In-vivo hippocampus cell multi-photon imaging device

Abstract

The application discloses a kind of in vivo hippocampus cell multiphoton imaging device, first by brain injection system IRDye 800CW 2-DG marker is injected into the first target area (depth greater than 1.4mm, and contain hippocampus cell) of live animal, then by multiphoton imaging system using 1700nm waveband excitation its generation two-photon fluorescence signal, finally two-photon fluorescence signal is detected to obtain microscopic image.Wherein, the microscopic image shows the hippocampus cell of brain surface of live animal to preset depth (for example, 2060 μm depth).That is, the prior art uses the near-infrared fluorescent substance with higher effectiveness to inject into the target area, then excites by the soliton pulse signal of specific waveband range, and carries out microscopic imaging based on the generated multiphoton fluorescence signal, improves the effectiveness of imaging hippocampus cell (depth greater than 1.4mm), and verifies the cell type of live animal brain of IRDye 800CW 2-DG marker by the second near-infrared fluorescent substance.

Description

Technical Field

[0001] This invention relates to the field of optical imaging technology, specifically to a live hippocampal somatic cell multiphoton imaging device. Background Technology

[0002] Currently, the visualization of deep brain structures in live mice using 1700nm multiphoton imaging technology remains a significant challenge, particularly for the complete observation of hippocampal cells. Existing multiphoton imaging techniques can only effectively image superficial hippocampal cells (depth < 1.4 mm) in live mice. However, for hippocampal cells deeper than 1.4 mm (such as those in the dentate gyrus or the basal region of the hippocampus), imaging capabilities are insufficient due to limitations such as optical attenuation, enhanced scattering, and reduced signal-to-noise ratio.

[0003] In neuroscience research, visualizing deep brain structures is crucial for understanding brain function. The hippocampus, a key brain region involved in memory formation and neural circuit regulation, is located at a relatively deep level, making it difficult for traditional optical microscopy to achieve high-resolution imaging of the entire hippocampus. In recent years, two-photon fluorescence microscopy has made significant progress in in vivo tissue imaging; however, its penetration depth is limited by tissue scattering and optical attenuation, limiting its application to imaging relatively shallow structures.

[0004] Therefore, the relevant technologies need to be improved. Summary of the Invention

[0005] The main objective of this invention is to propose a live hippocampal somatic cell multiphoton imaging device, which aims to at least solve the technical problem of insufficient effectiveness of imaging devices in imaging hippocampal somatic cells with a depth of more than 1.4 mm in related technologies.

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

[0007] In a first aspect, the present invention provides a live hippocampal somatic cell multiphoton imaging device, the live hippocampal somatic cell multiphoton imaging device comprising a multiphoton imaging system and a brain injection system;

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

[0009] The multiphoton imaging system is used to perform multiphoton imaging on the first target region that has been marked, and to 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 to a predetermined depth of a live animal.

[0010] Based on the first aspect, the multiphoton imaging system includes a first multiphoton imaging component and a second multiphoton imaging component; the second near-infrared phosphor further includes SR101 markers and ScAAV-hSyn-mCherry-WPREs markers.

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

[0012] The first multiphoton imaging component is used to perform multiphoton imaging on the marked first target region and obtain a microscopic image corresponding to the excited two-photon fluorescence signal.

[0013] The second multiphoton imaging component is used to perform multiphoton imaging on the marked second target region 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 it 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 a preset injection time and dose.

[0018] Based on the first aspect, the multiphoton imaging system further includes a fiber laser, a photonic crystal fiber, and an optical focusing assembly;

[0019] The fiber laser is used to generate a laser signal of 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 first and second target regions that have been labeled in the living animal cells, so as to excite two-photon fluorescence signals and three-photon fluorescence signals.

[0022] Based on the first aspect, the multiphoton 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 multiphoton imaging component and the second multiphoton imaging component respectively according to a preset path.

