroic mirror and then reflected by the second dichroic mirror to the head of the experimental animal so as to excite the calcium imaging virus of the experimental animal, and the reflected light of the experimental animal is received by the image sensor so as to acquire the neuroactive image of the experimental animal. The principle of exciting the calcium ion imaging virus is as follows: the change of the concentration of calcium ions in the neuron is represented by fluorescence intensity by using the calcium ion indicator, and the change of the activity of the neuron is reflected by rapid imaging of the change of the concentration of the calcium ions.

In one aspect, the first light source and the optogenetic optical pathway may be utilized to apply optogenetic stimulation to the experimental animal to achieve temporal modulation of neuronal activity by selecting different parameters, such as wavelength, light intensity, frequency, and duty cycle, and spatial modulation of neuronal activity by selectively illuminating a cell locally; on the other hand, neuro-active images of experimental animals can be acquired by using the second light source and the calcium imaging optical path, and the change of the optogenetic stimulus to neurons, nerve circuits or animal behaviors can be presented, so that the method is applied to animal behavioral studies (including feeding behaviors, rewarding behaviors, anxiety-depression behaviors, pain behaviors and the like).

In summary, the head-mounted microscope not only can be used for observing an isolated biological sample, but also can be worn on the head of an experimental animal (living animal) to move along with the experimental animal, and is applied to animal behavioural research, and the application range is wide.

In some alternative embodiments, the head-mounted microscope further comprises a first filter, a second filter, a first collimating lens, a second collimating lens, and an imaging lens group comprising a liquid lens and at least one achromatic lens;

in the optogenetic optical path, there are sequentially provided, along a propagation direction of light emitted from the first light source: the first filter, the first collimating lens, the first dichroic mirror, and the second dichroic mirror;

in the calcium imaging optical path, there are sequentially provided, along a propagation direction of light emitted from the second light source: the second filter, the second collimating lens, the first dichroic mirror, the second dichroic mirror; the reflected light propagation direction along the experimental animal is sequentially provided with: the imaging lens group and the image sensor.

The beneficial effect of this technical scheme lies in: on the one hand, the optogenetic optical path and the calcium imaging optical path can share part of components (such as a first dichroic mirror and a second dichroic mirror), so that the volume and the quality of the head-mounted microscope are reduced on the premise of ensuring the imaging effect, the free movement of small experimental animals is ensured, and the wearing burden on the small experimental animals is avoided; on the other hand, the optical filter can filter out unnecessary wave bands in the light source (the wave bands can influence experimental effect), and the collimating lens can converge and collimate the divergent light source, so that the light source is more uniformly and linearly converged. Compared with the way of re-collimation after the dichroic mirror, the performance is improved, the uniformity of the emergent light is better, and the power loss is smaller.

In some alternative embodiments, the at least one acromatic lens comprises: a first acromatic lens, a second acromatic lens, and a third acromatic lens;

in the calcium imaging optical path, there are sequentially provided, along a propagation direction of reflected light of the experimental animal: the first acromatic lens, the second acromatic lens, the liquid lens, the third acromatic lens, and the image sensor.

The beneficial effect of this technical scheme lies in: on the imaging (calcium imaging optical path) of the head-mounted microscope, the size of the field of view of the final imaging can be adjusted by adopting a combination of an achromatic lens and a liquid lens, wherein the achromatic lens can effectively inhibit the influence of chromatic aberration on the imaging quality, and is beneficial to improving the definition and resolution of microscopic images.

Existing head-mounted microscopes commonly use self-focusing lenses (Grin lenses), which have limited coupling efficiency to light, generally have large light energy loss, resulting in weak final light intensity, and the manner of achromatic lenses plus liquid lenses can achieve a larger numerical aperture than self-focusing lenses, thereby imaging a larger field of view.

In some alternative embodiments, the first light source emits light having a wavelength of 605nm and the second light source emits light having a wavelength of 470nm.

The beneficial effect of this technical scheme lies in: dichroic mirrors are characterized by being almost completely transparent to light of certain wavelengths and almost completely reflective to light of other wavelengths. The wavelength of the light emitted by the first light source may be 605 and n m, the wavelength of the light emitted by the second light source may be 470nm, the wavelengths of the light emitted by the first and second light sources being different such that the first dichroic mirror transmits the light emitted by the first light source and reflects the light emitted by the second light source; the second dichroic mirror reflects light emitted from the first light source, and reflects light emitted from the second light source.

In some alternative embodiments, the laboratory animal is any one of the following: mice, cats, dogs, pigs, cows, horses, monkeys, gorillas, and humans.

