Living hippocampus cell multiphoton imaging device

Abstract

The invention discloses a living body hippocampus cell multi-photon imaging device, which is characterized in that an IRDye 800CW 2-DG marker is injected into a first target area (the depth is greater than 1.4 mm, and hippocampus cells are included) of a living animal through a brain injection system, and then a two-photon fluorescence signal is generated by excitation of a 1700nm wave band through a multi-photon imaging system; and finally, detecting the two-photon fluorescence signal to obtain a microscopic image. Wherein the microscopic image shows hippocampus cells from the brain surface of the living animal to a preset depth (e.g., 2060 [mu] m depth). According to the technical scheme, the near-infrared fluorescent substance with higher effectiveness is injected into the target area in advance, then excitation is carried out through the soliton pulse signal in the specific wave band range, microscopic imaging is carried out based on the generated multi-photon fluorescence signal, the effectiveness of imaging of hippocampus cells (the depth is larger than 1.4 mm) is improved, and the imaging accuracy of the hippocampus cells (the depth is larger than 1.4 mm) is improved. And the type of the living animal brain cell marked by IRDye 800CW 2-DG is verified through a second near-infrared fluorescent substance.

Description

Technical Field

[0001] The present invention relates to the technical field of optical imaging, and particularly to a multi-photon imaging device for living hippocampal cells. Background Art

[0002] Currently, there are still important challenges in the visualization of deep brain structures in living mice using multi-photon imaging technology in the 1700 nm wavelength band, especially for the complete observation of hippocampal cells. The related multi-photon imaging technology can only effectively image the superficial hippocampal cells (depth < 1.4 mm) of living mice, but for hippocampal cells with a depth exceeding 1.4 mm (such as the dentate gyrus or the bottom region of the hippocampus), due to technical limitations such as optical attenuation, enhanced scattering, and reduced signal-to-noise ratio, the imaging ability is insufficient.

[0003] In brain science research, visualizing deep brain structures is crucial for understanding brain functions. The hippocampus, as a key region of the brain, is involved in memory formation and neural circuit regulation. However, due to its relatively deep location, it is difficult for traditional optical microscopy imaging technology to achieve high-resolution imaging of the entire hippocampal region. In recent years, two-photon fluorescence microscopy imaging technology has made important progress in the field of living tissue imaging. However, its penetration depth is limited by tissue scattering and optical attenuation, and it is only applicable to the imaging of relatively superficial structures.

[0004] Therefore, the related technology needs to be improved. Summary of the Invention

[0005] The main purpose of the present invention is to propose a multi-photon imaging device for living hippocampal cells, aiming to at least solve the technical problem of insufficient effectiveness when the imaging device images hippocampal cells with a depth exceeding 1.4 mm in the related technology.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a multi-photon imaging device for living hippocampal cells is provided. The multi-photon imaging device for living hippocampal cells includes a multi-photon imaging system and a brain injection system;

[0008] The brain injection system is used to label a first target area of a living animal with a first near-infrared fluorescent substance; wherein, the first near-infrared fluorescent substance includes an IRDye 800CW 2-DG label, and the first target area includes hippocampal cells;

[0009] The multi-photon imaging system is used to perform multi-photon imaging on the already labeled first target area and obtain a microscopic image corresponding to the excited two-photon fluorescence signal; wherein, the microscopic image is used to display hippocampal cells from the brain surface of the living animal to a preset depth.

[0010] Based on the first aspect, the multi - photon imaging system includes a first multi - photon imaging component and a second multi - photon imaging component; the second near - infrared fluorescent substance further includes an SR101 marker and a ScAAV - hSyn - mCherry - WPREs marker.

[0011] The brain injection system is used to label the hippocampal cells in the first target area with the IRDye 800CW 2 - DG marker by injection; wherein, the SR101 marker is labeled on the astrocytes in the second target area by smearing, and the ScAAV - hSyn - mCherry - WPREs marker is labeled on the nerve cells in the second target area by intravenous injection. The depth of the second target area is less than that of the first target area, and the first target area and the second target area are within the same field of view.

[0012] The first multi - photon imaging component is used to perform multi - photon imaging on the completed - labeled first target area and obtain a microscopic image corresponding to the excited two - photon fluorescence signal.

[0013] The second multi - photon imaging component is used to perform multi - photon imaging on the completed - labeled second target area and obtain a microscopic image corresponding to the excited three - photon fluorescence signal.

[0014] Based on the first aspect, the brain injection system includes a three - dimensional displacement stage system, a displacement stage controller, and an injection pump controller.

[0015] The displacement stage controller is used to control the three - dimensional displacement stage system according to the input injection position, angle, and depth.

[0016] The three - dimensional displacement stage system is used to control the movement of the microneedle in the preset X, Y, and Z directions to align with the hippocampal cells of the live animal at different injection stages.

[0017] The injection pump controller is used to apply a preset injection pressure and inject the IRDye 800CW 2 - DG marker into the hippocampal cells through the microneedle according to the preset injection time and dose.

[0018] Based on the first aspect, the multi - photon imaging system further includes a fiber laser, a photonic crystal fiber, and an optical focusing component.

[0019] The fiber laser is used to generate a laser signal with a preset first wavelength.

[0020] The photonic crystal fiber is used to convert the laser signal into a high-energy soliton laser signal with a preset second wavelength; wherein, the second wavelength is greater than the first wavelength and is within the target wavelength range;

[0021] The optical focusing component is used to focus the high-energy soliton laser signal onto the marked first target area and the second target area in the living animal cells to excite two-photon fluorescence signals and three-photon fluorescence signals.