[0024] Based on the first aspect, the multiphoton imaging system further includes a half-wave plate, a polarizing 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 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 multiphoton 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 multiphoton imaging component is a first photomultiplier tube, which, in conjunction with a first bandpass filter, detects 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 multiphoton imaging component is a second photomultiplier tube, and the first photomultiplier tube, in conjunction with a second bandpass filter, detects 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 assembly includes a scanning galvanometer, a scanning lens, a sleeve lens, and a water immersion objective.

[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 focus the high-energy soliton laser signal in the scanning path in the first stage, and together with the sleeve lens, expands the high-energy soliton laser signal.

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

[0036] The live hippocampal somatic cell multiphoton imaging device of the present invention first injects an IRDye800CW 2-DG marker into a first target region (deeper than 1.4 mm, containing hippocampal somatic cells) of a live animal via a brain injection system. Then, the device is excited using a 1700 nm wavelength using a multiphoton imaging system to generate a two-photon fluorescence signal, which is then detected to obtain a microscopic image. This microscopic image is used to display hippocampal somatic cells from the brain surface of the live animal to a predetermined depth (e.g., 2060 μm). In other words, this technical solution combines a multiphoton imaging system with a brain injection system, pre-injecting a more effective near-infrared fluorescent material into the target region, then exciting the region with a soliton pulse signal within a specific wavelength range emitted by the multiphoton imaging system, and performing microscopic imaging on the generated multiphoton fluorescence signal. This improves the effectiveness of imaging hippocampal somatic cells (deeper than 1.4 mm), and verifies the type of live animal brain cells labeled with the first near-infrared fluorescent material using a second near-infrared fluorescent material. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 A schematic diagram of the modules of the live hippocampal somatic cell multiphoton imaging device provided in the embodiments of this application;

[0039] Figure 2 A schematic diagram of the specific structure of the live hippocampal somatic cell multiphoton imaging device provided in the embodiments of this application;

[0040] Figure 3This is a spectrum of a high-energy soliton laser signal in an embodiment of this application;

[0041] Figure 4 This is a 2PF (two-photon fluorescence) imaging image of the brain of a live mouse in an embodiment of this application;

[0042] Figure 5 This is a schematic diagram illustrating the colocalization labeling of various near-infrared fluorescent substances in cells in the embodiments of this application.

[0043] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0044] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

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

[0046] Among related technologies, multiphoton imaging technology using the 1700nm band still faces significant challenges in visualizing the deep structures of the mouse brain, especially for the complete observation of hippocampal cells. Existing multiphoton imaging techniques can only effectively image superficial hippocampal cells (depth <1.4mm) in live mice. However, for hippocampal cells deeper than 1.4mm (such as the dentate gyrus or the basal region of the hippocampus), the imaging capability is insufficient due to technical limitations such as optical attenuation, enhanced scattering, and reduced signal-to-noise ratio.

[0047] In neuroscience research, visualizing brain structures, especially deep brain structures, is a primary task in understanding brain function. For example, the deep blood vessels and cellular structures of the brain are crucial for studying neural activity. Brain cells are complex and diverse, mainly including neurons, glial cells, endothelial cells, and pericytes, each of which can be further subdivided into several subclasses, such as astrocytes and oligodendrocytes among glial cells.

[0048] Multiphoton microscopy, as a tool capable of subcellular resolution and deep imaging, is widely used in the study of brain structures in living animals. In recent years, 1700nm multiphoton fluorescence imaging has made significant progress in the field of in vivo mouse cerebral vascular imaging. However, its application in the study of other brain structures, especially in imaging hippocampal cells in ordinary mice, is limited. Current research is mostly limited to using transgenic mice, using fluorescent protein labeling to image hippocampal cells. This limitation is mainly reflected in two aspects: first, the biological sample range of transgenic mice is limited, preventing its widespread application to non-transgenic animal models; second, the types and number of near-infrared fluorescent markers suitable for 1700nm excitation are far fewer than traditional green fluorescent markers. Therefore, compared to the excellent depth demonstrated by 1700nm multiphoton imaging in vascular imaging, its depth and resolution in hippocampal cell imaging are significantly limited. These technical bottlenecks significantly weaken the potential and application scope of 1700nm multiphoton imaging in deep brain cell research. In the future, solving the compatibility of fluorescent markers and improving imaging depth will become important directions for further promoting the application of this technology.