The beneficial effect of this technical scheme lies in: the head-wearing microscope can be applied to any experimental animal of mice, cats, dogs, pigs, cattle, horses, monkeys, gorilla and humans, and the excitation or inhibition state of a plurality of nerve cells in the experimental animal can be changed within a few milliseconds by utilizing the optogenetic technology, so that the behavior of the experimental animal moving freely is influenced, and the head-wearing microscope has important significance for researching normal brain functions and various brain diseases.

In a second aspect, the present application provides a microscopic imaging system comprising a data processing device, a camera, a first light source, a second light source, and any of the above head-mounted microscopes;

the camera is used for collecting behavioural images of experimental animals;

the data processing device is used for being electrically connected with the camera and the head-mounted microscope respectively.

In some alternative embodiments, the microimaging system further comprises a triple-core cable;

the data processing device further comprises a data acquisition board, and the data acquisition board is electrically connected with the head-mounted microscope by utilizing the three-core cable, so that the head-mounted microscope can realize the functions of electric energy transmission and data transmission.

In some alternative embodiments, the data acquisition board is a PCIe data acquisition card.

The beneficial effect of this technical scheme lies in: the current microscopic imaging system commonly uses multicore (more than three-core) cables to transmit electric energy respectively, leads to the cable to be thick and hard again, causes the restriction to the action of experimental animal, influences experimental effect, and the coaxial cable of this application adopts three-core cable, realizes the function of transmission electric energy and communication simultaneously for the cable quality is light and soft, does not influence experimental animal's activity.

In some alternative embodiments, the microimaging system further comprises an interaction device;

the interaction device is used for receiving user operation, generating a user interaction instruction and outputting the user interaction instruction;

the data processing device is used for receiving the user interaction instruction and controlling the light emitted by the first light source and/or the second light source.

The beneficial effect of this technical scheme lies in: the interaction component can be used for receiving user operation, and corresponding user interaction instructions are generated according to the needs of a user, so that corresponding parameters of light emitted by the first light source and/or the second light source, such as wavelength, light intensity, frequency and duty ratio, are controlled.

In some alternative embodiments, the interaction means comprises one or more of: keyboard, mouse, microphone assembly, touch screen, keys and knobs.

Drawings

The present application is further described below with reference to the drawings and examples.

Fig. 1 is a schematic optical structure of a head-mounted microscope according to an embodiment of the present application.

Fig. 2 is an electrical block diagram of a head mounted microscope provided in an embodiment of the present application.

Fig. 3 is a schematic structural diagram of a microscopic imaging system according to an embodiment of the present application.

Fig. 4 is an enlarged schematic view at S in fig. 3.

Fig. 5 is a block diagram of a microscopic imaging system according to an embodiment of the present application.

In the figure: 10. a first light source; 11. a first optical filter; 12. a first collimating lens; 13. a first dichroic mirror; 14. a liquid lens; 15. a first achromatic lens; 16. a second achromatic lens; 17. a third achromatic lens; 20. a second light source; 21. a second optical filter; 22. a second collimating lens; 23. a second dichroic mirror; 30. an image sensor; 100. a head-mounted microscope; 200. a data processing device; 300. a coaxial cable; 400. a first display; 500. and a second display.

Detailed Description

The present application will be further described with reference to the drawings and detailed description, which should be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.

In the embodiments of the present application, "at least one" means one or more, and "a plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a alone, a and B together, and B alone, wherein a, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, at least one (one) of a, b or c may represent: a, b, c, a and b, a and c, b and c, a and b and c, wherein a, b and c can be single or multiple. It is noted that "at least one" may also be interpreted as "one (a) or more (a)".

It is also noted that, in the embodiments of the present application, words such as "exemplary" or "such as" are used to mean serving as an example, instance, or illustration. Any implementation or design described as "exemplary" or "e.g." in the examples of this application should not be construed as preferred or advantageous over other implementations or designs. Rather, the use of words such as "exemplary" or "such as" is intended to present related concepts in a concrete fashion.

In the following, first, one of the application fields (i.e., optogenetic) of the embodiments of the present application will be briefly described.

Optogenetics (optogenetics) is a technology that combines optics and genetics (genetics) to precisely control the activity of specific types of neurons in the brain, spinal cord, peripheral nerves of living animals, even free-moving animals. Optogenetics can be achieved with time accuracy on the order of milliseconds and space accuracy on the order of individual cells. The technology of optogenetics is widely applied in the field of neuroscience at present, and can be applied to the treatment of various nerve and mental diseases, such as Parkinson's disease, alzheimer's disease, epilepsy, spinal cord injury, schizophrenia and the like in the future.