[0022] Based on the first aspect, the multi-photon imaging system further includes a beam splitter;

[0023] The beam splitter is used to transmit the three-photon fluorescence signal and the two-photon fluorescence signal to the first multi-photon imaging component and the second multi-photon imaging component respectively along a preset path.

[0024] Based on the first aspect, the multi-photon imaging system further includes a half-wave plate, a polarization beam splitter and a first lens group;

[0025] The half-wave plate is used to adjust the polarization state of the laser signal, the polarization beam splitter is used to separate light beams with different polarization directions, and the first lens group is used to focus and shape the laser signal.

[0026] Based on the first aspect, the multi-photon imaging system further includes a second lens group, a low-pass filter and a neutral density filter;

[0027] The second lens group is used to collimate or expand the high-energy soliton laser signal output from the photonic crystal fiber;

[0028] The low-pass filter is used to filter the high-energy soliton laser signal;

[0029] The neutral density filter is used to adjust the laser intensity of the high-energy soliton laser signal.

[0030] Based on the first aspect, the first multi-photon imaging component is a first photomultiplier tube, and the first photomultiplier tube cooperates with a first band-pass filter to detect the two-photon fluorescence signal to obtain a microscopic image corresponding to the two-photon fluorescence signal.

[0031] Based on the first aspect, the second multi-photon imaging component is a second photomultiplier tube, and the first photomultiplier tube cooperates with a second band-pass filter to detect the three-photon fluorescence signal to obtain a microscopic image corresponding to the three-photon fluorescence signal.

[0032] Based on the first aspect, the optical focusing component includes a scanning galvanometer, a scanning lens, a sleeve lens and a water immersion objective lens;

[0033] The scanning galvanometer is used to control the transmission of the high-energy soliton laser signal according to a preset scanning path;

[0034] The scanning lens is used to perform a first-stage focusing on the high-energy soliton laser signal in the scanning path, and expand the beam of the high-energy soliton laser signal together with the sleeve lens;

[0035] The water immersion objective lens is used to perform a second-stage focusing on the expanded high-energy soliton laser signal, so as to focus on the hippocampal somatic cells in the first target area, the astrocytes in the second target area and the nerve cells in the living animal, and generate three-photon fluorescence signals and two-photon fluorescence signals.

[0036] The multi-photon imaging device for living hippocampal somatic cells of the present invention first injects the IRDye800CW 2-DG marker into the first target area (with a depth greater than 1.4 mm and containing hippocampal somatic cells) of the living animal through the brain injection system, and then uses the multi-photon imaging system to excite it at a wavelength band of 1700 nm to generate two-photon fluorescence signals, and detects the two-photon fluorescence signals to obtain microscopic images. Among them, the microscopic images are used to display the hippocampal somatic cells from the brain surface of the living animal to a preset depth (for example, a depth of 2060 μm). That is, through the combination of the multi-photon imaging system and the brain injection system, the present technical solution pre-injects a near-infrared fluorescent substance with higher effectiveness into the target area, and then is excited by the soliton pulse signal within a specific wavelength band range emitted by the multi-photon imaging system, and performs microscopic imaging on the generated multi-photon fluorescence signals, improving the effectiveness of imaging hippocampal somatic cells (with a depth greater than 1.4 mm), and verifying the types of brain cells of the living animal labeled with the first near-infrared fluorescent substance through the second near-infrared fluorescent substance. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is a module schematic diagram of the multi-photon imaging device for living hippocampal somatic cells provided by the embodiment of the present application;

[0039] Figure 2 It is a specific structure schematic diagram of the multi-photon imaging device for living hippocampal somatic cells provided by the embodiment of the present application;

[0040] Figure 3It is the spectrogram of the high-energy soliton laser signal in the embodiment of the present application;

[0041] Figure 4 It is the 2PF (two-photon fluorescence signal) imaging diagram of the brain of a living mouse in the embodiment of the present application;

[0042] Figure 5 It is the schematic diagram of the co-localization labeling of various near-infrared fluorescent substances in cells in the embodiment of the present application.

[0043] The realization, functional features and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the accompanying drawings. Detailed implementation manners

[0044] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] It should be noted that related terms such as "first", "second", etc. can be used to describe various components, but these terms do not limit the components. These terms are only used to distinguish one component from another. For example, without departing from the scope of the present invention, the first component can be called the second component, and similarly, the second component can also be called the first component. The term "and / or" refers to any combination of one or more of the related items and the described items.

[0046] In the related art, the multi-photon imaging technology in the 1700nm band still has important challenges in the visualization of the deep brain structure of living mice, especially for the complete observation of hippocampal cells. The existing multi-photon imaging technology can only effectively image the shallow-layer cells (depth < 1.4mm) of the hippocampus of living mice, but for hippocampal cells with a depth exceeding 1.4mm (such as the dentate gyrus or the bottom area of the hippocampus), due to technical limitations such as optical attenuation, enhanced scattering, and reduced signal-to-noise ratio, the imaging ability is insufficient.

[0047] In brain science research, visualizing the brain structure, especially the deep brain structure, is the primary task for understanding brain functions. For example, the blood vessels and cell structures in the deep brain are crucial for studying the neural activities of the brain. The cell types in the brain are complex and diverse, mainly including neurons, glial cells, endothelial cells, pericytes, etc. Each major category can be further divided into multiple subcategories. For example, among glial cells, there are astrocytes and oligodendrocytes, etc.