[0049] In summary, the imaging devices in the related technologies have technical problems with insufficient effectiveness when imaging hippocampal cells with a depth exceeding 1.4 mm.

[0050] To resolve the above technical issues, please refer to Figure 1 and Figure 2 The live hippocampal somatic cell multiphoton imaging device of this 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 (e.g., IRDye 800CW 2-DG marker) in a first target region (e.g., a region with a depth greater than 1.4 mm and containing hippocampal somatic cells) in a live animal during the injection phase, so that the hippocampal somatic cells in the first target region are labeled.

[0052] The main function of the multiphoton imaging system is to perform multiphoton imaging on a labeled first target region (deep region) and a second target region (shallow region) during the imaging phase. Specifically, the first and second target regions are irradiated with a preset wavelength (e.g., 1665 nm, which falls within the 1700 nm band) to excite the fluorescent molecules in the labeled hippocampal cells, causing them to generate two-photon fluorescence signals, and obtaining the corresponding microscopic images. These microscopic images are used to display hippocampal cells from the surface of the brain of a live animal to a preset depth (e.g., a preset depth of 2060 μm).

[0053] It should be noted that tissues absorb and scatter light in the 1700nm band, resulting in minimal light attenuation. Therefore, using laser signals with wavelengths in the 1700nm band for multiphoton fluorescence signal excitation is the best choice for deep multiphoton imaging.

[0054] Furthermore, it should be noted that IRDye 800CW 2-DG, as a near-infrared fluorescent glucose analog, has shown great potential in labeling and imaging of mouse hippocampal somatic cells (960-2100 μm: CA1 region 960-1390 μm; dentate gyrus 1390-2100 μm). 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 effective cross-section in 1700 nm two-photon excitation. This efficient two-photon absorption characteristic provides a strong signal at lower laser power, thereby significantly improving the sensitivity and resolution of deep tissue imaging while reducing photodamage to living tissue. The hippocampus is a deep brain region, requiring extremely high tissue penetration and fluorescence stability. With its low light absorption and scattering characteristics in the near-infrared optical window, the IRDye 800CW 2-DG can provide clear imaging with a high signal-to-noise ratio in deep tissues, while its high optical stability ensures continuous and reliable signal, providing an efficient and non-invasive tool for the study of deep brain structures.

[0055] The live hippocampal somatic cell multiphoton imaging device of the present invention first injects the IRDye800CW 2-DG marker into a first target region (depth greater than 1.4 mm) of live animal hippocampal somatic cells via a brain injection system. Then, a multiphoton imaging system excites the cells at a 1700 nm wavelength to generate a two-photon fluorescence signal, and the two-photon fluorescence signal is detected to obtain a microscopic image. This microscopic image is used to display hippocampal somatic cells from the brain surface of a live animal to a predetermined depth (e.g., 2060 μm). This technical solution combines a multiphoton imaging system with a brain injection system. A more effective near-infrared fluorescent material is pre-injected into the first target region. Then, a soliton pulse signal within a specific wavelength range emitted by the multiphoton imaging system excites the region, and the resulting multiphoton fluorescence signal is microscopically imaged. This improves the effectiveness of imaging cells at different depths in the hippocampus, maintaining high efficiency and stable imaging quality even in live hippocampal cell imaging at depths exceeding 1.4 mm. Furthermore, the types of live animal brain cells labeled with the IRDye800CW 2-DG marker are verified using a second near-infrared fluorescent material (SR101 marker and ScAAV-hSyn-mCherry-WPREs marker).

[0056] In an optional embodiment of this example, the multiphoton imaging system includes a first multiphoton imaging component and a second multiphoton imaging component; the second near-infrared fluorescent material further includes SR101 marker and ScAAV-hSyn-mCherry-WPREs marker, and the second target region (shallow region) includes astrocytes and nerve cells.

