Imaging method and system, storage medium and computing device

Abstract

The invention provides an imaging method and system, a storage medium and computing device.The method comprises the steps that an oxygen-sensitive film which is prepared in advance and provided with an oxygen-sensitive probe is installed on to-be-detected tissue of a to-be-detected object; the to-be-detected tissue contains specific tissue cells sensitive to calcium signals; alternately irradiating the multiple types of exciting light on the to-be-detected tissue according to a target interval through the imaging path; according to the target interval, different cameras are used for continuously collecting all frames of first exciting light signals on the oxygen sensitive film, all frames of second exciting light signals of the specific tissue cells and all frames of reflected light signals of blood oxygen in the tissue to be detected; generating a tissue oxygen partial pressure image of the to-be-detected tissue according to the continuously collected frames of first excitation light signals, generating a calcium signal image of the to-be-detected tissue according to the continuously collected frames of second excitation light signals, and generating a blood oxygen signal image of the to-be-detected tissue according to the continuously collected frames of reflected light signals.

Description

Technical Field

[0001] This application relates to the field of biomedical imaging technology, and more specifically, to an imaging method, system, storage medium, and computing device. Background Technology

[0002] The normal functioning of various internal physiological tissues in an organism often depends on the coordinated and precise physiological activities within those tissues. To deeply understand the impact of different tissue diseases on internal physiological tissues, it is necessary to collect data on changes in physiological activity within the tissues during the occurrence of disease. These changes typically require imaging techniques for direct and quantifiable observation. For example, the normal functioning of brain tissue requires precise coordination between neural activity, blood supply, and energy metabolism. To deeply understand the pathological mechanisms of brain diseases such as epilepsy and stroke, it is necessary to observe the complete causal chain among these three factors. Observing this causal chain is usually achieved by imaging the physiological activities within brain tissue.

[0003] Because the generation and changes of diseases have a chain effect on physiological activities, existing technologies often rely on different imaging devices to image different physiological activities separately, and then perform comprehensive analysis based on the results of each imaging session to collect data on changes in physiological activities within physiological tissues. However, this approach has significant bottlenecks in terms of integration, spatiotemporal resolution, and long-term stability, resulting in considerable drawbacks. Summary of the Invention

[0004] In view of this, this application provides an imaging method, system, storage medium, and computing device that significantly improves the integration, spatiotemporal resolution, and long-term stability of tissue imaging by simultaneously performing tissue imaging in multiple modalities.

[0005] Specifically, this application is implemented through the following technical solution: In a first aspect, embodiments of this application provide an imaging method, including: An oxygen-sensitive film with a pre-prepared oxygen-sensitive probe is mounted on the tissue to be tested; the tissue to be tested contains specific tissue cells that are sensitive to calcium signals. Through the imaging pathway, multiple types of excitation light are alternately irradiated onto the tissue to be detected according to the target interval; According to the target interval, different cameras are used to continuously acquire each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected. Based on the first excitation light signal acquired in each consecutive frame, a tissue oxygen partial pressure image of the tissue to be detected is generated; based on the second excitation light signal acquired in each consecutive frame, a calcium signal image of the tissue to be detected is generated; and based on the reflected light signal acquired in each consecutive frame, a blood oxygen signal image of the tissue to be detected is generated.

[0006] Secondly, embodiments of this application also provide an imaging system, the system including an oxygen-sensitive thin film and an imaging unit, the imaging unit including an illumination module, an acquisition module and a generation module; The oxygen-sensitive membrane is used to be installed on the tissue to be tested; the tissue to be tested has specific tissue cells that are sensitive to calcium signals. The illumination module is used to alternately irradiate the tissue to be detected with multiple types of excitation light at target intervals through the imaging path; The acquisition module is used to continuously acquire, according to the target interval, each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected using different cameras. The generation module is used to generate a tissue oxygen partial pressure image of the tissue to be detected based on each frame of the first excitation light signal acquired continuously, to generate a calcium signal image of the tissue to be detected based on each frame of the second excitation light signal acquired continuously, and to generate a blood oxygen signal image of the tissue to be detected based on each frame of the reflected light signal acquired continuously.

[0007] Thirdly, an optional implementation of this application also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, performs the steps described in the first aspect above.

[0008] Fourthly, an optional implementation of this application also provides a computer device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the program to perform the steps described in the first aspect above.

[0009] The imaging method, system, storage medium, and computing device provided in this application provide a basis for simultaneous and long-term acquisition of three modalities of information: oxygen partial pressure, blood supply, and calcium content of specific tissue cells, by mounting a pre-prepared flexible oxygen-sensitive film integrating oxygen-sensitive probes onto the tissue to be detected. Through the imaging pathway, excitation light of different wavelengths is alternately irradiated onto the same area of ​​the tissue to be detected at target time intervals, achieving separation of different wavelength optical signals in the time dimension, laying the foundation for subsequent differentiation between the acquired excitation and reflection signals. According to the target interval, different cameras are used to continuously and synchronously acquire the first excitation signal from the oxygen-sensitive film, the second excitation signal from specific tissue cells, and the reflection signal from the tissue to be detected, ensuring efficient separation and synchronous capture of the three signals and effectively avoiding interference from spectral overlap. Finally, based on the continuously acquired excitation and reflection signals, tissue oxygen partial pressure signal images, calcium signal images, and blood oxygenation signal images are generated synchronously, achieving multimodal synchronous and long-term continuous imaging. In summary, by integrating a flexible oxygen-sensitive film with an oxygen-sensitive probe into the tissue to be tested, which carries specific tissue cells, and by designing alternating irradiation with excitation light of various wavelengths according to target intervals, and by efficiently and collaboratively acquiring and imaging excitation and reflection signals, high spatiotemporal resolution and long-term stability synchronous real-time imaging of various physiological activities reflected by various signals within the tissue to be tested are achieved. Furthermore, analysis and detection are performed based on the synchronously obtained multimodal images. This overcomes the technical bottleneck of existing tissue imaging technologies, which rely on independent acquisition of a single modality and then integration of multimodal images for analysis and detection, thus failing to completely and accurately capture the precise physiological activities within the components to be tested.

[0010] For a description of the effects of the aforementioned imaging system, computer-readable storage medium, and computer equipment, please refer to the description of the imaging method above; it will not be repeated here.

[0011] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0012] Figure 1 This is a flowchart illustrating a tissue imaging method according to an exemplary embodiment of this application; Figure 2 This is a schematic diagram illustrating an exemplary embodiment of the present application of an oxygen-sensitive thin film installation. Figure 3 This is a schematic diagram illustrating an imaging module according to an exemplary embodiment of this application; Figure 4This is a schematic diagram illustrating a specific process for acquiring different optical signals, as shown in an exemplary embodiment of this application. Figure 5 This is a schematic diagram of a composite structure of dendritic mesoporous silica nanoparticles loaded with an oxygen-sensitive probe, as illustrated in an exemplary embodiment of this application. Figure 6 This is a schematic diagram illustrating the specific preparation process of an oxygen-sensitive thin film according to an exemplary embodiment of this application; Figure 7 This is a schematic diagram illustrating the effect of an oxygen-sensitive thin film prepared according to an exemplary embodiment of this application; Figure 8 This is a schematic diagram illustrating an imaging system according to an exemplary embodiment of this application; Figure 9 This is a schematic diagram illustrating a specific application implementation of the imaging method and system provided in this application, as shown in an exemplary embodiment of this application. Figure 10 This is a schematic diagram illustrating a specific application implementation two of the imaging method and system provided in this application, as shown in an exemplary embodiment of this application. Figure 11 This is a schematic diagram of the structure of a computer device shown in an exemplary embodiment of this application. Detailed Implementation

[0013] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0014] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0015] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0016] Research has revealed that the normal functioning of brain tissue, as a highly complex internal organ, relies on the close coupling of neural electrical activity, local blood flow supply, and cellular energy metabolism. Therefore, to deeply elucidate the pathological mechanisms of brain diseases such as epilepsy, stroke, neurodegenerative diseases, and even brain tumors, it is essential to rely on techniques for synchronous, real-time, and long-term observation of the dynamic, multi-scale, and mutually causal physiological chain of "neural activity-vascular response-energy metabolism." Such techniques must be able to simultaneously capture calcium signals (neuronal firing), blood oxygen changes (neurovascular coupling), and brain tissue oxygen partial pressure (tissue energy metabolism level). However, existing imaging techniques are mostly limited to single-modality signal capture, or suffer from significant bottlenecks in integration, spatiotemporal resolution, and long-term stability, making it difficult to meet the requirements for synchronous capture. Moreover, existing imaging techniques have significant limitations when capturing signals under various individual modalities. For example, in calcium signal monitoring (i.e., neural activity monitoring), two-photon or wide-field fluorescence imaging based on gene-encoded calcium indicator proteins (such as the green fluorescent calmodulin-M13 peptide fusion protein (GCaMP)) has become mainstream. This technology indirectly reflects neuronal firing by detecting changes in fluorescence intensity induced by calcium ion influx. However, this method is limited to acquiring calcium signals associated with neuronal activity and cannot access metabolic states. In blood oxygenation monitoring, blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) and near-infrared spectroscopy (NIRS) are two widely used blood oxygenation detection techniques. Although BOLD-fMRI can achieve whole-brain imaging based on the paramagnetic effect of deoxyhemoglobin, it suffers from problems such as expensive equipment, low spatiotemporal resolution due to indirect contrast mechanisms based on blood oxygen sensitivity, and inability to distinguish between neural activity and vascular signals. While NIRS technology utilizes the absorption difference of oxygenated / deoxygenated hemoglobin in the near-infrared band (such as around 730 nm) for detection, and has the advantages of being portable and low-cost, it suffers from low spatial resolution due to shallow penetration depth. More importantly, both blood oxygenation monitoring methods are indirect measurement methods. Although they can provide important information on neural activity and various diseases, they are difficult to achieve quantitative blood oxygenation detection, which is crucial for cellular metabolic levels.In the field of oxygen partial pressure monitoring, while technologies such as electron paramagnetic resonance (EPR) and positron emission tomography (PET) can directly detect oxygen molecule concentration or consumption rate, they generally rely on exogenous probes or tracers, resulting in bulky and expensive equipment with extremely low temporal resolution (typically on the order of minutes to hours). Furthermore, limitations such as tracer metabolism, phototoxicity, or radioactivity may prevent their use for long-term, high-frequency dynamic observations. Currently, a foundation for long-term, stable imaging of tissue oxygen partial pressure has not been established, thus necessitating a low-cost imaging technique capable of long-term, stable acquisition. Further research has revealed that even in the field of optical sensing capable of directly detecting oxygen molecules, high-performance phosphorescent oxygen-sensitive probes, such as ruthenium (Ru) complexes, are typically only used in solution form for in vitro testing due to issues such as molecular hydrophilicity and aggregation quenching in the solid state. It is difficult to fabricate solid-state sensing films that can be stably attached to living tissues for extended periods. The lack of this key material form poses a fundamental obstacle to the direct and stable integration of high-performance oxygen-sensitive probes into tissue interfaces for long-term in-situ monitoring.