[0048] As a tool capable of achieving subcellular resolution and deep imaging, multiphoton microscopy has been widely used in the study of the brain structure of living animals. In recent years, multiphoton fluorescence imaging technology in the 1700 nm band has made significant progress in the field of cerebrovascular imaging in living mice. However, this technology has limited applications in the study of other brain structures, especially in the imaging of hippocampal cells in ordinary mice. Current research is mostly limited to using transgenic mice to image hippocampal cells through fluorescent protein labeling. This limitation is mainly reflected in two aspects: First, as a research object, transgenic mice have a limited range of biological samples and cannot be widely extended to non-transgenic animal models; second, the types and sample quantities of near-infrared fluorescent markers suitable for excitation in the 1700 nm band are much less than those of traditional green fluorescent markers. Therefore, compared with the excellent depth shown by the 1700 nm band multiphoton technology in vascular imaging, the depth and resolution of this technology in hippocampal cell imaging are significantly limited. These technical bottlenecks have significantly weakened the potential and application scope of the 1700 nm band multiphoton imaging technology in the study of deep brain cells. In the future, how to solve the adaptability of fluorescent markers and improve the imaging depth will become an important direction to further promote the application of this technology.

[0049] In summary, there is a technical problem of insufficient effectiveness in the related art when the imaging device images hippocampal cells with a depth exceeding 1.4 mm.

[0050] To solve the above technical problems, please refer to Figure 1 and Figure 2 , the multiphoton imaging device for living hippocampal cells in the embodiments of the present application includes a multiphoton imaging system and a brain injection system 200.

[0051] The main function of the brain injection system 200 is to label a first near-infrared fluorescent substance (such as an IRDye 800CW 2-DG marker) in a first target area (for example, an area with a depth greater than 1.4 mm and containing hippocampal cells) in a living animal during the injection stage, so that the hippocampal cells in the first target area are completed with labeling.

[0052] The main function of the multiphoton imaging system is to perform multiphoton imaging on the already labeled first target area (deep area) and the second target area (shallow area) during the imaging stage. Specifically, the first and second target areas are irradiated with a preset wavelength (for example, 1665 nm, which belongs to the 1700 nm band range) to excite the fluorescent molecules of the labeled hippocampal cells, so that they generate two-photon fluorescence signals, and a microscopic image corresponding to the excited two-photon fluorescence signals is obtained; among them, the microscopic image is used to display the hippocampal cells from the brain surface of the living animal to a preset depth (for example, the preset depth is 2060 μm).

[0053] It should be noted here that the light attenuation caused by the absorption and scattering of light by tissues in the 1700 nm wavelength band is the smallest. Therefore, using a laser signal with a wavelength in the 1700 nm wavelength band for multi-photon fluorescence signal excitation is a better choice for deep multi-photon imaging.

[0054] In addition, it should also be noted that the IRDye 800CW 2-DG marker, as a near-infrared fluorescently labeled glucose analog, has demonstrated great potential in imaging the labeling of hippocampal somatic cells in the mouse brain (960 - 2100 μm: 960 - 1390 μm in the CA1 region; 1390 - 2100 μm in the dentate gyrus). IRDye 800CW 2-DG combines the advantages of the IRDye 800CW near-infrared fluorescent dye and 2-deoxy-D-glucose (2-DG). Its near-infrared optical properties (excitation wavelength 774 nm, emission wavelength 791 nm) enable it to have a significant cross-section in two-photon excitation in the 1700 nm wavelength band. This efficient two-photon absorption property can provide strong signals at lower laser powers, thus significantly improving the sensitivity and resolution of deep tissue imaging while reducing light damage to living tissues. The hippocampus is a deep region of the brain with extremely high requirements for tissue penetration and fluorescence stability. Thanks to its low light absorption and scattering characteristics in the near-infrared optical window, IRDye 800CW 2-DG can provide clear imaging with a high signal-to-noise ratio in deep tissues, and its high light stability ensures the continuous reliability of the signal, providing an efficient and non-invasive tool for the study of deep brain structures.

[0055] The in-vivo hippocampal somatic cell multi-photon imaging device of the present invention first injects the IRDye800CW 2-DG marker into the first target area (depth greater than 1.4 mm) of the hippocampal somatic cells of a living animal through a brain injection system, and then uses a multi-photon imaging system to excite it to generate a two-photon fluorescence signal at the 1700 nm wavelength band, and detects the two-photon fluorescence signal to obtain a microscopic image. Among them, the microscopic image is used to display the hippocampal somatic cells from the brain surface of the living animal to a preset depth (for example, a depth of 2060 μm). That is, through the combination of the multi-photon imaging system and the brain injection system, this technical solution first injects a more effective near-infrared fluorescent substance into the first target area, and then is excited by the soliton pulse signal in a specific wavelength band emitted by the multi-photon imaging system, and performs microscopic imaging on the generated multi-photon fluorescence signal, improving the effectiveness of imaging hippocampal cells at different depths, so that in the imaging of in-vivo hippocampal somatic cells with a depth exceeding 1.4 mm, it can still maintain high-efficiency and stable imaging quality, and verify the cell types of the living animal brain labeled with the IRDye800CW 2-DG marker through the second near-infrared fluorescent substances (SR101 marker and ScAAV-hSyn-mCherry-WPREs marker).