[0057] This brain injection system is used to label hippocampal cells in a first target region via injection, and to label astrocytes in a second target region via smearing with SR101, a second near-infrared fluorescent material. Furthermore, it labels neurons in the second target region via intravenous injection with ScAAV-hSyn-mCherry-WPREs, a second near-infrared fluorescent material. The depth of the labeled astrocytes and neurons (i.e., the second target region) is less than the depth of the labeled hippocampal cells (i.e., the first target region), and both target regions are located within the same field of view. This ensures that target cells in both the first and second target regions at different depths in a live animal are labeled.

[0058] It should be noted that "within the same field of view" can refer to the area within a field of view of a multiphoton imaging system, meaning that 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. Also, the depth of the first target region is 0-2060 μm, and the depth of the second target region is 0-1400 μm.

[0059] The first multiphoton imaging component primarily targets the first target region for multiphoton imaging. 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 multiphoton imaging component targets the second target region, acquiring three-photon fluorescence signals and generating a microscopic image.

[0060] Through the above embodiments, multiple labeling of IRDye 800CW 2-DG marker with SR101 marker and ScAAV-hSyn-mCherry-WPREs marker was achieved, and cell colocalization imaging was realized respectively.

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

[0062] In an optional embodiment of this example, 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 stage controller 2001 controls the three-dimensional stage system 2002 based on the injection position, angle, and depth input by the user. The three-dimensional stage system 2002 controls the movement of the microneedle in preset X, Y, and Z directions to align it with the target area (hippocampal cells) of the live animal during the injection phase. The infusion pump controller 2003 applies a preset injection pressure and injects the near-infrared fluorescent material (IRDye 800CW 2-DG marker) into the first target area via the microneedle according to the preset injection time and dose. In other words, the brain injection system 200, by employing a three-dimensional stage system, a stage controller, and an infusion pump controller, ensures precise injection of the fluorescent material, improving imaging stability and repeatability.

[0064] The brain injection system 200 can also be equipped with an adjustable angle stage of ±15 degrees, combined with a flexible installation design of a 30-degree base, enabling continuous adjustment of the injection angle within a range of ±90 degrees to adapt to different experimental needs. The microneedles required for injection are prepared using a microelectrode puller (P97, Sutter), and then the dye is precisely introduced into the pulled microneedles using a 10μL pipette. Before injection, the dye-filled microneedles are pressure-tested to check their integrity and whether the dye can be smoothly expelled. If the microneedles cannot inject dye normally in air, it indicates that they cannot function properly in brain tissue and therefore need to be replaced or repaired. After confirming the microneedles are in good condition, their movement is precisely controlled using a three-dimensional translation stage. When the microneedles are inserted into the brain, special attention must be paid to adjusting the angle stage of the injection system to ensure that the angle between the microneedles and the platform matches the angle of the translation stage. If the angles do not match, the microneedles will enter the brain laterally, which not only fails to achieve precise injection but also causes serious damage to brain tissue. Therefore, ensuring precise calibration of the system angle is crucial for achieving high-quality brain injection procedures.

[0065] In addition, to achieve imaging of live hippocampal somatic cells, an injection procedure was performed on the brain. Specifically, a three-dimensional displacement stage was precisely controlled to ensure smooth advancement of the microneedle during brain insertion. During this process, the microneedle was not stopped at a specific depth; instead, positive pressure (ranging from 4-5 bar) was continuously applied. When the needle tip reached a depth of 2200 μm below the brain surface, the positive pressure was stopped, and the needle tip was slowly withdrawn. The entire injection process, from start to finish, including closing the cranial window, takes approximately 30 to 60 minutes. This time provides sufficient time for the injected IRDye 800CW 2-DG to diffuse within the brain tissue and achieve effective labeling of hippocampal somatic cells.

[0066] The following describes the devices included in a multiphoton imaging system:

[0067] In an optional embodiment of this invention, 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; and the optical focusing assembly is used to focus the high-energy soliton laser signal onto the first and second target regions that have been marked in the live animal to excite two-photon fluorescence signals and three-photon fluorescence signals.