[0017] Meanwhile, at the system level, the systematic integration of multimodal imaging technologies through hardware not only faces physical and cost limitations, but also suffers from spectral overlap between excitation and reflection spectra when combining blood oxygenation and fluorescence imaging, resulting in severe crosstalk and signal-to-noise ratio degradation. Therefore, there is an urgent need for an imaging system capable of simultaneously realizing tissue oxygen partial pressure imaging, calcium fluorescence imaging, and blood oxygenation imaging.

[0018] Based on the above research, this application proposes an integrated multimodal tissue imaging method based on a flexible oxygen-sensitive film and multi-spectral temporal separation imaging. At the material level, a hydrophilic ruthenium-based phosphorescent oxygen-sensitive probe is loaded onto dendritic mesoporous silica nanoparticles as a carrier, and a flexible oxygen-sensitive film that can be adhered to the surface of the tissue to be tested is prepared, solving the problem of the immobilization stability and functional durability of high-performance oxygen-sensitive probes in a living environment. At the system level, through an optical architecture of multi-wavelength excitation at target intervals, precise spectral separation, and multi-channel synchronous acquisition, the precise spatiotemporal separation and synchronous acquisition of calcium fluorescence, oxygen phosphorescence, and blood oxygen reflection signals are achieved, fundamentally avoiding multimodal signal crosstalk. In application, the oxygen-sensitive film is adhered to the tissue to be tested, and tissue imaging is performed using the above optical architecture. High spatiotemporal resolution images of tissue oxygen partial pressure, calcium, and blood oxygen signals can be obtained simultaneously, thus fully revealing the dynamic coupling relationship of "neural activity-vascular response-energy metabolism," providing an unprecedented integrated observation method for the study of brain disease mechanisms.

[0019] The shortcomings of the above solutions are the result of the inventor's practical experience and careful research. Therefore, the discovery process of the above problems and the solutions proposed in this application below should be considered as the inventor's contributions to this application.

[0020] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0021] It is understood that before using the technical solutions disclosed in the various embodiments of this application, users should be informed of the types, scope of use, and usage scenarios of the personal information involved in this application in an appropriate manner in accordance with relevant laws and regulations, and user authorization should be obtained.

[0022] To facilitate understanding of this embodiment, a detailed description of the imaging method disclosed in this application embodiment will be provided first. The imaging method provided in this application embodiment is generally executed by a terminal device or other processing device with certain computing capabilities. The terminal device can be a user equipment (UE), mobile device, user terminal, terminal, personal digital assistant device (PDA), handheld device, computer device, etc. In some possible implementations, the imaging method can be implemented by the processor calling computer-readable instructions stored in the memory.

[0023] The imaging method provided in this application embodiment will be described below using the imaging client as an example of the executing entity.

[0024] like Figure 1 The flowchart shown is a tissue imaging method provided in an embodiment of this application, which may include the following steps: S101: The oxygen-sensitive membrane with the pre-prepared oxygen-sensitive probe is installed on the tissue to be tested; the tissue to be tested contains specific tissue cells that are sensitive to calcium signals.

[0025] Here, an oxygen-sensitive probe is a molecular probe that can specifically respond to changes in the concentration of oxygen in the environment through changes in its optical signal intensity. Specifically, this application may use tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, i.e. , as an oxygen-sensitive probe.

[0026] Specifically, the oxygen-sensitive probe is a metal-organic complex that detects oxygen based on the phosphorescence quenching effect. When excited by light of a specific wavelength, the oxygen-sensitive probe molecules are excited to a triplet excited state and emit phosphorescence with a peak wavelength of approximately 630 nanometers. Oxygen molecules in the environment act as efficient quenchers, inducing nonradiative transitions in the excited probe molecules through collisions, resulting in a significant decrease in phosphorescence intensity as oxygen partial pressure increases. This process establishes a calibrable, quantitative inverse relationship between phosphorescence intensity and oxygen partial pressure, enabling optical quantitative measurement of the local oxygen partial pressure in the tissue being tested.

[0027] The oxygen-sensitive film contains the aforementioned oxygen-sensitive probe ( The thin film, which is solid, ultrathin (approximately tens of micrometers thick), uniform, and flexible, can serve as an implantable optical sensing interface, mounted (attached or implanted) on the living tissue to be tested for long-term, in-situ, and real-time oxygen partial pressure monitoring. The pre-preparation process for the oxygen-sensitive thin film with the oxygen-sensitive probe will be detailed later and will not be repeated here.

[0028] The subject of the study can be a living organism with the necessary physiological activity for research. It may have the capability for implantation of an oxygen-sensitive membrane, and the internal tissues may contain specific calcium-sensitive cells and a pre-defined pathological state. For example, the subject may include, but is not limited to, specific animals such as live rats / rat or live rabbits. By simulating a disease environment within the tissues of the subject and conducting multimodal optical imaging studies, multimodal information detection under specific diseases can be achieved. Specific diseases include, for example, epilepsy simulated through electrical stimulation or drug induction, focal cerebral ischemia simulated through transient middle cerebral artery occlusion (tMCAO), and intracranial tumors simulated through implantation of glioma cell line 261 (GL261). Specific tissue cells may include those expressing a green fluorescent calcium indicator based on M13 peptide and protein (Variant 6, fast kinetics, GCaMP6f) or similar calcium indicator proteins. Understandably, within the scope of this application, the target organism can be further expanded to include biological systems sensitive to calcium signals, including but not limited to various model organisms such as zebrafish, provided their tissue structure and physiological characteristics are suitable for implementing the multimodal imaging method provided in this application.

[0029] The tissue to be tested is a living tissue region within the subject body exhibiting a predetermined pathological state. This tissue must contain specific cells sensitive to calcium signals (calcium fluorescence characteristics, excitation light at 470 nm, emission light at 530 nm) and simultaneously meet the requirements for three optical modal detections: oxygen partial pressure sensing, calcium signal acquisition, and blood oxygenation detection. A key characteristic of the tissue is that it must contain specific cells expressing calcium-sensitive fluorescent proteins (such as GCaMP), thereby providing detectable optical signals for calcium transients related to neural activity. Simultaneously, the tissue must possess suitable anatomical structure and physiological state to allow the oxygen-sensitive membrane to be stably mounted (e.g., adhered to or implanted) on its surface or superficial layer, ensuring stable optical and physical coupling between the membrane and the tissue. Preset pathological states include, but are not limited to, states induced by hypoxia, anesthesia, stroke, tumors, or epileptic activity.

[0030] For example, the tissues to be tested include, but are not limited to, cerebral cortex tissue with a preset pathological state of the subject to be tested, other brain region tissues (such as hippocampus, thalamus, etc.), and specific structural tissues in the peripheral nervous system (such as retina), gastrointestinal tissue, tumor area tissue, and cardiac and cardiovascular-related neural tissue.

[0031] In specific implementation, a pre-prepared oxygen-sensitive probe ( After the flexible oxygen-sensitive film is applied, a mounting window for the oxygen-sensitive film can be set according to the location of the tissue to be detected. The oxygen-sensitive film, loaded on a specific carrier, is then tightly attached to the tissue through a safety window to lay the foundation for subsequent imaging. The specific carrier can be, for example, a glass slide or glass plate, and the mounting window can be, for example, a cranial window corresponding to brain tissue. The shape and size of the specific carrier are consistent with the safety window, and the size of the mounting window can be set empirically; this application does not impose specific limitations. The size of the oxygen-sensitive film can be less than or equal to the size of the specific carrier. The specific carrier serves two purposes: firstly, to isolate the tissue from the external environment, and secondly, to allow light to pass through, thereby enabling the acquisition of the excitation light signal from the oxygen-sensitive film.

[0032] Optionally, in order to ensure the reliability and continuity of imaging results during multimodal imaging, the object to be detected can be fixed so that the tissue to be detected covered with an oxygen-sensitive membrane is fully exposed under the imaging module.