[0056] In an alternative embodiment of the present embodiment, the multi-photon imaging system includes a first multi-photon imaging component and a second multi-photon imaging component; the second near-infrared fluorophore further includes an SR101 marker and a ScAAV-hSyn-mCherry-WPREs marker, and the second target region (shallow region) includes astrocytes and neurons.

[0057] The brain injection system is used to label hippocampal cells in the first target region with the IRDye 800CW 2-DG marker by injection, and to label astrocytes in the second target region with the SR101 marker in the second near-infrared fluorophore by smearing, and to label neurons in the second target region with the ScAAV-hSyn-mCherry-WPREs marker in the second near-infrared fluorophore by intravenous injection. The depth of the region where the labeled astrocytes and neurons are located (i.e., the second target region) is less than the depth of the region where the labeled hippocampal cells are located (i.e., the first target region). At the same time, the first target region and the second target region are within the same field of view. Thus, the target cells in the first target region and the second target region at different depths in the living animal are all labeled.

[0058] It should be noted that "within the same field of view" may mean within one field of view of the multi-photon imaging system, that is, in a single imaging or observation process, the first target region and the second target region can be observed simultaneously without adjusting the field of view or repositioning the sample. And, the depth of the first target region: 0 - 2060 μm, the depth of the second target region: 0 - 1400 μm.

[0059] The first multi-photon imaging component mainly performs multi-photon imaging on the first target region. Specifically, it uses an excitation light source to excite fluorescent molecules and collects two-photon fluorescence signals through an optical filtering system to generate a microscopic image corresponding to the excited two-photon fluorescence signals. The second multi-photon imaging component mainly targets the second target region, acquires three-photon fluorescence signals and generates microscopic images.

[0060] Through the above embodiment, multiplex labeling of the IRDye 800CW 2-DG marker with the SR101 marker and the ScAAV-hSyn-mCherry-WPREs marker is achieved, and cell co-localization imaging is respectively realized.

[0061] It should also be noted here that for the SR101 marker and the ScAAV-hSyn-mCherry-WPREs marker: The SR101 marker (Sulforhodamine 101) mainly labels astrocytes, while the ScAAV-hSyn-mCherry-WPREs marker selectively labels neurons through an AAV (adeno-associated virus) vector. These two three-photon markers are used to verify the cell type labeling effect of IRDye 800CW 2-DG (two-photon) in the brains of living animals.

[0062] In an alternative embodiment of the present embodiment, the brain injection system 200 includes a three-dimensional displacement stage system 2002, a displacement stage controller 2001, and an injection pump controller 2003.

[0063] Specifically, the displacement stage controller 2001 is used to control the three-dimensional displacement stage system 2002 according to the injection position, angle, and depth input by the user. The three-dimensional displacement stage system 2002 is used to control the movement of the micro-needle in the preset X, Y, and Z directions to align with the target area (hippocampal cells) of the living animal during the injection stage. The injection pump controller 2003 is used to apply a preset injection pressure and inject the near-infrared fluorescent substance (IRDye 800CW 2-DG marker) into the first target area through the micro-needle according to the preset injection time and dose. That is, the brain injection system 200 ensures the precise injection of the fluorescent substance by using a three-dimensional displacement stage system, a displacement stage controller, and an injection pump controller, improving the imaging stability and repeatability.

[0064] In this brain injection system 200, an angle adjustment stage adjustable by ±15 degrees can also be equipped, combined with the flexible installation design of a 30-degree base, enabling the system to achieve continuous adjustment of the injection angle within a range of ±90 degrees to meet different experimental requirements. The micro-needle required for injection is prepared using a microelectrode puller (P97, Sutter), and then the dye is precisely introduced into the pulled micro-needle using a 10 μL pipette. Before injection, the micro-needle filled with the dye needs to be pressure-tested to check its integrity and whether the dye can be smoothly discharged. If the micro-needle cannot inject the dye normally in the air, it means that it cannot work smoothly in brain tissue either, so it needs to be replaced or repaired. After confirming that the micro-needle is in good condition, the movement of the micro-needle is precisely controlled through a three-dimensional translation stage. When the micro-needle penetrates the brain, special attention should be paid to adjusting the angle adjustment stage of the injection system to make the angle between the micro-needle and the platform consistent with the angle of the displacement stage. If the angles do not match, the micro-needle will enter the brain in a lateral piercing manner, which not only cannot achieve precise injection but also causes serious damage to brain tissue. Therefore, ensuring the precise calibration of the system angle is the key to achieving high-quality brain injection operations.

[0065] In addition, to achieve imaging of living hippocampal somatic cells, an injection operation was performed on the brain. Specifically, by precisely controlling the three-dimensional displacement stage, the micropipette was steadily advanced during the process of piercing the brain. During this process, the micropipette did not stay at a specific depth for injection, but instead continuously applied positive pressure (the air pressure range can be 4 - 5 bar). When the tip position reached a depth of 2200 μm below the brain surface, the application of positive pressure was stopped, and the tip was slowly withdrawn. The entire injection process, from start to finish, including closing the cranial window, took approximately 30 to 60 minutes. This period provided sufficient time for the IRDye 800CW 2-DG injected into the brain to diffuse in the brain tissue and effectively label the hippocampal somatic cells.