[0068] The fiber laser 10 may be a femtosecond pulsed laser (FLCPA-02CSZU, Calmar laser) capable of emitting a wavelength of 1550nm and a repetition frequency of 1MHz, which is used to generate a laser signal of a first wavelength (the wavelength may be 1550nm) 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. Based on the soliton self-frequency shift effect, the rod-shaped photonic crystal fiber converts a 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 (e.g., 1665 nm, which falls within the target wavelength range of -1700 nm), and transmits it to the optical focusing component 30. Specifically, the photonic crystal fiber utilizes its special waveguide structure to broaden the laser signal of the first wavelength through nonlinear effects and generate a soliton pulse signal of the second wavelength (spectral representation as shown in the image). Figure 3 (As shown). Optimized fiber parameters ensure that the second wavelength of the high-energy soliton laser signal is stable at 1665 nm, while having sufficient pulse energy to meet the excitation requirements of subsequent multiphoton fluorescence signals.

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

[0071] In an optional embodiment of this example, the multiphoton 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 multiphoton imaging component 40 and the second multiphoton 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 multiphoton imaging component 40 via a preset optical path for efficient detection and image acquisition. Simultaneously, the three-photon fluorescence signal is transmitted to the second multiphoton imaging component 50 via another preset optical path to achieve high-resolution imaging.

[0073] In an optional embodiment of this example, the multiphoton 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 it at different angles, so as to adapt it to the needs of the optical path system and subsequent optical components. By optimizing the polarization state, the energy utilization rate of the laser when passing through the optical components 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 between P-polarized light and S-polarized light, so that P-polarized light is transmitted and S-polarized light is reflected, thereby achieving optical path optimization and beam separation. This function helps to enhance signal contrast, reduce background light interference, and improve the detection sensitivity of fluorescence signals.

[0076] The first lens group 130 is used to focus and shape the light beam to ensure that the optical signal maintains optimal optical quality during transmission, thereby improving the resolution and signal collection efficiency of the multiphoton imaging system.

[0077] In summary, the multiphoton imaging system uses a half-wave plate 110, a polarization beam splitter 120, and a 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, thereby improving the stability of the laser signal before it enters the photonic crystal fiber 20.

[0078] In an optional embodiment of this example, the multiphoton 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 divergent characteristics 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 high-energy soliton laser signals, primarily because high-energy soliton laser signals may contain high-frequency noise components. If directly used for two-photon or three-photon excitation, this could affect the stability of the fluorescence signal. Therefore, the low-pass filter 150 removes unnecessary high-frequency components, retaining the effective excitation wavelength range suitable for bioimaging and ensuring 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 multiphoton microscopy relies on high-power ultrafast pulsed lasers, excessively high laser power 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 imaging, while optimizing the signal-to-noise ratio of fluorescence signal acquisition.

[0082] In an optional embodiment of this example, the first multiphoton imaging component 40 is a first photomultiplier tube (H7422-50, Hamamatsu). The first photomultiplier tube, together with the first bandpass filter 170 (FF01-855 / 210-25, Semrock), detects the two-photon fluorescence signal corresponding to the first target region to obtain a microscopic image corresponding to the two-photon fluorescence signal (2PF).

[0083] In an optional embodiment of this example, the second multiphoton imaging component 50 is a second photomultiplier tube. The second photomultiplier tube (H7422p-50, Hamamatsu) works in conjunction with the second bandpass filter 180 (FF01-630 / 92-25, Semrock) to detect the three-photon fluorescence signal (3PF) corresponding to the second target region, thereby obtaining a microscopic image corresponding to the three-photon fluorescence signal.

[0084] In an optional embodiment of this example, the optical focusing assembly 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 the preset scanning path, the scanning lens is used to perform the first stage focusing of the high-energy soliton laser signal in the scanning path, and expands the high-energy soliton laser signal to a beam ratio of 1:4 with the sleeve lens, and the water immersion objective 80 is used to perform the second stage focusing of the expanded high-energy soliton laser signal to focus it on the labeled cells in the first target region and the second target region in the live animal, and generate three-photon fluorescence signal and / or two-photon fluorescence signal.

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

[0087] In an optional embodiment of this example, the entire multiphoton imaging system may further include a reflector 301 and a mirror 305.