[0033] Alternatively, if the oxygen-sensitive film is not loaded onto a specific carrier, after the oxygen-sensitive film is tightly mounted onto the tissue to be tested through the mounting window, a glass slide matching the window can be installed at the mounting window. On the one hand, the cover glass acts as a sealed physical barrier, effectively isolating the external environment, preventing the tissue to be tested from being infected by bacteria or viruses, and maintaining its normal physiological pressure and humidity environment, ensuring the stability of the vital signs of the subject under long-term oxygen-sensitive film mounting. On the other hand, by using a transparent material with high optical quality and a specific thickness (such as a cover glass), while achieving effective sealing, the glass slide can minimize the absorption, scattering, and refraction interference of various wavelengths of excitation and emission light, providing a stable, consistent, and low-distortion optical interface for subsequent multimodal optical imaging. This ensures a clear and reliable optical path from the surface of the tissue to the image, meeting the technical requirements of high-precision, long-term dynamic imaging.

[0034] like Figure 2 The diagram shown is a specific schematic diagram of the installation of an oxygen-sensitive membrane according to an embodiment of this application. A transgenic mouse expressing the GCaMP6f calcium indicator protein is used as the test subject. A craniotomy is performed on the test subject to expose its cerebral cortex tissue as the test tissue. A pre-prepared oxygen-sensitive membrane containing Ru(dpp)3Cl2 is installed inside the craniotomy of the mouse, ensuring it adheres tightly to the surface of the mouse's cerebral cortex. A glass slide perfectly matching the size of the craniotomy is then placed over the oxygen-sensitive membrane. This completes the installation of the oxygen-sensitive membrane on the test tissue (cerebral cortex tissue) of the test subject (transgenic mouse).

[0035] S102: Through the imaging pathway, multiple types of excitation light are alternately irradiated onto the tissue to be detected according to the target interval.

[0036] Here, the imaging path can be located within the imaging unit, which can be an imaging system comprising a tri-color Köhler illumination module, a polarization beam splitter module, and at least two cameras. The imaging path is the path composed of the tri-color Köhler illumination module and the polarization beam splitter module within the imaging unit. The tri-color Köhler illumination module of the imaging unit is constructed based on the Köhler illumination principle to achieve uniform and controllable multi-wavelength excitation; the polarization beam splitter module achieves physical isolation between the excitation and emission light paths, thereby maximizing the signal-to-noise ratio of signal acquisition. The at least two cameras are used to achieve synchronous, parallel, and optimized acquisition of different modal optical signals at the physical detection level.

[0037] The multiple types of excitation light include at least three types of excitation light with different wavelengths and spectral separation, used to excite optical signals corresponding to different physiological parameters to be measured based on the dissimilarity of wavelength and optical properties. In this application, the multiple types of excitation light include at least blue light, red light, and infrared light. Blue light is used to excite calcium-sensitive fluorescent proteins to generate calcium fluorescence signals and to excite oxygen-sensitive probes to generate phosphorescence signals, with a wavelength range of 450–495 nm; in application, blue light with a wavelength of 470 nm can be selected. Red light is used to interact with tissue hemoglobin to obtain reflected light signals, with a wavelength range of 620–750 nm; in application, red light with a wavelength of 630 nm can be selected. Infrared light is also used to interact with tissue hemoglobin to obtain reflected light signals, with a wavelength range of 700 nm–1 mm; in application, infrared light with a wavelength of 730 nm can be selected. For example, the multiple types of excitation light include blue light with a wavelength of 470 nm, red light with a wavelength of 630 nm, and red light with a wavelength of 730 nm. The combination and wavelength selection of this multi-band excitation light are designed to maximize the matching between each excitation spectrum and its corresponding absorption spectrum, while minimizing the spectral overlap between different excitation lights and between them and each reflected signal, thus laying the foundation for subsequent high-specificity, low-crosstalk multi-mode signal separation and acquisition.

[0038] The target interval refers to the temporal parameter that controls the sequential on- and off-off of various types of excitation light. The size of the target interval (e.g., milliseconds) can be adaptively set according to the physiological characteristics of the object to be detected (e.g., heart rate and respiratory cycle of different species), specific detection requirements (e.g., required temporal resolution), and the physiological or pathological process being studied. For example, to capture the millisecond-level rapid coupling of nerves, blood vessels, and metabolism in a mouse epilepsy model, a high temporal resolution is required, and the target interval can be set to approximately 3 milliseconds to achieve full-modal synchronous acquisition of 30-40 frames per second. When monitoring slow processes such as tumor growth, the target interval can be extended to hundreds of milliseconds or even seconds to prioritize improving the signal-to-noise ratio per frame for observing trends from hourly to dayly levels. By adjusting the target interval, various research needs from acute onset to chronic evolution can be flexibly adapted. In this application, for the same round or the same time series of imaging, the target interval used when different types of excitation light are alternately excited is strictly consistent, that is, blue light, red light, and infrared light are alternately excited according to a fixed, periodic time interval. In this application, different types of excitation light can be driven by a timing controller to alternately irradiate at a preset target interval. This can also be achieved through other feasible hardware or hardware / software combinations, such as, but not limited to: using a motor-driven mechanical filter wheel to sequentially switch different wavelengths of excitation light; using an acousto-optic tunable filter or electro-optic modulator to rapidly select and switch the wavelength of the excitation source; or deploying multiple independently controllable light source modules and having them illuminate in turn at preset target intervals. This target interval design ensures that the acquisition of signals from different modalities is strictly synchronized at equal intervals on the time axis, providing a crucial timing reference for subsequent precise timing alignment and causal correlation analysis of time-series data of different physiological parameters (calcium signal, oxygen partial pressure, blood oxygen), thereby effectively avoiding multimodal data time misalignment and resolution errors caused by asynchronous excitation timing.

[0039] It should be noted that in this application, the excitation light intensity of different types of excitation light can be set according to the following requirements: 1. For the same type (i.e., a specific wavelength) of excitation light, the set excitation light intensity must remain consistent throughout the entire process of alternating irradiation at target intervals. This constant control of the same light intensity over time ensures that the intensity differences of the physiological optical signals (such as calcium fluorescence, phosphorescence, or specific reflected light) excited by the excitation light at different times can truly reflect the changes in physiological parameters, rather than originating from the instability of the light source itself, thereby guaranteeing the temporal consistency and quantitative accuracy of data acquisition. 2. For different types (i.e., different wavelengths) of excitation light, the set light intensity can be the same or different, and the specific light intensity can be independently configured according to the actual detection needs. This design achieves the stability of each light source in the time dimension, while allowing independent adjustment of intensity in the spectral dimension, together constituting an illumination control strategy that balances data reliability and system flexibility.

[0040] In practice, the illumination module in the imaging unit can be used to alternately and cyclically irradiate the surface of the test tissue of the test object that has been fitted with an oxygen-sensitive film, according to a preset and fixed target interval, through the imaging path composed of the three-color Köhler illumination module and the polarization beam splitting module.

[0041] In one embodiment, regarding the imaging path in S102 above, which includes a Köhler illumination module and a polarization beam splitting module, the excitation of the beam flow in the imaging path can be performed as follows: Multiple types of excitation light are flashed alternately at target intervals, and each type of excitation light passes through the Köhler illumination module and the polarization beam splitting module in sequence to irradiate the tissue to be tested.

[0042] The Köhler illumination module, also known as the tri-color Köhler illumination module, is used to create a uniform, glare-free illumination area on the sample surface through the cooperation of optical elements. Specifically, it can include multiple light sources, aperture stops, field stops, multiple filters, and dichroic mirrors—core components for achieving uniform and controllable illumination. The light source generates the excitation beam and provides illumination; the aperture stop is conjugate to the back focal plane of the objective lens, controlling the aperture angle of the illumination beam to adjust the coherence and resolution of the illumination; the field stop is conjugate to the sample plane (i.e., the tissue to be examined with an oxygen-sensitive film) and the final image plane, strictly limiting the size of the illumination area and effectively eliminating stray light outside the field of view; the dichroic mirror selectively reflects and transmits light of different wavelengths, ensuring effective beam transmission.

[0043] The polarization beam splitter module is used for efficient separation of the excitation and emission light paths. It specifically includes a polarization beam splitter and cooperating optical components such as filters and dichroic mirrors, all integrated into the acquisition light path. Specifically, the polarization beam splitter module reflects the excitation light from the Köhler illumination module, which has been modulated to a specific polarization direction (e.g., S-polarization), and guides it to the objective lens, thus illuminating the sample. Simultaneously, the emission signal light generated by the sample (including calcium fluorescence, oxygen phosphorescence, and reflected light), after being collected and returned by the objective lens, can largely pass through the polarization beam splitter because its polarization state has changed or depolarized relative to the excitation light. This light is then guided to the first and second cameras at the rear for acquisition. Building upon spectral filtering, the polarization beam splitter module further significantly suppresses strong background noise generated by the reflection of excitation light at the optical interface from the polarization dimension, preventing it from directly entering the acquisition channel. This significantly improves the signal-to-noise ratio of weak physiological optical signals (especially phosphorescence signals), providing a clean signal source for the subsequent generation of high-quality multimodal images.

[0044] In practice, the timing controller drives the multicolor light source to alternately illuminate blue light (approximately 470 nm), red light (approximately 620 nm), and infrared light (approximately 730 nm) at preset target intervals. For each illuminated excitation beam, it first enters the Köhler illumination module, and then passes sequentially through an aperture stop conjugate to its rear focal plane and a field stop conjugate to its sample plane to shape the beam. Subsequently, the beam continues to pass through the polarization beam splitting module, which reflects it and guides it to the objective lens. Finally, after passing through the objective lens, it forms a clear and uniformly bright illumination spot on the sample plane, which accurately and uniformly illuminates the surface of the tissue to be tested, on which the oxygen-sensitive film has been installed.