The optogenetics uses molecular biology, virus biology and other means to introduce exogenous photosensitive protein genes into living cells, and the photosensitive channel proteins are expressed on the cell membrane structure; then, the activation and the closing of the light-sensitive channel protein on the cell membrane structure are controlled by the irradiation of light with specific wavelength; activation and closure of the light-sensitive protein can control opening and closing of ion channels on the cell membrane, thereby altering changes in cell membrane voltage, such as depolarization and hyperpolarization of the membrane. When the membrane voltage depolarization exceeds a certain threshold, the neuron is induced to generate a conductive electric signal, namely the neuron is activated; conversely, when the membrane voltage hyperpolarizes to a certain level, the generation of the action potential of the neuron, that is, the inhibition of the neuron, is inhibited. Neuronal biologists often use this technique to study the function of the neural network by optically controlling the activity of specific neurons without or with low damage, and is particularly useful in vivo, even awake animal behavioural experiments.

Compared with traditional electrophysiological stimulation and pharmacological stimulation, the optogenetic technology has the characteristics of stronger specificity, better sensitivity, low toxicity, rapidness, accuracy, high reversibility and the like, and the high space-time resolution is almost the same as that of the somatic nerve cell activity process, and can reach the sub-millisecond and millisecond level.

Optogenetic technology has been widely used in many fields such as molecular biology, clinical medicine, neuroscience, etc. Animal behaviours are a discipline for studying the functions, mechanisms, developments and evolutions of various behaviors of animals, and play an important role in the development of neuroscience. The optogenetic technology greatly promotes the development of neuroscience, also greatly overcomes the defect that electrophysiology cannot identify specific neurons, and becomes a new technology widely applied to multiple fields and subjects, and is widely applied to animal behavioural research.

There are many techniques for studying brain neural activity, and techniques for imaging neurons in the brain of animals using a microscopic imaging system combining calcium imaging techniques and microscopy are now quite mature. However, the microimaging system can only passively record the brain activities of animals, and the animals are stimulated and intervened in the experimental process in some active modes, so that the optogenetic function is added on the basis of the microimaging system to perform optogenetic stimulation on the animals, and the soundness of the experiment can be well improved.

Referring to fig. 1, fig. 1 is a schematic optical structure of a head-mounted microscope according to an embodiment of the present application.

The head-mounted microscope is used for being worn on the head of an experimental animal, the brain area of the experimental animal is injected with the photo-genetic virus and the calcium imaging virus, and the head-mounted microscope is used for forming a photo-genetic optical path and a calcium imaging optical path;

the head-mounted microscope includes a first dichroic mirror 13, a second dichroic mirror 23, and an image sensor 30;

in the optogenetic optical path, the light emitted from the first light source 10 is transmitted through the first dichroic mirror 13 and then reflected to the head of the experimental animal through the second dichroic mirror 23, so as to apply optogenetic stimulation to the experimental animal;

in the calcium imaging optical path, the light emitted from the second light source 20 is reflected by the first dichroic mirror 13 and then reflected by the second dichroic mirror 23 to the head of the experimental animal, so as to excite the calcium imaging virus of the experimental animal, and the reflected light of the experimental animal is received by the image sensor 30, so as to collect the neuro-active image of the experimental animal.

Therefore, the method can inject the photo-genetic virus and the calcium imaging virus into the brain region of the experimental animal in advance, wherein the process of injecting the photo-genetic virus is the process of introducing the exogenous light-sensitive protein gene into the target cells, and the process of injecting the calcium imaging virus is the process of introducing the calcium ion indicator into the target cells. As the optogenetic virus is injected into the brain region of the experimental animal, the light-sensitive channel protein is expressed on the cell membrane structure.

In the optogenetic optical path, the light emitted by the first light source 10 is transmitted through the first dichroic mirror 13 and then reflected to the head of the experimental animal through the second dichroic mirror 23, and along with the irradiation of the light, the activation and the closing of the light-sensitive channel proteins on the cell membrane structure can be controlled, and the activation and the closing of the light-sensitive proteins can control the opening and the closing of the ion channels on the cell membrane, so as to change the voltage change of the cell membrane, such as the depolarization and the hyperpolarization of the membrane. When the membrane voltage depolarizes to a certain level, the neuron is induced to generate a conductive electric signal, namely the neuron is activated; in contrast, when the membrane voltage is hyperpolarized to a certain level, the generation of the action potential of the neuron, namely the inhibition of the neuron, is inhibited, and the activation and inhibition of the neuron is realized, namely the optogenetic stimulation.