[0088] Specifically, reflector 301 can be selectively positioned at the output end of neutral density filter 160 to change the direction of the laser beam path, reflecting the high-energy soliton laser signal filtered by neutral density filter 160 to scanning galvanometer 302, facilitating subsequent beam scanning and sample excitation. Its high reflectivity surface effectively reduces light loss and ensures the stability of energy transmission of the excitation light. Reflector 305 primarily functions as a beam path adjuster in the optical system, ensuring that the laser enters the immersion objective 80 along a predetermined trajectory, achieving precise live-body imaging.

[0089] It should be noted that the live animal surgical procedures involved in the embodiments of this application include the following: All animal experiments were conducted in accordance with the "Guidelines for the Care and Use of Experimental Animals of Shenzhen University" and approved by the Animal Ethics Committee of the School of Medicine, Shenzhen University (Approval No.: IACUC-202300036). All mice were obtained from the Guangdong Provincial Medical Experimental Animal Center, China. Adult female mice (C57BL / 6J, 8-10 weeks old) were used for imaging. Mice were anesthetized with a gas anesthesia system (Matrix VIP3000, Midmark) and isoflurane. The animal body temperature was maintained at 36.5℃ using a heating pad, and 50 μL of h20 ... -1A 5% glucose solution was administered. A craniotomy with a diameter of 3 mm was performed, centered 2 mm lateral to the reticular fossa. A self-made metal sheet was tightly bonded to the skull with dental cement, and the cranial window was sealed with a 5 mm diameter coverslip. Before imaging, mice were injected with ~1 μL LIRDye 800CW 2-DG to label hippocampal somatic cells at the center of the cranial window. SR101 at a concentration of 1 mmol / L was applied to the surface of the mouse brain, left for 5 minutes, and then rinsed with physiological saline to label astrocytes; 200 μL ScAAV-hSyn-mCherry-WPREs were injected into the orbit to label nerve cells.

[0090] The live hippocampal somatic cell multiphoton imaging device described in this application can be used to perform imaging experiments on IRDye 800CW 2-DG mice that have undergone brain injection labeling. In specific implementation, the excitation light power is controlled by a variable medium-density attenuation plate (Thorlabs, model NDC-50-4M), thereby enabling imaging of brain cells at different depths under different power conditions (e.g., ...). Figure 4 (As shown). At an excitation wavelength of 1665 nm, hippocampal somatic cell imaging from the surface of the mouse brain to a depth of 2060 μm was successfully achieved.

[0091] Through a specific implementation process, a three-dimensional reconstruction of 2PF cell imaging images at a depth of 2100 μm below the brain surface was obtained. Figure 4 (Left side of the brain). At a depth of 2 mm to the side and posterior to the fontanelle, the depth of the subsurface white matter (WM) layer in the mouse brain ranges from 800 to 960 μm, and the hippocampal regions range from CA1 (960-1390 μm) to the dentate gyrus (1390-2060 μm). Figure 4 The right side (af) shows two-dimensional 2PF images at different imaging depths below the brain surface: (a) cells of the cerebral cortex; (b) cells of the white matter layer; (c) cells of the CA1 region of the hippocampus; (df) cells of the dentate gyrus of the hippocampus.

[0092] Specific implementation results: The deepest cell that this imaging technology can resolve is located 2060 μm below the surface of the mouse brain. Figure 4 (f) on the right side. This indicates that under 1665nm excitation conditions, 2PF imaging can penetrate the entire neocortex and white matter (WM), and further extend to the bottom of the hippocampus, providing strong technical support for deep brain tissue imaging.

[0093] In this embodiment, mice that had been labeled with IRDye 800CW 2-DG cells were subjected to multiplex cell labeling using SR101 and ScAAV-hSyn-mCherry-WPREs, respectively. Simultaneously, 2PF and 3PF imaging was performed on brain cells at different depths under different power conditions and at 1665nm wavelength excitation.