[0045] like Figure 3The diagram shown is a specific schematic of an imaging module provided in an embodiment of this application. The imaging module includes independent light source components (which can be multi-wavelength LEDs or lasers) for light source 1, light source 2, and light source 3. The emitted light beam first passes through an aperture stop, which is precisely conjugated to the back focal plane of the objective lens to control the illumination aperture angle and resolution. The light beam then passes through a field stop, which is conjugated to the sample plane (i.e., the tissue to be detected) and the image plane to define the illumination area and reduce stray light. After being collimated by the lens in the lens barrel, the light beam enters a polarization beam splitting module composed of a filter and a dichroic mirror / polarization beam splitter. The filter is used to select excitation light of a specific wavelength (such as blue light for calcium excitation, blue light for oxygen-sensitive film excitation, etc.), while the dichroic mirror, based on the wavelength-selective reflection characteristic, efficiently reflects the excitation light from the light source by 90 degrees, causing it to be redirected and precisely guided into the objective lens, and finally focused on the sample plane (i.e., the brain tissue with the oxygen-sensitive film installed). On the signal acquisition side, multimodal emitted light (calcium fluorescence, oxygen phosphorescence, and reflected light) from the tissue under test returns along the same optical path. When the emitted light passes through the same polarization beam splitter, it is precisely split into two orthogonally polarized beams. After spectral separation, these beams are guided to the imaging channels of at least two cameras, ultimately forming a clear, spectrally pure image on image plane 1 or image plane 2. This optical architecture, strictly adhering to the Kohler illumination principle, achieves multi-wavelength beam excitation and multi-channel synchronous acquisition through meticulous filtering and beam splitting design. This ensures illumination uniformity and imaging quality while fundamentally avoiding spectral crosstalk between different modal signals, laying a core foundation for high-fidelity, multi-parameter synchronous imaging.

[0046] S103: According to the target interval, use different cameras to continuously acquire each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected.

[0047] Here, the signal acquisition intervals of the camera are consistent with the alternating target illumination intervals of different types of excitation light, which can be uniformly implemented by the same timing controller or hardware trigger. This ensures that the acquisition time of each frame of image precisely corresponds to the illumination window of a specific excitation light (e.g., phosphorescence signals are only acquired under blue light illumination, and calcium fluorescence signals are only acquired under blue light illumination), thereby avoiding incorrect matching between the signal and the excitation source. If the timing of the two is inconsistent, it will lead to crosstalk between different modal signals and time axis misalignment, destroying the synchronization and causal relationship of the data. Therefore, maintaining strict consistency between the excitation and acquisition intervals is the foundation for ensuring the authenticity and alignment of multimodal data and enabling subsequent quantitative analysis.

[0048] The different cameras are at least two physically independent image sensors with separate spectral acquisition channels, used to achieve physical separation and synchronous parallel acquisition of multimodal optical signals at the hardware level. Specifically, by guiding reflected light signals of different wavelength ranges (such as calcium fluorescence, oxygen phosphorescence, and blood oxygen reflected light) to different dedicated cameras via beam splitters, crosstalk between different modal signals is completely isolated at the spatial and detection hardware levels. This configuration not only ensures the spectral purity and high signal-to-noise ratio of the data from each channel, but also ensures that all measured physiological parameters can be recorded synchronously, eliminating the time delay and misalignment inherent in time-division acquisition, and providing an indispensable hardware foundation for the subsequent generation of spatiotemporally aligned multi-parameter image data.

[0049] The first excitation light signal is a phosphorescent signal from the oxygen-sensitive thin film. Specifically, when excitation light of a specific wavelength (blue light, specifically 470 nm) irradiates the oxygen-sensitive thin film, the oxygen-sensitive probe fixed in the film... Upon excitation, the phosphorescent signal emitted exhibits an intensity inversely proportional to the local oxygen partial pressure. That is, the higher the oxygen partial pressure in the tissue being tested, the weaker the phosphorescent signal emitted by the film. The phosphorescent signal is not a simple reflection of excitation light on the film surface, but rather a signal with a specific lifetime. The characteristic emission light emitted by the probe molecule itself when excited. Phosphorescent signals, for example, can be red light signals.

[0050] The second excitation light signal is a calcium fluorescence signal from specific tissue cells. Specifically, when excitation light of a specific wavelength (blue light, specifically 470 nm) is irradiated onto the tissue to be tested, the labeled tissue cells (which may be labeled by a gene encoding a calcium indicator protein (such as GCaMP)) are excited by the influx of calcium ions, producing a characteristic fluorescence signal. This fluorescence signal is a direct optical response to changes in intracellular calcium ion concentration, and its intensity is correlated with the level of neural electrical activity. The calcium fluorescence signal can be, for example, a green light signal.

[0051] The reflected light signal is an optical signal used to calculate blood oxygen saturation. Specifically, the reflected light signal is the light signal returned after the tissue being tested is irradiated by infrared light and red light of a specific wavelength (specifically, 630 nm red light and 730 nm infrared light), and the light interacts with hemoglobin (including oxyhemoglobin and deoxyhemoglobin) in the tissue. The reflected light signal can be further divided into reflected red light signal or reflected infrared light signal. The intensity of the reflected light signal is simultaneously modulated by the tissue scattering characteristics and the hemoglobin absorption characteristics, and its intensity ratio or change under the two excitation lights is related to the oxygenation status of hemoglobin within the tissue.

[0052] Specifically, in this application, the imaging module can be configured with two cameras for image acquisition. The first camera is used to acquire the phosphorescence signal and blood oxygen reflection signal of the oxygen-sensitive film, and the second camera is configured to specifically acquire the calcium fluorescence signal. Alternatively, three cameras can be used to independently and synchronously acquire the phosphorescence signal, the reflection signal, and the fluorescence signal.

[0053] In practice, different cameras can be used to continuously acquire frames of oxygen phosphorescence signals from the oxygen-sensitive membrane, frames of calcium fluorescence signals from specific tissue cells, and frames of reflected light signals from blood oxygen in the tissue under test, according to target intervals. Specifically, when irradiated with blue light, two cameras are used to acquire red oxygen phosphorescence signals from the oxygen-sensitive membrane and green calcium fluorescence signals from labeled neurons, respectively; when irradiated with infrared and red light, a single camera is used to sequentially acquire tissue reflected light signals (reflected red light signals or reflected infrared light signals) for blood oxygen calculation. By controlling different types of excitation light to alternately irradiate the tissue under test at target intervals, and simultaneously using multiple cameras to synchronously acquire the corresponding optical signals, the effectiveness of the acquired optical signals can be guaranteed.

[0054] In one embodiment, the various types of excitation light may include red light, infrared light, and blue light; the different cameras may include at least one first camera and one second camera; the first excitation light signal may include an oxygen phosphorescence signal excited by blue light based on the oxygen quenching effect; the second excitation light signal may include a calcium fluorescence signal excited by blue light based on the calcium fluorescence characteristics of specific tissue cells; the reflected light signal may include reflected red light signal and reflected infrared light signal of blood oxygen in the tissue to be detected.

[0055] Regarding the above S103, the following steps can be taken: According to the target interval, the first camera continuously acquires each frame of oxygen phosphorescence signal on the oxygen-sensitive film, the second camera continuously acquires each frame of calcium fluorescence signal of specific tissue cells, and the first camera continuously acquires each frame of reflected red light signal or reflected infrared light signal of blood oxygen in the tissue to be tested.

[0056] In practice, following the same target interval (e.g., 3ms) as the alternating excitation light irradiation, when blue light irradiates (470nm), the first camera acquires the oxygen phosphorescence signal emitted by the oxygen-sensitive membrane after excitation; the second camera simultaneously acquires the calcium fluorescence signal generated by specific cells in the tissue (e.g., neurons expressing GCaMP6f) after being excited by blue light; when red light (630nm) irradiates, the first camera correspondingly acquires the reflected red light signal from the tissue to be detected; when infrared light (630nm) irradiates, the first camera correspondingly acquires the reflected infrared light signal from the tissue to be detected.

[0057] like Figure 4The diagram shown is a specific schematic of a different optical signal acquisition process provided in an embodiment of this application. Blue light with a wavelength of 470 nm is used as the excitation light to irradiate the tissue under test. This excitation light simultaneously induces two characteristic emission signals: one is a calcium fluorescence signal with a wavelength of 530 nm emitted by a specific calcium indicator protein within the tissue under test; the other is a 630 nm wavelength signal emitted by an implanted oxygen-sensitive membrane, the intensity of which depends on the partial pressure of local blood oxygen. abbreviation The oxygen phosphorescence signal was detected. At the acquisition end, a second camera was configured specifically to receive and record calcium fluorescence signals; the first camera was configured to receive and record oxygen phosphorescence signals. Additionally, the tissue under test was irradiated with red light at a wavelength of 630 nm and infrared light at a wavelength of 730 nm to obtain reflected light signals, and the first camera acquired the blood oxygen reflected light signals excited by red and infrared light, respectively. Figure 4 In this process, different optical signals are acquired through three parallel optical paths: (a) a blue light excitation path, using blue light with a wavelength of 470 nm as the excitation light, in the oxygen-sensitive thin film. Upon excitation, the light emits a phosphorescent signal with a wavelength of 630 nm, which is captured by the first camera; labeled tissue cells (such as those labeled with GCaMP) in the tissue to be tested are excited to produce a characteristic fluorescence signal with a wavelength of 530 nm, which is captured by the second camera. (b) Red light excitation pathway: Red light with a wavelength of 630 nm is used as the excitation light. After passing through the tissue to be tested, a reflected light signal with a wavelength of 630 nm is generated, which is captured by the first camera; (c) Infrared light excitation pathway: Infrared light with a wavelength of 730 nm is used as the excitation light. After passing through the tissue to be tested, a reflected light signal with a wavelength of 730 nm is generated, which is captured by the first camera.