[0066] The devices included in the multiphoton imaging system are described below:

[0067] In an alternative embodiment of the present embodiment, the multiphoton imaging system further includes a fiber laser 10, a photonic crystal fiber 20, and an optical focusing assembly. Specifically, the fiber laser is used to generate a laser signal of a preset first wavelength; the photonic crystal fiber is used to convert the laser signal into a high-energy soliton laser signal of a preset second wavelength; the optical focusing assembly is used to focus the high-energy soliton laser signal onto the first target area and the second target area that have been labeled in a living animal to excite two-photon fluorescence signals and three-photon fluorescence signals.

[0068] The fiber laser 10 can be a femtosecond pulsed laser (FLCPA - 02CSZU, Calmar laser) that can emit laser signals with a wavelength of 1550 nm and a repetition frequency of 1 MHz, which is used to generate a laser signal of a first wavelength (the wavelength can be 1550 nm) and transmit it to the photonic crystal fiber 20.

[0069] The photonic crystal fiber 20 can be a rod-shaped photonic crystal fiber (PCrod, SC - 1500 / 100 - Si - ROD, NKT Photonics) with a length of 44 cm and a core diameter of 100 μm. The rod-shaped photonic crystal fiber converts the laser signal of the first wavelength (1550 nm, not within the target wavelength range) into a high-energy soliton laser signal of a preset second wavelength (for example, 1665 nm, which belongs to the target wavelength range of 1700 nm band range) based on the soliton self-frequency shift effect and transmits it to the optical focusing assembly 30. Specifically, the photonic crystal fiber uses its special waveguide structure to broaden the laser signal of the first wavelength and generate a soliton pulse signal of the second wavelength through non-linear effects (the spectrum is as Figure 3 shown). The optimized fiber parameters ensure that the second wavelength of the high-energy soliton laser signal is stable at 1665 nm and has sufficient pulse energy to meet the excitation requirements of subsequent multiphoton fluorescence signals.

[0070] The optical focusing component can be a combination of lenses, galvanometric mirrors, etc., mainly used to accurately focus the high-energy soliton laser signal of the second wavelength onto the target area with shallow position marking completed and the target area with deep position marking completed. By optimizing the numerical aperture and focal position of the focusing lens, it is ensured that the high-energy soliton laser signal effectively excites the marked cells in the first target area and the second target area at different depths in the living animal, thereby generating corresponding two-photon fluorescence signals and three-photon fluorescence signals.

[0071] In an alternative embodiment of this embodiment, the multi-photon imaging system further includes a beam splitter 70; the beam splitter 70 is used to transmit the three-photon fluorescence signal and the two-photon fluorescence signal to the first multi-photon imaging component 40 and the second multi-photon imaging component 50 respectively according to a preset path.

[0072] Specifically, through the beam splitter 70, the two-photon fluorescence signal is transmitted to the first multi-photon imaging component 40 through a preset optical path for efficient detection and image acquisition. At the same time, the three-photon fluorescence signal is transmitted to the second multi-photon imaging component 50 through another preset optical path to achieve high-resolution imaging.

[0073] In an alternative embodiment of this embodiment, the multi-photon imaging system further includes a half-wave plate 110, a polarization beam splitter 120, and a first lens group 130.

[0074] The half-wave plate 110 is used to adjust the polarization state of the laser signal, that is, the half-wave plate (λ / 2 wave plate) can change the polarization direction of the incident laser by rotating different angles to make it adapt to the requirements of the optical path system and subsequent optical elements. By optimizing the polarization state, the energy utilization rate of the laser passing through the optical element can be improved, and the optical loss caused by polarization can be reduced.

[0075] The polarization beam splitter 120 is used to separate beams with different polarization directions, that is, the polarization beam splitter is used to distinguish P-polarized light and S-polarized light, so that the P-polarized light is transmitted and the S-polarized light is reflected, thereby realizing optical path optimization and beam separation. This function helps to enhance the signal contrast, reduce background light interference, and improve the detection sensitivity of the fluorescence signal.

[0076] The first lens group 130 is used to focus and shape the beam to ensure that the optical signal maintains the best optical quality during transmission, so as to improve the resolution and signal collection efficiency of the multi-photon imaging system.

[0077] In summary, the multi-photon imaging system uses the half-wave plate 110, the polarization beam splitter 120, and the first lens group 130 to optimize the polarization state of the laser signal, separate the beam, and efficiently couple the light into the optical fiber to improve the stability of the laser signal before entering the photonic crystal fiber 20.

[0078] In an alternative embodiment of the present embodiment, the multi-photon imaging system further includes a second lens group 140, a low-pass filter 150, and a neutral density filter 160.

[0079] The second lens group 140 is used to collimate the high-energy soliton laser signal output from the photonic crystal fiber 20. Since the high-energy soliton laser signal has a divergent characteristic after being output from the photonic crystal fiber, the second lens group 140 is responsible for collimating the laser beam to ensure parallel propagation of the beam and improve the transmission efficiency of the optical system.

[0080] The low-pass filter 150 is used to filter the high-energy soliton laser signal. It is mainly considered that the high-energy soliton laser signal may contain high-frequency noise components, which may affect the stability of the fluorescence signal if directly used for two-photon or three-photon excitation. Thus, the low-pass filter 150 can remove unnecessary high-frequency components and retain the effective excitation wavelength range suitable for biological imaging to ensure more stable laser output.