In the calcium imaging optical path, the light emitted from the second light source 20 is reflected by the first dichroic mirror 13 and then reflected by the second dichroic mirror 23 to the head of the experimental animal to excite the calcium imaging virus of the experimental animal, and the reflected light (fluorescence signal) of the experimental animal is received by the image sensor 30 to collect the neuroactive image of the experimental animal. The principle of exciting the calcium ion imaging virus is as follows: the change of the concentration of calcium ions in the neuron is represented by fluorescence intensity by using the calcium ion indicator, and the change of the activity of the neuron is reflected by rapid imaging of the change of the concentration of the calcium ions.

In one aspect, the first light source 10 and the optogenetic optical pathway may be utilized to apply optogenetic stimulation to experimental animals to achieve temporal modulation of neuronal activity by selecting different parameters, such as wavelength, light intensity, frequency and duty cycle, and spatial modulation of neuronal activity by selectively illuminating a cell locally; on the other hand, neuro-active images of experimental animals may be acquired using the second light source 20 and the calcium imaging optical path, and the change of the optogenetic stimulus to neurons, neural circuits, or animal behaviors may be presented, thereby being applied to animal behavioral studies (including eating behaviors, rewarding behaviors, anxiety-depression behaviors, pain behaviors, etc.).

A head mounted microscope (head stage) is a device that can perform dynamic fluorescence imaging over a wide range on animals that are awake and free to move. Cell activity can be visualized using a head-mounted microscope, synchronized behaviours, and long-term tracking of neural loop activity.

Dichroic Mirrors (dichroics Mirrors), also known as Dichroic Mirrors, are commonly used in laser technology. When the light is incident at 45 degrees or at a large angle, a light source is separated to obtain a specific spectrum, and the specific spectrum is changed to the direction of a part of a spectrum light path, so that the light source is commonly used for sensor systems such as an enzyme-labeled instrument, a fluorescent microscope system, a projection light engine system, a laser lamp, an optical instrument beam splitting system, video glasses and the like.

The dichroic mirror has the characteristics of almost completely transmitting light with certain wavelength, almost completely reflecting light with other wavelengths, high transmittance, accurate wavelength positioning, small light energy loss and the like.

In the present embodiment, the first dichroic mirror 13 is configured to transmit light emitted from the first light source 10 and emit light emitted from the second light source 20; the second dichroic mirror 23 is for reflecting the light emitted from the first light source 10, and emitting the light emitted from the second light source 20. That is, the first light source 10 will undergo once the transmission of the first dichroic mirror 13 and once the reflection of the second dichroic mirror 23 (transmission+reflection) in the optogenetic optical path, and the second light source 20 will undergo once the reflection of the first dichroic mirror 13 and once the reflection of the second dichroic mirror 23 (twice reflection) in the calcium imaging optical path.

The principle of optogenetic stimulation is: a tool virus vector is used to transfer a light-sensitive gene (such as ChR2, eBR, npHR3.0, arch or OptoXR, etc.) into cells of a specific type of nervous system for expression of a specific ion channel or GPCR. The photoinduction channel can respectively generate selectivity to the passing of cations or anions under the illumination stimulation of different wavelengths, such as Cl-, na+, H+ and K+, thereby changing the membrane potential at two sides of the cell membrane and achieving the purpose of selectively exciting or inhibiting the cell.

The principle of calcium ion imaging is as follows: the concentration of calcium ions in the tissue is detected using a calcium ion indicator. Calcium ion imaging techniques are mainly used in neurological studies, where calcium ion changes suggest neuronal activity. Calcium ion imaging technology belongs to the field of optogenetic technology, and the actual detection is that the concentration of Ca2+ in cells or tissues changes, so that the calcium concentration change is converted into a fluorescent signal, and the cell electrical activity is converted into a recordable optical signal.

The embodiment of the application does not limit the calc imaging virus and the photo genetic virus, and the calc imaging virus can be, for example, chr2 or Chrismson; the optogenetic virus may be, for example, GCaMP6 or RCaMP1.

In one specific application, the calcium imaging virus uses chrisson and the optogenetic virus uses GCaMP6.

In some alternative embodiments, the collection and stimulation may be performed at a predetermined collection frequency and stimulation frequency, respectively, and the collection and stimulation may be performed simultaneously, or may be performed first and then the stimulation may be performed first and then the collection may be performed.

The first light source 10 and the second light source 20 are not limited in this embodiment, and any one of the first light source 10 and the second light source 20 may be a light source such as a laser, an LED lamp, a halogen lamp, a xenon lamp, or a mercury lamp. The angle between the direction of the incident light of the first light source 10 in the optogenetic optical path and the direction of the incident light of the second light source 20 in the calcium imaging optical path may be 90 degrees.

In some alternative embodiments, the wavelength of the light emitted by the first light source 10 is 605nm, the wavelength of the light emitted by the second light source 20 is 470nm, and the wavelength of the light reflected by the experimental animal is 525nm.