[0058] S104: Generate a tissue oxygen partial pressure image of the tissue to be tested based on each frame of the first excitation light signal acquired continuously; generate a calcium signal image of the tissue to be tested based on each frame of the second excitation light signal acquired continuously; and generate a blood oxygen signal image of the tissue to be tested based on each frame of the reflected light signal acquired continuously.

[0059] Here, the tissue oxygen partial pressure image is used to reflect the oxygen partial pressure at various points within the tissue under test in two-dimensional space. Quantitative distribution refers to the spectral reconstruction based on the inverse relationship between phosphorescence intensity and oxygen partial pressure. Calcium signal images reflect the calcium ion concentration within the tissue being tested. These are reconstructed based on the correlation between fluorescence intensity and intracellular calcium ion concentration, reflecting the spatiotemporal distribution and intensity of electrical activity in calcium-sensitive cells (such as neurons). Blood oxygenation signal images reflect the spatial distribution of blood oxygen saturation within the tissue being tested. These are calculated based on the functional relationship between the intensity of reflected red and infrared light signals and the oxygenation status of hemoglobin. These three types of images together constitute a simultaneous, quantitative, and visual representation of the three key physiological dimensions of the target region: neural activity, blood supply, and energy metabolism.

[0060] In this application, the calculation of blood oxygen saturation can be based on Beer-Lambert's law, which describes the relationship between the attenuation of light in a scattering medium (such as biological tissue) and the concentration of light-absorbing substances. Specifically, the tissue to be tested is alternately irradiated with infrared light at a wavelength of 730 nm and red light at a wavelength of 620 nm. Since oxyhemoglobin and deoxyhemoglobin have significantly different molar absorption coefficients at these two characteristic wavelengths, the intensity of the reflected light from the tissue to be tested will vary differently with changes in blood oxygen saturation. By continuously acquiring and recording the intensity of the reflected light signals corresponding to each pair of sequentially irradiated infrared and red light using a first camera, the light intensity attenuation values ​​at the two wavelengths can be obtained. Substituting these two attenuation values ​​into the inversion model established based on the dual-wavelength Beer-Lambert law, the blood oxygen saturation value reflecting the degree of hemoglobin oxygenation can be calculated. This calculation process is performed in real time after each new pair of reflected light signals is acquired, ultimately generating blood oxygen signal images of each frame. A pair of reflected light signals includes either reflected red light signals or reflected infrared light signals acquired before and after the acquisition.

[0061] In practice, for continuous oxygen phosphorescence signal frames from the first camera, the grayscale values ​​of each pixel are converted into absolute oxygen partial pressure values ​​in real time using pre-stored phosphorescence intensity and oxygen partial pressure calibration curves, thereby reconstructing tissue oxygen partial pressure images frame by frame. For continuous calcium fluorescence signal frames from the second camera, the relative fluorescence change value reflecting the level of neuronal activity is calculated by subtracting the background and comparing it with the baseline fluorescence intensity, generating calcium signal images in real time. For paired red and infrared light reflection signal frames from the first camera, the blood oxygen saturation value of each pixel is calculated in real time based on the dual-wavelength calculation model established by Lambert-Beer's law, generating blood oxygen signal images. The three sets of image sequences are output in real time with identical timestamps and spatial coordinates, thus synchronously and intuitively presenting the spatiotemporal dynamic changes of neural electrical activity, tissue metabolic level, and blood oxygenation status in the target brain region in a living state.

[0062] In this way, by integrating a flexible oxygen-sensitive thin film with an oxygen-sensitive probe, and designing alternating irradiation with excitation light of various wavelengths according to the target interval, and efficiently and synergistically acquiring and imaging excitation and reflection signals, high spatiotemporal resolution and long-term stable synchronous real-time imaging of the three key physiological processes of energy metabolism, neural activity and blood supply in the tissue to be detected are achieved. This overcomes the technical bottleneck of existing tissue imaging technologies, which cannot fully capture the causal chain of "neural activity - vascular response - energy metabolism" due to single-modality or loose multimodal technology.

[0063] In one embodiment, the oxygen-sensitive film in S101 can be prepared by the following steps A1 to A3: A1: Dendritic mesoporous silica nanoparticles (DMSN) were prepared using hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water.

[0064] Here, DMSNs prepared using a synergistic template method with cetyltrimethylammonium p-toluenesulfonate (CTATos) as the main directing agent and triethanolamine (TEA) as the co-directing agent have a core advantage: they form a unique dendritic radial mesoporous structure. This structure possesses extremely high specific surface area and pore volume, providing ample sites for subsequent efficient loading of oxygen-sensitive probes. The open channels inherent in DMSNs greatly facilitate... The diffusion and fixation of probe molecules effectively prevented... Blockage or accumulation within the pores. Simultaneously, the DMSNs obtained by this method have surfaces rich in active silanol groups, readily interacting with ruthenium-based probe molecules. The strong interaction between the polymer matrix and the composite material significantly enhances the stability of the composite. Therefore, these DMSNs, as nanocarriers, can stably confine hydrophilic probes in a monodisperse form. Among them, dendritic mesoporous silica nanoparticles are spherical, so they can be called dendritic mesoporous silica nanospheres.

[0065] In practice, DMSN can be prepared by thoroughly mixing hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water.

[0066] In one embodiment, A1 above can be implemented through the following steps A1-1 to A1-4: A1-1: According to the first preparation ratio, a first mass of hexadecyltrimethylammonium p-toluenesulfonate, a second mass of triethanolamine, and a first volume of deionized water are mixed and stirred at a first temperature for a first time to obtain a first solution.

[0067] Here, the first preparation ratio is a predetermined molar ratio between hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water. This first preparation ratio must be followed when synthesizing the hydrothermal reaction precursor solution for dendritic mesoporous silica nanoparticles (DMSNs) using hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water. For example, the first preparation ratio can be 0.96 g: 0.105 g: 50 ml.

[0068] The first mass refers to the mass of CTATos added based on the first preparation ratio. CTATos acts as the primary surfactant template (main structure directing agent) to guide the formation of mesoporous structures. The second mass refers to the mass of TEA added based on the first preparation ratio. TEA acts as a co-structure directing agent and base catalyst, responsible for regulating reaction kinetics and synergistically guiding the generation of hierarchical structures. The first volume refers to the volume of deionized water added, which, as the reaction solvent, determines the concentration and scale of the entire reaction system. The ratio between the first and second masses determines the effectiveness of the template effect, while the ratio of their respective masses to the first volume (volume of water) jointly determines the concentration of reactants. This set of parameters ensures that the reaction can proceed in a controllable and reproducible chemical microenvironment, thereby directionally synthesizing DMSNs nanocarriers with the desired morphology, size, and surface properties. For example, the first mass can be 0.96 g, the second mass can be 0.105 g, and the first volume can be 50 mL.

[0069] The first temperature and the first duration constitute the key thermodynamic and kinetic conditions for the pre-reaction and homogenization of a mixed solution of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water. The first temperature refers to the constant reaction temperature maintained during the mixing and stirring process. The first temperature range is 70–90°C, specifically 80°C. This temperature setting aims to achieve two purposes: firstly, to provide the necessary thermodynamic driving force for the full formation and self-assembly of surfactant micelles; and secondly, to create a suitable thermal environment for the subsequent hydrolysis and polycondensation steps of the silicon source. The first duration refers to the duration of stirring at the first temperature. The first duration range is 50–70 min, specifically 60 min. Its function is to ensure the stability of the mixture (CTATos, TEA, ...). The precursor system achieves sufficient dispersion, homogeneous mixing, and necessary pre-equilibrium at the molecular scale, thereby forming a stable and homogeneous reaction precursor system. These two parameters, together with the aforementioned first mass, second mass, and first capacity, work synergistically to constitute the controllable and reproducible initial reaction environment necessary for the synthesis of DMSNs with ideal mesoporous structure and morphology.

[0070] The first solution refers to a micelle solution, specifically a homogeneous and stable micelle solution formed by self-assembly in deionized water using hexadecyltrimethylammonium p-toluenesulfonate as the main structure directing agent and triethanolamine as the co-structure directing agent. The first solution is an essential templated reaction medium for the synthesis of DMSNs. The micelle structure formed within it provides a pre-defined nanoscale template for the subsequent hydrolysis and condensation of the silicon source, thereby directly determining the morphology, size, and order of the porous structure in the final product.

[0071] In specific implementation, according to the first preparation ratio, weigh 0.96 g of CTATos, 0.105 g of TEA, and 50 mL of deionized water. Add CTATos and TEA to the deionized water and mix. Stir at 80°C for 1 hour to obtain the first solution.

[0072] A1-2: The second volume of tetraethyl silicate and the first solution are stirred and mixed at a second temperature for a second time to obtain the DMSN precursor.

[0073] Here, the second volume refers to the volume of added silicon source tetraethyl orthosilicate (TEOS). This volume parameter is one of the key variables determining the density, size, and mesoporous structure development of the final silica nanosphere framework. The second volume is related to the first preparation ratio; for example, the second volume can specifically be 7.8 mL.

[0074] The second temperature refers to the constant reaction temperature maintained during the TEOS hydrolysis and polycondensation reaction, ranging from 70 to 90°C, specifically 80°C. This temperature parameter is the core thermodynamic condition for controlling the reaction kinetics, thereby guiding the controlled deposition and curing of the silica network on the surface of the pre-assembled micelle template.