[0094] Specific implementation results showed that IRDye 800CW 2-DG labeled cells (such as...) were observed at a depth of 196 μm below the surface of the mouse brain. Figure 5 (b) shows the colocalization of cells with SR101-labeled cells (as shown in the image). Figure 5 (as shown in (a)). SR101 is known for its excellent labeling specificity and is mainly used for the specific labeling of astrocytes. This specific implementation result demonstrates that IRDye 800CW2-DG can label astrocytes. This discovery lays the technical foundation for further research on the distribution and function of deep astrocytes in the hippocampus. Combining the multiphoton imaging advantages of IRDye 800CW 2-DG in the 1700nm band, high-resolution, deep multiphoton imaging of astrocytes in the hippocampus can be achieved in the future, advancing research on the function of glial cells in deep brain structures.

[0095] Furthermore, at a depth of 294 μm below the brain surface, experiments observed IRDye 800CW 2-DG (such as...) Figure 5 (d) shown) and ScAAV-hSyn-mCherry-WPREs-labeled cells (e.g.) Figure 5 (c) shows the colocalization. ScAAV-hSyn-mCherry-WPREs are a tool for specifically labeling mature neurons and are widely used to study neuronal distribution, function, and network connectivity. The colocalization results indicate that IRDye800CW 2-DG can label not only astrocytes but also mature neurons, and its labeling ability in tissues exhibits good stability and specificity.

[0096] This application's embodiments comprehensively validate the dual function of IRDye 800CW 2-DG in labeling astrocytes and mature neurons through specific implementation results. Furthermore, combined with its multiphoton imaging characteristics in the 1700nm band, it demonstrates its broad application potential in imaging studies of deep brain regions. Particularly in the study of deep structures such as the hippocampus, this probe's advantage lies in its ability to penetrate the cortex and white matter layer, reaching deep into the basal region of the hippocampus and achieving simultaneous imaging of astrocytes and neurons. This provides powerful tool support for exploring the interactions and functional connections of different cell types in deep brain regions, opening up new directions for brain science research.

[0097] In summary, this application's embodiments detailed the application performance and cell labeling potential of IRDye 800CW 2-DG in 1700nm multiphoton imaging. Co-localization labeling of astrocytes and mature neurons was achieved by combining SR101 and ScAAV-hSyn-mCherry-WPREs, and experimental results validated the dual labeling capability and deep imaging stability of IRDye 800CW 2-DG. The study shows that IRDye 800CW 2-DG can penetrate the neocortex and white matter layer, providing high-resolution multiphoton imaging of the basal hippocampus, offering powerful tool support for the visualization of deep brain structures. In the future, IRDye 800CW 2-DG may become a key tool for studying deep brain cell interactions and neural network pathological mechanisms.

[0098] The live hippocampal somatic cell multiphoton imaging device of the present invention first injects a first near-infrared fluorescent material into a first target region (for labeling hippocampal somatic cells) of a live animal via a brain injection system. Then, the first target region is imaged using a multiphoton imaging system, ultimately obtaining a microscopic image corresponding to the two-photon fluorescence signal. In other words, this technical solution improves the effectiveness of hippocampal somatic cell imaging by combining a multiphoton imaging component with a brain injection system, thereby maintaining high efficiency and stable imaging quality even in live hippocampal somatic cell imaging at depths exceeding 1.4 mm (its beneficial effects have been demonstrated by experimental data).

[0099] As can be seen, this invention combines 1700nm multiphoton microscopy, IRDye 800CW 2-DG fluorescent labeling, and brain injection technology to explore its potential application in cell imaging of deep structures in the mouse brain, especially the hippocampus. Furthermore, multiple labeling of IRDye 800CW 2-DG with SR101 and ScAAV-hSyn-mCherry-WPREs was achieved, enabling co-localization imaging of cells. The application of brain injection technology significantly improved the accuracy of labeling, allowing the fluorescent probe to accurately target the brain region while avoiding interference with surrounding tissues. Moreover, combined with the characteristics of IRDye 800CW 2-DG, this method overcomes the limitations of traditional labeling methods, providing important support for efficient labeling and imaging of the hippocampus. This technique, which combines brain injection with 1700nm multiphoton imaging, provides new tools to support the study of cell type distribution, function, and interactions 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 different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0101] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A live hippocampal somatic cell multiphoton imaging device, characterized in that, The live hippocampal somatic cell multiphoton imaging device includes a multiphoton imaging system and a brain injection system; The brain injection system is used to label a first target region of a live animal with a first near-infrared fluorescent substance; wherein the first near-infrared fluorescent substance includes IRDye 800CW 2-DG marker, and the first target region includes hippocampal cells. The multiphoton imaging system is used to perform multiphoton imaging on the first target region that has been marked, and to 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 to a predetermined depth of a live animal.