Thus, a dichroic mirror is characterized by being almost completely transparent to light of a certain wavelength and almost completely reflective to light of other wavelengths. The wavelength of light emitted from the first light source 10 may be 605nm, the wavelength of light emitted from the second light source 20 may be 470nm, the wavelengths of light emitted from the first light source 10 and the second light source 20 are different, such that the first dichroic mirror 13 transmits the light emitted from the first light source 10, and reflects the light emitted from the second light source 20; the second dichroic mirror 23 reflects the light emitted from the first light source 10, and reflects the light emitted from the second light source 20.

In some embodiments, the image sensor 30 may employ a CMOS image sensor 30 or a CCD image sensor 30.

The CCD sensor is a novel photoelectric conversion device capable of storing signal charges generated by light. When a pulse with a specific time sequence is applied to the CCD, the stored signal charges can be directionally transmitted in the CCD to realize self-scanning. It mainly comprises a photosensitive unit, an input structure, an output structure and the like. The photoelectric conversion device has the functions of photoelectric conversion, information storage, time delay and the like, and is high in integration level and low in power consumption. The CCD has an area array and a linear array, wherein the area array is a device for arranging CCD pixels into 1 plane; and a linear array is a device in which CCD pixels are aligned in 1 line.

The CMOS image sensor 30 is a typical solid-state imaging sensor, and is generally composed of an image sensor cell array, a row driver, a column driver, timing control logic, an AD converter, a data bus output interface, a control interface, and the like, which are generally integrated on the same silicon chip. The working process can be generally divided into resetting, photoelectric conversion, integration and reading. The CMOS image sensor 30 has several advantages:

1) Random window read capability. The random window read operation is one aspect of the CMOS image sensor 30 that is functionally superior to a CCD, also referred to as region of interest selection. In addition, the highly integrated nature of the CMOS image sensor 30 makes it easy to implement the function of simultaneously opening multiple tracking windows.

2) Radiation resistance. Overall, the potential radiation resistance of the CMOS image sensor 30 is significantly enhanced relative to the CCD performance.

3) System complexity and reliability. The system hardware structure can be greatly simplified by using the CMOS image sensor 30.

4) Non-destructive data readout mode.

5) And (5) optimized exposure control. It is noted that the CMOS image sensor 30 also has several drawbacks, mainly two indicators of noise and fill rate, due to the integration of multiple functional transistors in the pixel structure. In view of the relatively superior performance of the CMOS image sensor 30, the CMOS image sensor 30 is widely used in various fields.

In some alternative embodiments, the head-mounted microscope further comprises a first filter 11, a second filter 21, a first collimating lens 12, a second collimating lens 22, and an imaging lens group comprising a liquid lens 14 and at least one acromatic lens;

in the optogenetic optical path, there are sequentially provided, along a propagation direction of light emitted from the first light source 10: the first filter 11, the first collimating lens 12, the first dichroic mirror 13, and the second dichroic mirror 23;

In the calcium imaging optical path, there are sequentially provided, along the propagation direction of the light emitted from the second light source 20: the second filter 21, the second collimating lens 22, the first dichroic mirror 13, the second dichroic mirror 23; the reflected light propagation direction along the experimental animal is sequentially provided with: the imaging lens group and the image sensor 30.

Thus, on one hand, the optogenetic optical path and the calcium imaging optical path can share part of components (such as the first dichroic mirror 13 and the second dichroic mirror 23), so that the volume and the quality of the head-mounted microscope are reduced on the premise of ensuring the imaging effect, the free movement of the small experimental animal is ensured, and the wearing burden on the small experimental animal is avoided; on the other hand, the optical filter can filter out unnecessary wave bands in the light source (the wave bands can influence experimental effect), and the collimating lens can converge and collimate the divergent light source, so that the light source is more uniformly and linearly converged. Compared with the way of re-collimation after the dichroic mirror, the performance is improved, the uniformity of the emergent light is better, and the power loss is smaller.

In some alternative embodiments, the at least one acromatic lens comprises: first acromatic lens 15, second acromatic lens 16, and third acromatic lens 17;

In the calcium imaging optical path, there are sequentially provided, along a propagation direction of reflected light of the experimental animal: the first acromatic lens 15, the second acromatic lens 16, the liquid lens 14, the third acromatic lens 17, and the image sensor 30.

Thus, on the imaging (calcium imaging optical path) of the head-mounted microscope, the combination of the achromatic lens and the liquid lens 14 can be used to adjust the size of the field of view of the final imaging, wherein the achromatic lens can effectively suppress the influence of chromatic aberration on the imaging quality, which is beneficial to improving the definition and resolution of the microscopic image.