[0075] The second duration is the precise duration of mixing and reaction between TEOS and the first solution, ranging from 1.5 to 2.5 hours, specifically 2 hours. This time parameter is the core kinetic condition to ensure that the silica precursor completes a sufficient and controllable hydrolysis-condensation process on the micelle template surface, thereby forming a silica coating layer with a complete structure and defined morphology.

[0076] The DMSN precursor is a key intermediate in the synthesis of the final dendritic mesoporous silica nanospheres (DMSNs). Specifically, it is a silica-surfactant composite nanostructure formed after TEOS undergoes hydrolysis and condensation in a first solution composed of a micelle template, still containing an organic template within its structure. The DMSN precursor has initially replicated the complex morphology of the micelle template, but the silica network is not yet fully solidified and still encapsulates the organic template.

[0077] In practice, 7.8 mL of TEOS can be added to the first solution and stirred at 80°C for 2 hours to obtain the DMSN precursor.

[0078] A1-3: Place the pretreated DMSN precursor into the sintering apparatus and raise the temperature of the sintering apparatus to the first target temperature at a preset heating rate.

[0079] Here, pretreatment refers to operations including but not limited to separation, purification, and drying performed on the DMSN precursor before high-temperature sintering. This pretreatment is a necessary prerequisite to ensure the effectiveness and controllability of the subsequent high-temperature sintering process. It eliminates the uncontrollable decomposition, structural damage, or contamination that impurities may cause at high temperatures, and ensures that the precursor powder can be uniformly heated and fully reacted in the sintering furnace, ultimately obtaining pure and structurally regular DMSNs.

[0080] A sintering apparatus is a specialized device used for high-temperature heat treatment of pretreated DMSN precursors to achieve organic decomposition and inorganic framework consolidation. Specifically, a muffle furnace can be used for sintering DMSN precursors. However, other devices capable of programmed heating, controllable temperature control, and high-temperature heat treatment can also be employed. For example, tube furnaces can achieve similar functions under specific gas conditions (such as air or nitrogen); vacuum sintering furnaces can perform heat treatment in low-pressure or oxygen-free environments, suitable for applications with special requirements for the sintering gas; furthermore, microwave sintering furnaces and other devices utilizing different heating principles can also be used as alternative sintering apparatuses, as long as they meet the process requirements of removing the organic template and consolidating the silica framework. Sintering DMSN precursors using a sintering apparatus can thoroughly and uniformly remove the organic template from the precursor, while simultaneously promoting further condensation and crystallization of the silica framework and activating surface silanol groups, ultimately yielding pure, structurally stable DMSNs with abundant surface silanol groups.

[0081] The preset heating rate refers to a pre-set process parameter used to precisely control the temperature rise per unit time. The heating rate can be selected in the range of 2~4℃ / min, and this application uses 3℃ / min as an example.

[0082] The first target temperature refers to a pre-set specific high temperature threshold that the sintering process is required to reach and maintain. The first target temperature can be selected in the range of 450~600℃, and this application uses 550℃ as an example.

[0083] In practice, the DMSN precursor is effectively separated from the reaction mother liquor by centrifugation. Unreacted monomers, ionic byproducts, and solvent impurities are thoroughly removed by washing. Then, it is dried at room temperature to remove residual liquid media, obtaining a pure and physically stable DMSN precursor solid powder. Next, to remove the organic template (CTATos / TEA), enhance the structural stability of the mesoporous silica framework, and activate surface silanol groups, DMSN is sintered in air using a muffle furnace. Specifically, the precursor powder can be programmed to rise from room temperature to a specific first target temperature (e.g., 550°C) in air at a rate of 3°C / min using a muffle furnace.

[0084] A1-4: Maintain the first target temperature until the third duration is met, then remove the heating and cool to room temperature to obtain the prepared DMSN.

[0085] Here, the third duration is a pre-set process parameter used to control the duration of continuous heating of the material at the first target temperature. It provides the necessary and sufficient time for the complete decomposition of the organic matter and the full consolidation of the silica framework, ensuring that the entire nanostructure reaches a thermodynamically and chemically stable state. The feasible range for this duration is 4 to 8 hours. The third duration is determined through comprehensive optimization based on the organic loading of the precursor, the preset heating rate, and the first target temperature; for example, it can be 5 hours.

[0086] In practice, after reaching 550°C, sintering is continued for 5 hours. After ensuring complete decomposition of organic matter and solidification of the silica network, the generated DMSNs are allowed to cool freely to room temperature in the sintering apparatus.

[0087] A2: Utilizing a silica suspension prepared based on DMSN and an aqueous sodium hydroxide solution, and tris(4,7-phenyl-1,10-o-diazaphenanthroline)ruthenium(II) diester. A mixed solution of anhydrous ethanol and anhydrous ethanol was used to prepare a solution loaded with... Dendritic mesoporous silica nanoparticles .

[0088] In practice, the DMSNs obtained after pretreatment and sintering are first dispersed in an aqueous sodium hydroxide solution to form a silica suspension. Then, tris(4,7-phenyl-1,10-o-diazaphenanthroline)ruthenium(II) diester, i.e. Dissolve it in anhydrous ethanol to prepare a mixed solution. Then, add the mixed solution to the above silica suspension and stir continuously, so that... Uniform loading of DMSNs through physical adsorption and chemical interaction yields loaded with... Composite nanomaterials of DMSNs, namely .

[0089] In one embodiment, A2 above can be implemented through the following steps A2-1 to A2-3: A2-1: According to the second preparation ratio, the third mass of DMSN and the third volume of sodium hydroxide aqueous solution are mixed and stirred continuously for a fourth time to obtain a silica suspension.

[0090] Here, the second preparation ratio is the mixing ratio between the volumes of DMSNs and sodium hydroxide aqueous solution, specifically 1 gram (g): 20 milliliters (mL). Based on this second preparation ratio, on the one hand, the DMSNs are ensured to be uniformly and stably dispersed, avoiding aggregation; on the other hand, the suitable sodium hydroxide concentration is... Probe molecules are efficiently and uniformly loaded onto the abundant mesopores and surfaces of DMSNs through ion exchange or coordination, creating a chemical environment.

[0091] The third mass and third volume are two parameters determined based on the second preparation ratio. The third mass is the precise mass of DMSNs added to achieve the second preparation ratio, used to quantitatively determine the total amount of functional nanocarriers participating in the surface activation and loading reactions. The third volume is the precise volume of an aqueous sodium hydroxide solution with a specific concentration (0.01 mol / L) added to achieve the second preparation ratio, used to provide the necessary alkaline chemical environment and liquid dispersion medium. For example, the first mass can be 1 g, and the third volume can be 20 ml.

[0092] The fourth duration is a preset continuous stirring time parameter, ranging from 14 to 30 minutes, and 15 minutes is used in this application. Stirring for the fourth duration provides the necessary and sufficient time window for DMSNs to achieve adequate and uniform dispersion in the sodium hydroxide aqueous solution and for the effective activation of their surface silanol groups.

[0093] In practice, 1 g of DMSNs was dispersed in 20 mL of 0.01 mol / L NaOH aqueous solution at a ratio of 1:20 g / mL, and stirred continuously for 15 minutes to obtain a silica suspension.

[0094] A2-2: According to the third preparation ratio, the fourth mass... Mix with the fourth volume of anhydrous ethanol solution to obtain the second solution.

[0095] Here, the third preparation ratio refers to A specific mixing ratio between the mass of the product and the volume of anhydrous ethanol is used to form a mixture that allows... The probe molecules are fully dissolved and at an appropriate concentration. A solution mixed with ethanol is used for subsequent... The probe is efficiently and uniformly loaded onto DMSNs to provide suitable reactant concentrations and fluid volume conditions. The third preparation ratio can be 100 mg: 5 mL.

[0096] The fourth mass is the oxygen-sensitive probe added based on the third preparation ratio. The mass is used to quantitatively determine the total amount of reactive oxygen species-sensitive probe molecules participating in the loading reaction; the fourth capacity is based on the volume of anhydrous ethanol added according to the third preparation ratio, used as a solvent to provide dissolution. The probe molecules form the medium required to form a homogeneous solution. For example, the fourth mass can be 100 mg and the fourth volume can be 5 mL.

[0097] The second solution is A clear and stable mixture of probe molecules and anhydrous ethanol.

[0098] In specific implementation, 100mg can be prepared according to the third preparation ratio. Dissolve in 5 mL of anhydrous ethanol to prepare a second solution.

[0099] A2-3: The second solution was added to the silica suspension, and after continuous stirring for five hours, vacuum filtration was performed to obtain... .

[0100] Here, the fifth duration is a pre-set time. The feasible range for the fifth duration is 10 to 30 minutes; 15 minutes is selected as an example in this application. The core function of the fifth duration is to provide... The probe molecules provide the necessary and sufficient time window for adequate diffusion and stable loading onto the surface and within the pores of dendritic mesoporous silica nanospheres (DMSNs), thereby ensuring uniform loading and stable bonding. Composite nanomaterials.

[0101] In practice, the pre-prepared The entire anhydrous ethanol mixture was added to the silica suspension, and the resulting mixture was then stirred continuously at room temperature for 15 minutes. The probe molecules (i.e., components) diffuse fully and adsorb uniformly onto the surface and pores of DMSNs. After loading, the mixture is separated into solid and liquid phases using a vacuum filtration device. The solid product is collected and thoroughly washed multiple times with anhydrous ethanol to remove physically adsorbed free probe molecules and residual alkaline solvent, ultimately obtaining uniformly loaded and firmly bound probes. .