[0081] The neutral density filter 160 is used to adjust the laser intensity of the high-energy soliton laser signal. Since multi-photon microscopy imaging relies on high-power ultrafast pulsed lasers, if the laser power is too high, it may cause photobleaching of fluorescent molecules or tissue damage. The neutral density filter 160 can flexibly adjust the laser power to ensure that the laser intensity is maintained within a safe range during the imaging process and optimize the signal-to-noise ratio of the fluorescence signal acquisition.

[0082] In an alternative embodiment of the present embodiment, the first multi-photon imaging component 40 is a first photomultiplier tube (H7422-50, Hamamatsu). The first photomultiplier tube cooperates with the first band-pass filter 170 (FF01-855 / 210-25, Semrock) to detect the two-photon fluorescence signal corresponding to the first target area and obtain a microscopic image corresponding to the two-photon fluorescence signal (2-photon fluorescence, 2PF).

[0083] In an alternative embodiment of the present embodiment, the second multi-photon imaging component 50 is a second photomultiplier tube. The second photomultiplier tube (H7422p-50, Hamamatsu) cooperates with the second band-pass filter 180 (FF01-630 / 92-25, Semrock) to detect the three-photon fluorescence signal (3-photon fluorescence, 3PF) corresponding to the second target area and obtain a microscopic image corresponding to the three-photon fluorescence signal.

[0084] In an alternative embodiment of the present embodiment, the optical focusing component includes a scanning galvanometer 302, a scanning lens 303, a sleeve lens 304, and a water immersion objective lens 80.

[0085] Specifically, the scanning galvanometer is used to control the transmission of the high-energy soliton laser signal according to a preset scanning path. The scanning lens is used to perform the first-stage focusing on the high-energy soliton laser signal in the scanning path, and together with the sleeve lens, expand the beam of the high-energy soliton laser signal to a spot ratio of 1:4. The water immersion objective lens 80 is used to perform the second-stage focusing on the expanded high-energy soliton laser signal, so as to focus it on the labeled cells in the first target area and the second target area in the living animal, and generate three-photon fluorescence signals and / or two-photon fluorescence signals.

[0086] In addition, a dichroic mirror 60 may be provided before the input end of the water immersion objective lens 80. The dichroic mirror 60 is mainly used to separate the excitation light and the emission light, and guide the signal light to subsequent filters and detectors to achieve efficient optical detection or imaging.

[0087] In an alternative embodiment of this embodiment, the entire multi-photon imaging system may further include a reflector 301 and a mirror 305.

[0088] Specifically, the reflector 301 may be selectively disposed at the output end of the neutral density filter 160, and is used to change the direction of the laser light path, and reflect the high-energy soliton laser signal filtered by the neutral density filter 160 to the scanning galvanometer 302, facilitating subsequent beam scanning and sample excitation. Its highly reflective surface can effectively reduce light loss and ensure the stability of the energy transmission of the excitation light. The mirror 305 mainly plays a role in adjusting the beam path in the optical path system, ensuring that the laser enters the water immersion objective lens 80 along a predetermined trajectory to achieve accurate in-vivo imaging.

[0089] It should be noted that the surgical procedures for living animals involved in the embodiments of this application include the following: All animal experiments were carried out in accordance with the "Shenzhen University Guide for the Care and Use of Laboratory Animals" and approved by the Animal Ethics Committee of the School of Medicine of Shenzhen University (Approval No.: IACUC-202300036). All mice were from the Guangdong Provincial Medical Laboratory Animal Center in China. Adult female mice (C57BL / 6J, 8-10 weeks old) were used for imaging. The mice were anesthetized using a gas anesthesia system (Matrix VIP3000, Midmark) and isoflurane. A heating pad was used to maintain the animal's body temperature at 36.5°C, and 50 μL of h -15% glucose solution. A craniotomy with a diameter of 3 mm was performed centered 2 mm posterior-lateral to the bregma. A self-made metal sheet was tightly adhered to the skull with dental cement, and the cranial window was sealed with a coverslip with a diameter of 5 mm. Before imaging, the mouse was injected with ~1 μL of IRDye 800CW 2-DG into the brain to label the hippocampal cells, and the injection site was at the center of the cranial window. SR101 with a concentration of 1 mmol / L was applied to the surface of the mouse brain, left for 5 min, and then rinsed with saline to label the astrocytes; 200 μL of ScAAV-hSyn-mCherry-WPREs was injected into the orbit to label the neurons.

[0090] Using the in vivo hippocampal cell multiphoton imaging device of the embodiment of the present application, imaging experiments can be carried out on mice injected with labeled IRDye 800CW 2-DG through brain injection. In the specific implementation process, the excitation light power is regulated by a variable neutral density attenuation filter (Thorlabs, model NDC-50-4M), so as to image brain cells at different depths under different power conditions (as Figure 4 shown). At an excitation wavelength of 1665 nm, successful imaging of hippocampal cells from the surface of the mouse brain to a depth of 2060 μm was achieved.

[0091] Through the specific implementation process, a three-dimensional reconstruction of the 2PF cell imaging image at a depth of 2100 μm below the brain surface was obtained ( Figure 4 the left part in). At 2 mm lateral and posterior to the bregma, the depth range of the white matter (WM) layer under the surface of the mouse brain is 800 - 960 μm, and the range of the hippocampal region is: CA1 region (960 - 1390 μm), dentate gyrus (1390 - 2060 μm). Figure 4 The right part (a - f) of shows the two-dimensional 2PF images at different imaging depths below the brain surface: (a) are cerebral cortical cells; (b) are white matter layer cells; (c) are hippocampal CA1 region cells; (d - f) are hippocampal dentate gyrus cells.