2. The live hippocampal somatic cell multiphoton imaging device as described in claim 1, characterized in that, The multiphoton imaging system includes a first multiphoton imaging component and a second multiphoton imaging component; the second near-infrared phosphor also includes SR101 markers and ScAAV-hSyn-mCherry-WPREs markers. The brain injection system is used to label the IRDye 800CW 2-DG marker into the hippocampal cells in the first target region by injection. The SR101 marker is applied to astrocytes in the second target region by smearing, and the ScAAV-hSyn-mCherry-WPREs marker is applied to nerve cells in the second target region by intravenous injection. The depth of the second target region is less than the depth of the first target region, and the first target region and the second target region are located in the same field of view. The first multiphoton imaging component is used to perform multiphoton imaging on the marked first target region and obtain a microscopic image corresponding to the excited two-photon fluorescence signal. The second multiphoton imaging component is used to perform multiphoton imaging on the marked second target region and obtain a microscopic image corresponding to the excited three-photon fluorescence signal.

3. The live hippocampal somatic cell multiphoton imaging device as described in 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 microneedle in the preset X, Y and Z directions to align it with the hippocampal cells of the live animal at different injection stages. 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 a preset injection time and dose.

4. The live hippocampal somatic cell multiphoton imaging device as described in claim 2, characterized in that, The multiphoton imaging system also includes a fiber laser, a photonic crystal fiber, and an optical focusing assembly; 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 with a preset second wavelength; wherein the second wavelength is greater than the first wavelength and is within the target wavelength range; The optical focusing component is used to focus the high-energy soliton laser signal onto the first and second target regions that have been labeled in the living animal cells, so as to excite two-photon fluorescence signals and three-photon fluorescence signals.

5. The live hippocampal somatic cell multiphoton imaging device as described in claim 4, characterized in that, The multiphoton imaging system also includes a beam splitter; The beam splitter is used to transmit the three-photon fluorescence signal and the two-photon fluorescence signal to the first multiphoton imaging component and the second multiphoton imaging component respectively according to a preset path.

6. The live hippocampal somatic cell multiphoton imaging device as described in claim 4, characterized in that, The multiphoton imaging system also includes a half-wave plate, a polarizing 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 beams with different polarization directions, and the first lens group is used to focus and shape the laser signal.

7. The live hippocampal somatic cell multiphoton imaging device as described in claim 6, characterized in that, The multiphoton imaging system also 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 live hippocampal somatic cell multiphoton imaging device as described in claim 4, characterized in that, The first multiphoton imaging component is a first photomultiplier tube. The first photomultiplier tube, in conjunction with a first bandpass filter, detects the two-photon fluorescence signal to obtain a microscopic image corresponding to the two-photon fluorescence signal.

9. The live hippocampal somatic cell multiphoton imaging device as described in claim 8, characterized in that, The second multiphoton imaging component is a second photomultiplier tube. The second photomultiplier tube, in conjunction with a second bandpass filter, detects the three-photon fluorescence signal to obtain a microscopic image corresponding to the three-photon fluorescence signal.

10. The live hippocampal somatic cell multiphoton imaging device as described in claim 4, characterized in that, The optical focusing assembly includes a scanning galvanometer, a scanning lens, a sleeve lens, and a water immersion objective. 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 focus the high-energy soliton laser signal in the scanning path in the first stage, and together with the sleeve lens, expands the high-energy soliton laser signal. The water immersion objective is used to perform a second-stage focusing of the expanded high-energy soliton laser signal to focus on the hippocampal cells of the first target region, the astrocytes of the second target region, and the nerve cells in a living animal, and to generate three-photon fluorescence signals and two-photon fluorescence signals.

Images