[0102] like Figure 5 The figure shows a schematic diagram of a composite structure of dendritic mesoporous silica nanoparticles loaded with oxygen-sensitive probes, provided in an embodiment of this application. As can be seen in the figure, DMSNs (whose size can be characterized by a scale bar of approximately 200 nm) with radially branched channels serve as nanocarriers, and their surface and abundant mesopores are uniformly loaded with ruthenium-based phosphorescent probe molecules (in order to...). (This is indicated). This composite structure combines the high specific surface area, open channels, and abundant surface silanol groups of DMSN, as well as... Leveraging the probe's superior oxygen quenching sensitivity, a high-performance solution-state oxygen-sensitive probe was successfully transformed into a solid, stable nanocomposite.

[0103] A3: Utilize Oxygen-sensitive films were prepared using polydimethylsiloxane (PDMS) matrix and PDMS curing agent.

[0104] In practice, the pre-prepared The composite nanomaterial was mixed with a liquid polydimethylsiloxane (PDMS) matrix (a two-component thermosetting polydimethylsiloxane material, Sylgard184 Part A) to obtain a homogeneous premix. Subsequently, a PDMS curing agent (a two-component thermosetting polydimethylsiloxane crosslinking agent, Sylgard184 Part B) was added to the premix, and the mixture was heated and cured to finally form a uniform, flexible, and transparent oxygen-sensitive film.

[0105] In one embodiment, A3 above can be implemented through the following steps A3-1 to A3-5: A3-1: According to the fourth preparation ratio, the fifth mass... The sixth mass of PDMS matrix was mixed with the fifth volume of toluene solution until it became transparent, thus obtaining the third solution.

[0106] Here, the fourth preparation ratio is A specific mixing ratio between the mass of the composite nanomaterial and the mass of the PDMS matrix. This fourth preparation ratio can be used to determine the relative content of the functional nanofiller in the polymer matrix, thereby precisely controlling the oxygen sensing sensitivity, mechanical flexibility, and optical uniformity of the final oxygen-sensitive film. Specifically, the fourth preparation ratio can be 1:10 (i.e., 20 mg / L). (corresponding to 200 mg PDMS matrix), based on a comprehensive balance of nanofiller dispersibility, film mechanical integrity and sensing performance, the feasible range of this ratio can be extended from 1:8 to 1:12.

[0107] The fifth quality refers to the added The mass of the composite nanomaterial is used to quantitatively determine the total amount of reactive oxygen sensing units introduced into the thin film, directly affecting the final oxygen sensing sensitivity and response intensity of the film. The sixth mass is the precise mass of the added PDMS matrix, used to provide the total amount of polymer material to form a continuous, flexible thin film matrix, determining the basic mechanical properties and molding ability of the film. For example, the fifth mass can be 20 mg, and the sixth mass can be 200 mg.

[0108] The fifth volume refers to the precise volume of toluene solvent added. Toluene (mass fraction not less than 99.9%) serves as a temporary organic solvent to significantly reduce the viscosity of the PDMS prepolymer matrix during the initial mixing stage, thereby facilitating the achievement of… The highly uniform, non-agglomerated physical dispersion of the nanocomposite throughout the polymer precursor creates the necessary hydrodynamic conditions. The fifth capacity can specifically be 2 ml, and its size can be determined jointly by the fifth and sixth masses.

[0109] The third solution is composed of the fifth mass. A homogeneous and transparent composite slurry is formed by thoroughly mixing composite nanomaterials, a sixth mass of liquid PDMS matrix, and a fifth volume of toluene solvent. This slurry serves as a direct precursor material for preparing oxygen-sensitive films. The toluene solvent temporarily reduces the viscosity of the PDMS prepolymer, enabling the nanofiller to achieve a highly uniform and non-agglomerated dispersion.

[0110] In practice, 20mg can be used. The solution was thoroughly mixed with 200 mg of PDMS matrix (Sylgard184 Part A) in 2 mL of high-purity toluene until it became transparent, thus obtaining the third solution.

[0111] A3-2: After ultrasonic treatment of the third solution for a period of six hours, the solution is continuously magnetically stirred at a first preset speed for a period of seven hours at room temperature to obtain the initial composite material.

[0112] Here, the sixth duration is a pre-set time length, which can be between 4 and 10 minutes; 5 minutes is used as an example in this application. Utilizing the cavitation effect of ultrasound, the nano-aggregates in the third solution can be initially broken up and dispersed, thus laying the foundation for highly uniform distribution of the nanofiller through subsequent long-term magnetic stirring.

[0113] The first preset speed is a pre-set parameter used to control the rotational speed of the magnetic stirrer rotor. At the first preset speed, the magnetic stirrer provides moderate and continuous shear force and convection to the third solution containing toluene solvent, promoting uniform and gentle evaporation of the solvent while ensuring... The nanofiller achieves and maintains uniform dispersion at the molecular level throughout the PDMS prepolymer matrix, preventing sedimentation or re-agglomeration. The first preset rotation speed can be in the range of 450 revolutions per minute (RPM) to 700 RPM; this application uses 500 RPM as an example.

[0114] The seventh duration is also a pre-set time length, which can range from 45 hours to 72 hours; this application uses 48 hours as an example. By continuously stirring the third solution with magnetic force for the third duration, complete evaporation of the toluene solvent can be achieved. A thermodynamically stable uniform dispersion is achieved and maintained in PDMS prepolymer.

[0115] The initial composite material was a cross-linked, viscous polymer nanocomposite, specifically... The composite nanomaterials are uniformly dispersed in PDMS prepolymer, and the resulting nanocomposite is obtained after thorough solvent removal. This initial composite material serves as a stable precursor for oxygen-sensitive films, allowing for direct subsequent molding and curing. It exhibits highly uniform and stable dispersion of nanosensing units within the polymer matrix, with extremely low solvent content. This provides a material basis for ultimately obtaining a uniformly thick, flexible, transparent solid film with excellent oxygen-sensing properties through a curing reaction.

[0116] In practice, the third solution is first subjected to ultrasonic treatment at room temperature (ultrasonic treatment parameters, for example, 40 kHz, 100 W) for 5 minutes to achieve initial dispersion. Then, it is continuously magnetically stirred at 500 rpm at room temperature for 48 hours to ensure that the toluene solvent gradually evaporates until it is completely volatilized. The initial composite material was obtained by uniformly dispersing the material in a PDMS matrix.

[0117] A3-3: Add the seventh mass of PDMS curing agent to the composite material and degas it in a vacuum environment for eight hours to obtain the target composite material.

[0118] Here, the seventh mass refers to the precise mass of the added PDMS curing agent (Sylgard184 Part B). The PDMS curing agent forms a stable three-dimensional polymer network through a quantitative reaction with the active groups in the PDMS prepolymer (Part A). The seventh mass can specifically be 20 mg, and its exact value is related to the sixth mass. The commonly used mass ratio of the seventh mass to the sixth mass is 1:10, and the feasible range of this mass ratio is between 1:8 and 1:15.

[0119] The eighth time period is a preset duration, which can range from 10 to 30 minutes; this application uses 15 minutes as an example. The eighth time period specifies the duration of vacuum degassing of the composite material after adding the curing agent. Within this eighth time period, the composite material after adding the curing agent undergoes vacuum degassing to thoroughly remove residual trace amounts of solvent and air bubbles that may have been incorporated during mixing. This ensures that the resulting target composite material has a uniform density and extremely low internal defects, laying the foundation for obtaining a bubble-free, uniformly thick, high-quality oxygen-sensitive film in subsequent coating and curing steps.

[0120] The target composite material is a liquid precursor for preparing oxygen-sensitive films. Specifically, it is a homogeneous, bubble-free composite slurry that can be directly used for final molding and curing. As the ultimate liquid precursor for preparing oxygen-sensitive films that can be directly used for coating molding, it not only achieves highly uniform dispersion of nanofillers, but also completes the precise metering and homogeneous mixing of all necessary components (including PDMS prepolymer, curing agent, and oxygen-sensitive nanounits), and eliminates internal defects that affect the integrity of the film through degassing.

[0121] In practice, after the initial mixing and solvent evaporation steps, 20 mg of PDMS curing agent (Sylgard184 Part B) can be added to the initial composite material. The initial composite material with added PDMS curing agent is then degassed under vacuum and held for 15 minutes to remove any residual solvent and bubbles, thus obtaining the liquid precursor of the oxygen-sensitive film, i.e., the target composite material.

[0122] A3-4: After rotating the target composite material continuously at the second preset speed for nine hours, continue rotating at the third preset speed for ten hours.

[0123] Here, the second preset rotation speed is a pre-set first-stage speed parameter used to drive the substrate to rotate in the initial stage of the spin coating process. Its core function is to rapidly and uniformly spread the target composite material coated on the substrate to the entire target area through appropriate centrifugal force, laying the foundation for the formation of a uniform initial liquid film after subsequent high-speed spin coating, thereby ensuring the uniformity of film thickness. The second preset rotation speed can be in the range of 400 RPM to 800 RPM; this application uses 500 RPM as an example.

[0124] The ninth duration is a preset time length used to specify the duration required for spin coating at the second preset rotation speed. The ninth duration can be shifted from 4 seconds to 10 seconds; this application uses 5 seconds as an example.

[0125] The third preset rotational speed is a pre-set, second-level (higher) speed parameter used to drive the substrate rotation in the final stage of the spin coating process. The third preset rotational speed is typically greater than the second preset rotational speed, and its core function is to rapidly and controllably thin and level the initially wet film that has been uniformly spread on the substrate by applying a stronger centrifugal force, thereby precisely controlling and achieving the desired final film thickness. The third preset rotational speed can be in the range of 1800 RPM to 3000 RPM; this application uses 2000 RPM as an example.