An acromatic lens is a lens group that corrects chromatic aberration of light of three wavelengths (blue, green, and red) with the objective of adjusting the focus of the longest wavelength and the shortest wavelength to one position.

In some embodiments, first acromatic lens 15 and second acromatic lens 16 may be the same model as 45089 and third acromatic lens 17 may be 63691.

That is, the imaging lens group employs a combination of 3 acromatic lenses and 1 liquid lens 14, and in the calcium imaging optical path, reflected light from the laboratory animal passes through 2 acromatic lenses (first acromatic lens 15 and second acromatic lens 16, model 45089), liquid lens 14, 1 acromatic lens (third acromatic lens 17, model 63691) in that order, and reaches image sensor 30.

Wherein the light converging direction of the first acromatic lens 15 and the second acromatic lens 16 is the same as the propagation direction of the reflected light of the experimental animal, and the light converging direction of the third acromatic lens 17 is opposite to the propagation direction of the reflected light of the experimental animal.

Referring to fig. 2, fig. 2 is an electrical block diagram of a head-mounted microscope provided in an embodiment of the present application.

Wherein, coax FPD Link is a video signal protocol.

SERDES is an acronym for SERializer/deseriaalizer. The method is a Time Division Multiplexing (TDM) and point-to-point (P2P) serial communication technology, namely, a plurality of low-speed parallel signals are converted into high-speed serial signals at a transmitting end, and the high-speed serial signals are converted into low-speed parallel signals at a receiving end again through a transmission medium (an optical cable or a copper wire). The point-to-point serial communication technology fully utilizes the channel capacity of a transmission medium, reduces the number of required transmission channels and device pins, and improves the transmission speed of signals, thereby greatly reducing the communication cost.

GPIO (General-purpose input/output), P0-P3 with a function similar to 8051, the PINs of the GPIO can be used by program control freely, and the PIN can be used as General input (GPI) or General output (GPO) or General input and output (GPIO) according to practical considerations.

I2C is commonly referred to as the I2C bus, a simple, bi-directional two-wire synchronous serial bus developed by Philips corporation. Only two wires are required to transfer information between devices connected to the bus.

Therefore, the head-mounted microscope can be applied to any experimental animal of mice, cats, dogs, pigs, cattle, horses, monkeys, gorillas and humans, the excitation or inhibition state of a plurality of nerve cells in the experimental animal can be changed within a few milliseconds by utilizing the optogenetic technology, the behavior of the experimental animal which moves freely is influenced, and the head-mounted microscope has important significance for researching normal brain functions and various brain diseases.

Referring to fig. 3, fig. 4 and fig. 5, fig. 3 is a schematic structural diagram of a microscopic imaging system provided in an embodiment of the present application, fig. 4 is an enlarged schematic diagram at S in fig. 3, and fig. 5 is a block structural diagram of a microscopic imaging system provided in an embodiment of the present application.

The microscopic imaging system comprises a data processing device 200, a camera (not shown), a first light source (not shown), a second light source (not shown), and any of the head-mounted microscopes 100;

the camera is used for collecting behavioural images of experimental animals;

the data processing device 200 is configured to be electrically connected to the camera and the head-mounted microscope 100, respectively.

In some alternative embodiments, the microimaging system further comprises a tri-core cable 300 or a coaxial cable;

the three-core cable adopts a single power supply technology, and the coaxial cable adopts a POC power supply technology. Coaxial cables are single-core cables, which are light and flexible, but can provide limited power, while three-core cables can provide higher power while the cables are light and flexible in weight.

The data processing device 200 further includes a data acquisition board, and the data acquisition board is electrically connected to the head-mounted microscope 100 by using the three-core cable 300, so that the head-mounted microscope 100 can realize functions of power transmission and data transmission.

The data processing apparatus 200 may be a host (for example, may be a medical host), where the host includes a board card, a graphics card, a PC motherboard, an industrial power supply, and a hard disk, the graphics card is externally connected to a first display 400 and a second display 500, the first display 400 is used for optogenetic setting, the second display 500 is used for data acquisition processing, and the PC motherboard is externally connected to a mouse (keyboard and mouse) for interaction. The host computer can adopt the form of shallow, and the host computer bottom is provided with 4 universal wheels, and the user of being convenient for removes. The control function of the host may be implemented by MPU, MCU, DSP, FPGA or any combination thereof.

The board card is electrically connected to the head mounted microscope 100 by a three-wire cable 300.