[0092] Specific implementation results show that the deepest cells that can be resolved by this imaging technique are located 2060 μm below the surface of the mouse brain ( Figure 4 f in the right part). This indicates that under the excitation condition of 1665 nm, 2PF imaging can penetrate the entire neocortex and white matter layer (WM), and further penetrate to the bottom of the hippocampus, providing strong technical support for imaging deep brain tissues.

[0093] In the embodiments of the present application, for mice that have been labeled with IRDye 800CW 2-DG, SR101 and ScAAV-hSyn-mCherry-WPREs are respectively used for multiplex labeling of cells. Also under excitation at a wavelength of 1665 nm, 2PF and 3PF imaging are simultaneously performed on brain cells at different depths under different power conditions.

[0094] The specific implementation results show that at a depth of 196 μm below the mouse brain surface, the co-localization of IRDye 800CW 2-DG labeled cells (as shown in Figure 5 (b)) and SR101 labeled cells (as shown in Figure 5 (a)) is observed. SR101 is well-known for its good labeling specificity and is mainly used for specific labeling of astrocytes. This specific implementation result shows that IRDye 800CW 2-DG can label astrocytes. This discovery lays a technical foundation for further research on the distribution and function of deep astrocytes in the hippocampal region. Combining the multi-photon imaging advantages of IRDye 800CW 2-DG in the 1700 nm band, high-resolution and deep multi-photon imaging of astrocytes in the hippocampal region can be achieved in the future, promoting the research on the function of glial cells in deep brain structures.

[0095] In addition, at a depth of 294 μm below the brain surface, the co-localization of IRDye 800CW 2-DG (as shown in Figure 5 (d)) and ScAAV-hSyn-mCherry-WPREs labeled cells (as shown in Figure 5 (c)) is experimentally observed. ScAAV-hSyn-mCherry-WPREs is a tool for specifically labeling mature neurons and is widely used to study the distribution, function and their network connections of neurons. The co-localization results show that IRDye 800CW 2-DG can not only label astrocytes, but also be used for labeling mature neuron cells, and its labeling ability in tissues shows good stability and specificity.

[0096] The embodiments of the present application comprehensively verify the dual functions of IRDye 800CW 2-DG in labeling astrocytes and mature neurons through specific implementation results. At the same time, combined with its multi-photon imaging characteristics in the 1700 nm band, it demonstrates its wide application potential in imaging research of deep brain regions. Especially in the research of deep structures such as the hippocampus, the advantage of this probe is that it can penetrate the cortex and white matter layers, reach deep into the bottom region of the hippocampus, and achieve synchronous imaging of astrocytes and neurons. This provides strong tool support for exploring the interactions and functional connections of different cell types in deep brain regions, and opens up a new direction for brain science research.

[0097] In summary, the embodiments of the present application have elaborated in detail on the application performance and cell labeling potential of IRDye 800CW 2-DG in multi-photon imaging in the 1700 nm band. By combining SR101 and ScAAV-hSyn-mCherry-WPREs, co-localization labeling of astrocytes and mature neurons was achieved, and the dual-labeling ability and deep imaging stability of IRDye 800CW 2-DG were verified through experimental results. The research shows that IRDye 800CW 2-DG can penetrate the neocortex and white matter layers, providing high-resolution multi-photon imaging of the bottom region of the hippocampus, thus providing powerful tool support for the visualization research of deep brain structures. In the future, IRDye 800CW 2-DG may become a key tool for studying the interactions between deep brain cells and the pathological mechanisms of neural networks.

[0098] For the multi-photon imaging device for hippocampal cells in vivo of the present invention, first, a first near-infrared fluorescent substance is injected into the first target area of a living animal (for labeling hippocampal cells) through a brain injection system, and then the first target area is imaged through a multi-photon imaging system, and finally a microscopic image corresponding to the two-photon fluorescence signal is obtained. That is, through the combination of the multi-photon imaging component and the brain injection system in this technical solution, the effectiveness of imaging hippocampal cells is improved, so that high-efficiency and stable imaging quality can still be maintained in the in vivo imaging of hippocampal cells with a depth exceeding 1.4 mm (the actual beneficial effects have been proven by experimental data).

[0099] It can be seen that the present invention combines the multi-photon microscopy imaging technology in the 1700 nm band, the IRDye 800CW 2-DG fluorescence labeling, and the brain injection technology to explore its application potential in cell imaging of deep brain structures in mice, especially in the hippocampal region. In addition, through the multiple labeling of IRDye 800CW 2-DG with SR101 and ScAAV-hSyn-mCherry-WPREs, cell co-localization imaging is respectively achieved. The application of the brain injection technology significantly improves the accuracy of labeling, enabling the fluorescent probe to accurately act on the target brain area while avoiding interference with surrounding tissues. Moreover, combined with the characteristics of IRDye 800CW 2-DG, this method breaks through the limitations of traditional labeling means and provides important support for the efficient labeling and imaging of the hippocampal region. This technical solution combining brain injection and multi-photon imaging in the 1700 nm band provides new tool support for studying the distribution, functions, and interactions of cell types in deep brain regions, demonstrating the broad application prospects of IRDye 800CW 2-DG in brain science and neurological disease research.