In some embodiments, the microscopic imaging system is externally connected with 1 or more behavioural cameras to collect behaviors of the experimental animal in real time, and after the behavioural image videos are obtained, an upper computer software system installed on the host computer can analyze the behaviors in real time. In the analysis process, various real-time conditions such as real-time position state, current action, movement path and the like of the experimental animal (mouse) are calculated. After the real-time conditions of the plurality of experimental animals are obtained, the real-time conditions are analyzed through AI, the current most reasonable optogenetic stimulation mode is judged, and the mode is acted on the experimental animals. Similarly, the neural activity video acquired by the head-mounted microscope 100 can be analyzed in real time and fed back to the optogenetic stimulus according to the result.

Since video is analyzed and processed in real time, high-speed performance for image acquisition is highly demanded, and a function of acquiring images at high speed is realized in the following ways.

1. A high-definition image sensor is adopted, so that the image quality is ensured; 2. a Mipi csi-2 image sensor high-speed interface is adopted; 3. the three-core cable 300 is adopted for high-speed image transmission between boards; 4. and adopting an FPGA and PCIe4 to process and transmit high-speed data.

In the animal behavioural synchronization process, communication interaction among a plurality of external system devices is supported by adding DIO (Digital in and out, digital input output port) input and output of the system, so that a user can acquire communication signals among different devices in time. And with a variable threshold DI (Digital in, digital input port), it is possible to interface with the intra-industry microscopic imaging system to the greatest extent possible.

The characteristics of the microscopic imaging system DI are as follows: 1. the threshold value and hysteresis are selectable, and the sensing detection range is 3V to 20V; 2. induction input lightning protection; 3. detecting an overcurrent fault; 4. the maximum power consumption is automatically limited.

By analyzing both the neuro-active image and the behavioural image, the current state of the experimental animal can be accurately judged to be unsuitable for the optogenetic stimulation (compared with the situation that the experimental animal only depends on the neuro-active image), and the change of the optogenetic stimulation to the neuron, the nerve circuit or the animal behavior can be rapidly reflected while the experimental animal is collected during the stimulation.

From this, current microscopic imaging system generally uses multicore (more than three-core) cable to transmit the electric energy respectively, leads to the cable thick again hard, causes the restriction to the action of experimental animal, influences experimental effect, and the coaxial cable 300 of this application adopts three-core cable, is providing high power simultaneously for the cable quality is light and soft, does not influence experimental animal's activity.

Compared with a single-core coaxial cable, the POC power supply power of the coaxial cable is lower, and the three-core cable is additionally provided with 2 power supply lines, so that the power supply power is higher. The communication protocol of the POC power supply technology is V3LINK or FPD_LINK.

POC (Power Over Coaxia) is a technology based on coaxial video, coaxial control and power superposition. In the coaxial cable transmission, a high-definition video signal, a coaxial signal and a power supply are transmitted, that is, the high-definition video signal, the coaxial signal and the like are combined with the power supply and transmitted on one coaxial line.

The fpd_ LINK (Flat Panel Display Link) is a high-speed digital video interface standard, has the advantages of high bandwidth and low delay, and is mainly used for transmitting video data and supporting the transmission of the coaxial cable 300; V3L INK is a serializer/deserializer with ultra low latency that can aggregate video, clock, control, and GPIO data into a single wire bi-directional bridge between industry universal interfaces, with the advantages of enhanced signal integrity, reduced system size, weight, and power consumption.

In some embodiments, POC power does not exceed 150mA, avoiding interference with the transmitted signal.

In some embodiments, the data acquisition board may employ a PCIe high speed acquisition card, the image sensor may employ a CMOS image sensor and employ a MIPI CSI-2 interface.

PCIe (peripheral component interconnect express) is a high-speed serial computer expansion bus standard.

The PCIe high-speed acquisition card is an ideal tool for signal acquisition and analysis in the fields of radar, communication, satellite, lidar, photomultiplier, optical fiber sensing and the like, and the on-board FPGA has real-time signal processing capability and supports user-defined logic development. The board card provides a fast PCIEx8 data transmission interface and flexibly configured hardware combination, so that the best balance among performance, power consumption and cost is obtained, and the board card is particularly suitable for laboratories and off-site computer occasions.

MIPI CSI (Camera Serial Interface) is an interface standard specified by the Camera working group under MIPI alliance. CSI-2 is the MIPI CSI second version, which is a single or bi-directional differential serial interface, containing clock and data signals. The system mainly comprises an application layer, a protocol layer and a physical layer, and maximally supports 4-channel (Lane) data transmission, and the single-wire transmission speed is up to 1Gb/s. With the MIPI CSI-2 standard, image data can be sequentially passed through a single channel, which would employ two or four channels to connect an imaging chip or camera module. Wherein the maximum available bandwidth is linearly proportional to the number of channels, i.e. the available bandwidth when four channels are used is 2 times that when two channels are used.

the data processing device 200 is configured to receive the user interaction instruction and control light emitted by the first light source and/or the second light source.

Thus, the interaction component can receive user operation, and generate corresponding user interaction instructions according to the needs of a user, so as to control corresponding parameters of light emitted by the first light source and/or the second light source, such as wavelength, light intensity, frequency and duty ratio.

First, the wavelength depends on the type of light-sensitive protein, for example, chR2 may select an LED light source or laser light source of 473nm wavelength, npHR selects an LED light source or laser light source of 593nm wavelength. The choice of light intensity for an excitatory light-sensitive channel protein depends largely on its own characteristics, different light-sensitive proteins have different light-stimulation thresholds, depending on the magnitude of the inward current generated by the cells after light stimulation, the larger the current, the easier the explosion of action potential, and the lower the required light intensity (light intensity) is relatively. Too large a light intensity is easy to generate additional action potential, the stimulation time, i.e. the pulse width, is prolonged, the cation amount flowing into cells is also increased, and non-one-to-one photocurrent is also generated, which is contrary to the advantage of fine regulation of neurons by optogenetic technology, so that proper light intensity and stimulation pulse width are adopted.

The second is the selection of frequencies, since neurons of the brain all have their own appropriate firing frequencies, generally pyramidal neurons fire at frequencies around 10Hz, and inhibitory interneurons fire at frequencies above 40 Hz. At the same time, the stimulation frequency is also related to the dynamics of the light-sensitive protein, most action potentials of wild-type ChR2 are lost when light is stimulated above 40Hz, even an 'inhibition' phenomenon is presented, and ChETA which is a variant can still generate one-to-one response even if the light stimulation frequency is up to 200Hz if expressed on PV neurons. Whereas for inhibitory NpHR, arch or Mac, continuous light can eliminate the spontaneous activity of the cells well. Therefore, the type of neurons to be stimulated is considered first before the experiment, the proper photosensitive protein is selected, and various parameters of the optogenetic stimulation signals are determined.

In some alternative embodiments, the interaction means comprises one or more of: keyboard, mouse, microphone assembly, touch screen, keys and knobs. The microphone assembly may include a sound receiving unit and a voice recognition unit (voice recognition chip), among others.

The present application describes functional improvements and usage elements that are emphasized by the patent laws, and the above description and drawings are merely preferred embodiments of the present application and not limiting the present application, and therefore, all structures, devices, features, etc. that are similar and identical to those of the present application, i.e. all equivalents and modifications made by the patent application are intended to be within the scope of protection of the patent application of the present application.

Claims

1. A head-mounted microscope for wearing on the head of an experimental animal, the brain region of the experimental animal being injected with a photo-genetic virus and a calcium imaging virus, the head-mounted microscope being for forming a photo-genetic optical path and a calcium imaging optical path;

in the optogenetic optical path, light emitted by a first light source is transmitted through the first dichroic mirror and then reflected to the head of the experimental animal through the second dichroic mirror so as to apply optogenetic stimulation to the experimental animal;

in the calcium imaging optical path, light emitted by the second light source is reflected by the first dichroic mirror and then reflected by the second dichroic mirror to the head of the experimental animal so as to excite the calcium imaging virus of the experimental animal, and the image sensor is utilized to receive the reflected light of the experimental animal so as to collect the neuro-active image of the experimental animal.

2. The head-mounted microscope of claim 1, further comprising a first filter, a second filter, a first collimating lens, a second collimating lens, and an imaging lens group comprising a liquid lens and at least one acromatic lens;

3. The head-mounted microscope of claim 2, wherein the at least one acromatic lens comprises: a first acromatic lens, a second acromatic lens, and a third acromatic lens;

4. The head-mounted microscope of claim 2, wherein the first light source emits light having a wavelength of 605nm and the second light source emits light having a wavelength of 470nm.

5. The head-mounted microscope of any one of claims 1-4, wherein the laboratory animal is any one of: mice, cats, dogs, pigs, cows, horses, monkeys, gorillas, and humans.

6. A microscopic imaging system, characterized in that the microscopic imaging system comprises a data processing device, a camera, a first light source, a second light source, and the head-mounted microscope of any one of claims 1-5;

the camera is used for collecting behavioural images of experimental animals;

7. The microimaging system of claim 6, further comprising a triple-core cable;

8. The microscopic imaging system of claim 7, wherein the data acquisition board card is a PCIe data acquisition card.

9. The microscopy imaging system of claim 6, further comprising an interaction device;

10. The microscopic imaging system of claim 9, wherein the interaction means includes one or more of: keyboard, mouse, microphone assembly, touch screen, keys and knobs.