[0100] In the above embodiments, the descriptions of each embodiment have their own focuses. For the parts not detailed in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0101] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. A multi-photon imaging device for living hippocampal somatic cells, characterized in that, The in-vivo hippocampal somatic cell multi-photon imaging device includes a multi-photon imaging system and a brain injection system; The brain injection system is used to label a first target area of a living animal with a first near-infrared fluorescent substance; wherein, the first near-infrared fluorescent substance includes an IRDye 800CW 2-DG label, and the first target area includes hippocampal somatic cells; The multi-photon imaging system is used to perform multi-photon imaging on the already labeled first target area and obtain a microscopic image corresponding to the excited two-photon fluorescence signal; wherein, the microscopic image is used to display hippocampal somatic cells from the brain surface of a living animal to a preset depth.

2. The multi-photon imaging device for living hippocampal somatic cells according to claim 1, wherein The multi-photon imaging system includes a first multi-photon imaging component and a second multi-photon imaging component; the second near-infrared fluorescent substance also includes an SR101 label and a ScAAV-hSyn-mCherry-WPREs label, The brain injection system is used to label the hippocampal somatic cells in the first target area with the IRDye 800CW 2-DG label by injection; Wherein, the SR101 label is labeled on astrocytes in a second target area by smearing, and the ScAAV-hSyn-mCherry-WPREs label is labeled on nerve cells in the second target area by intravenous injection. The depth of the second target area is less than the depth of the first target area, and the first target area and the second target area are within the same field of view; The first multi-photon imaging component is used to perform multi-photon imaging on the already labeled first target area and obtain a microscopic image corresponding to the excited two-photon fluorescence signal; The second multi-photon imaging component is used to perform multi-photon imaging on the already labeled second target area and obtain a microscopic image corresponding to the excited three-photon fluorescence signal.

3. The multi-photon imaging device for living hippocampal somatic cells according to claim 2, characterized in that, The brain injection system includes a three-dimensional displacement stage system, a displacement stage controller, and an injection pump controller; The displacement stage controller is used to control the three-dimensional displacement stage system according to the input injection position, angle, and depth; The three-dimensional displacement stage system is used to control the movement of the micro needle in the preset X, Y, and Z directions to align with the hippocampal somatic cells of the living animal at different injection stages; The injection pump controller is used to apply a preset injection pressure and inject the IRDye 800CW 2-DG label into the hippocampal somatic cells through the micro needle according to the preset injection time and dose.

4. The multi-photon imaging device for in vivo hippocampal somatic cells according to claim 1, wherein, The multi-photon imaging system further includes a fiber laser, a photonic crystal fiber, and an optical focusing component; The fiber laser is used to generate a laser signal with a preset first wavelength; The photonic crystal fiber is used to convert the laser signal into a high-energy soliton laser signal with a preset second wavelength; wherein, the second wavelength is greater than the first wavelength and is within the target band range; The optical focusing component is used to focus the high-energy soliton laser signal onto the already labeled first target area and the second target area in the living animal cells to excite two-photon fluorescence signals and three-photon fluorescence signals.

5. The multi-photon imaging device for in vivo hippocampal somatic cells according to claim 4, characterized in that, The multi-photon imaging system further includes a beam splitter; The beam splitter is configured to transmit the three-photon fluorescence signal and the two-photon fluorescence signal to the first multi-photon imaging component and the second multi-photon imaging component respectively according to a preset path.

6. The multi-photon imaging device for living hippocampal somatic cells according to claim 4, wherein The multi-photon imaging system further includes a half-wave plate, a polarization beam splitter and a first lens group; The half-wave plate is used to adjust the polarization state of the laser signal, the polarization beam splitter is used to separate the light beams with different polarization directions, and the first lens group is used to focus and shape the laser signal.

7. The multi-photon imaging device for living hippocampal somatic cells according to claim 6, wherein The multi-photon imaging system further includes a second lens group, a low-pass filter and a neutral density filter; The second lens group is used to collimate or expand the high-energy soliton laser signal output from the photonic crystal fiber; The low-pass filter is used to filter the high-energy soliton laser signal; The neutral density filter is used to adjust the laser intensity of the high-energy soliton laser signal.

8. The multi-photon imaging device for living hippocampal somatic cells according to claim 4, characterized in that, The first multi-photon imaging component is a first photomultiplier tube, and the first photomultiplier tube cooperates with a first band-pass filter to detect the two-photon fluorescence signal to obtain a microscopic image corresponding to the two-photon fluorescence signal.

9. The multi-photon imaging device for living hippocampal somatic cells according to claim 8, wherein, The second multi-photon imaging component is a second photomultiplier tube, and the second photomultiplier tube cooperates with a second band-pass filter to detect the three-photon fluorescence signal to obtain a microscopic image corresponding to the three-photon fluorescence signal.

10. The multi-photon imaging device for in vivo hippocampal somatic cells according to claim 4, wherein The optical focusing component includes a scanning galvanometer, a scanning lens, a sleeve lens and a water immersion objective lens; The scanning galvanometer is used to control the transmission of the high-energy soliton laser signal according to a preset scanning path; The scanning lens is used to perform a first-stage focusing on the high-energy soliton laser signal in the scanning path, and expand the high-energy soliton laser signal together with the sleeve lens; The water immersion objective lens is used to perform a second-stage focusing on the expanded high-energy soliton laser signal to focus it on the hippocampal cells in the first target area, the astrocytes in the second target area and the nerve cells in the living animal, and generate a three-photon fluorescence signal and a two-photon fluorescence signal.

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