[0126] It should be noted that the specific values ​​of the second and third preset rotation speeds are not fixed, but are comprehensively optimized and determined based on at least one of the following factors: the target thickness of the oxygen-sensitive membrane, the specific requirements for membrane uniformity, and the physiological and anatomical characteristics of the subject being tested (e.g., cranial window size, cortical curvature, or tissue compliance of different animal models). Specifically, the target membrane thickness directly determines the centrifugal force required for the third preset rotation speed (high-speed phase); the requirement for membrane uniformity affects the duration of the two rotation speed phases and the smooth transition strategy of the speed combination; and the characteristics of the subject being tested (e.g., differences in brain size in mice or rats) may require adjustment of the parameters of the initial spreading phase (second preset rotation speed) to ensure complete coverage of the slurry on a substrate of a specific size.

[0127] The tenth time interval is a preset duration used to specify the duration of the spin-coating operation at the third preset rotation speed. It provides the necessary and precise time for the spread film to complete sufficient thinning, leveling, and final solvent evaporation under high-speed centrifugal force, thereby achieving precise control over the final film thickness and uniformity. The tenth time interval can range from 25 seconds to 60 seconds; this application uses 30 seconds as an example.

[0128] In practice, the target composite material is rotated at 500 rpm for 5 seconds to ensure uniform distribution; then it is rotated at 2000 rpm for 30 seconds to achieve the desired target film thickness (e.g., 40 micrometers).

[0129] It should be noted that the target thickness of the oxygen-sensitive film is determined by five key factors: optical performance, mechanical adhesion, sensing dynamics, biocompatibility, and controllability of the fabrication process. Optically, the thickness must balance sufficient phosphorescence signal intensity with minimizing excitation light attenuation and scattering. Mechanically, the thickness directly affects the film's flexibility and adhesion stability on uneven tissue surfaces. Sensing-wise, the thickness determines the diffusion response time of oxygen molecules and the spatial integration range of the measured oxygen partial pressure, affecting spatiotemporal resolution. In terms of biocompatibility, the thickness should be minimized to reduce physical pressure and metabolic interference on living tissues. Finally, the thickness must be within a range that can be stably and reproducibly achieved by fabrication processes such as spin coating. Based on the comprehensive optimization of these factors, the target thickness of the oxygen-sensitive film in this invention is typically between 10 and 100 micrometers, with approximately 40 micrometers being a preferred embodiment that balances high signal-to-noise ratio, good adhesion, rapid response, and minimally invasive implantation.

[0130] A3-5: The composite material after ten rotations is spin-coated onto a glass slide and cured at the second target temperature for the target duration to obtain an oxygen-sensitive film.

[0131] Here, the second target temperature is a pre-set temperature, a specific heat treatment temperature used to drive and complete the crosslinking and curing reaction of the PDMS matrix. The second target temperature provides the necessary thermal energy to the liquid composite slurry to activate the curing agent and promote sufficient chemical crosslinking of the PDMS prepolymer molecular chains, thereby transforming it into a solid oxygen-sensitive film with a stable three-dimensional network structure, predetermined mechanical properties (flexibility), and optical transparency. The second target temperature can be determined based on the curing kinetics of the PDMS system, the glass transition temperature of the cured film, and the thermal stability of the oxygen-sensitive nanounits therein; for example, the second target temperature could be 110°C.

[0132] The target duration is a pre-set duration parameter used to maintain the heat-treated state at a second target temperature. The target duration can range from 5.5 hours to 8 hours; this application uses 6 hours as an example.

[0133] The oxygen-sensitive thin film is an optical functional thin film, specifically using flexible PDMS as a continuous matrix, in which uniformly immobilized... A solid-state, ultrathin, and flexible optical functional film of nanocomposite sensing unit. It can be implanted or adhered to the surface of the tissue to be detected, achieving in-situ optical sensing through its internal structure. The oxygen quenching effect of the probe molecules converts the continuous changes in the oxygen partial pressure of the local tissue under test into optical signals of corresponding intensities in real time and quantitatively.

[0134] In practice, after obtaining the oxygen-sensitive spin-coated film of the target thickness, it is further cured at 110°C for 6 hours to complete the crosslinking of PDMS, ultimately obtaining a film with... Oxygen-sensitive membrane.

[0135] like Figure 6 The diagram shown illustrates the specific preparation process of an oxygen-sensitive thin film according to an embodiment of this application. First, DMSNs precursors are synthesized using CTATos and TEA as template agents, and purified DMSNs are obtained through high-temperature sintering. Subsequently, the ruthenium-based oxygen-sensitive probe is activated by sodium hydroxide alkaline solution and adsorbed into the solution. Stable loading is achieved on the surface and within the pores of DMSNs, forming a core-shell structured nanocomposite. Next, the composite nanomaterial was uniformly dispersed in a toluene solution of PDMS prepolymer. After prolonged stirring to evaporate the solvent, a homogeneous slurry third solution was formed. Following vacuum degassing to remove residual air bubbles, the solution was spin-coated onto a substrate. Finally, thermosetting at 110°C caused the PDMS to crosslink and form a solid film. This oxygen-sensitive film preparation process systematically solves the problems associated with hydrophilic ruthenium-based oxygen-sensitive probes. Overcoming the challenges of solidification, long-term stability, and in vivo compatibility, a functional oxygen-sensitive film that is implantable, flexible, transparent, and possesses high oxygen sensitivity was successfully fabricated, providing a key optical sensing interface for subsequent in vivo multimodal imaging. The specific fabrication process is detailed above and will not be repeated here. (About...) Figure 6 The specific implementation process of each step can be referred to the above embodiments, and will not be repeated here.

[0136] like Figure 7 The image shown is a schematic diagram illustrating the effect of an oxygen-sensitive thin film prepared according to an embodiment of this application. Figure 7 The top side shows the oxygen-sensitive films prepared based on the above preparation process, in the absence of oxygen (nitrogen gas, ... Environment and aerobic (oxygen) Luminous state under the following environment: The thin film exhibits bright phosphorescence, while... The phosphorescence in the medium was significantly reduced due to the quenching effect, which directly verifies that the thin film is based on... The oxygen concentration-dependent optical response characteristics of the probe. Figure 7The lower side illustrates the flexible fit of the oxygen-sensitive membrane, depicting how it achieves close, conformal contact with the brain tissue surface. This demonstrates that the oxygen-sensitive membrane not only possesses sensitive oxygen sensing capabilities but also excellent flexibility and bio-tissue adhesion, which is the key physical and functional basis for its ability to serve as an implantable interface for in vivo, long-term, in-situ brain tissue oxygen partial pressure imaging.

[0137] like Figure 8 The diagram shown is a specific schematic diagram of an imaging system provided in an embodiment of this application, including an oxygen-sensitive film 801 and an imaging unit, wherein the imaging unit includes an illumination module 802, an acquisition module 803 and a generation module 804. The oxygen-sensitive film 801 is used to be installed on the tissue to be tested; the tissue to be tested has specific tissue cells that are sensitive to calcium signals. The illumination module 802 is used to alternately irradiate the tissue to be detected with multiple types of excitation light at target intervals through the imaging path; The acquisition module 803 is used to continuously acquire, according to the target interval, each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected using different cameras. The generation module 804 is used to generate a tissue oxygen partial pressure image of the tissue to be detected based on each frame of the first excitation light signal acquired continuously, to generate a calcium signal image of the tissue to be detected based on each frame of the second excitation light signal acquired continuously, and to generate a blood oxygen signal image of the tissue to be detected based on each frame of the reflected light signal acquired continuously.

[0138] In one possible implementation, the system further includes a preparation module 805 for preparing the oxygen-sensitive film 801 by the following steps: Dendritic mesoporous silica nanoparticles (DMSN) were prepared using hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water. The silica suspension prepared based on the DMSN and sodium hydroxide aqueous solution, and tris(4,7-phenyl-1,10-o-diazaphenanthroline)ruthenium(II) diester were used. A mixed solution of anhydrous ethanol and anhydrous ethanol was used to prepare a solution loaded with... Dendritic mesoporous silica nanoparticles ; use The oxygen-sensitive film is prepared by using a polydimethylsiloxane (PDMS) matrix and a PDMS curing agent.

[0139] In one possible implementation, the preparation module 805, during the preparation of dendritic mesoporous silica nanoparticles (DMSN) using hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water, is used to: According to the first preparation ratio, a first mass of hexadecyltrimethylammonium p-toluenesulfonate, a second mass of triethanolamine, and a first volume of deionized water are mixed and stirred at a first temperature for a first time to obtain a first solution; The second volume of tetraethyl silicate and the first solution were stirred and mixed at a second temperature for a second time to obtain the DMSN precursor; The pretreated DMSN precursor is placed into a sintering apparatus, and the temperature of the sintering apparatus is raised to a first target temperature at a preset heating rate. The first target temperature is maintained until the third duration is met, then heating is stopped and the mixture is cooled to room temperature to obtain the prepared DMSN.

[0140] In one possible implementation, the preparation module 805, in the process of preparing a silica suspension based on the DMSN and an aqueous sodium hydroxide solution, and A mixed solution of anhydrous ethanol and a homogeneous loaded solution was prepared. Dendritic mesoporous silica nanoparticles When washing with ethanol, it is used for: According to the second preparation ratio, the third mass of DMSN and the third volume of sodium hydroxide aqueous solution are mixed and stirred continuously for a fourth time to obtain a silica suspension. According to the third preparation ratio, the fourth mass Mix with the fourth volume of anhydrous ethanol solution to obtain the second solution; The second solution was added to the silica suspension, and after continuous stirring for five hours, vacuum filtration was performed to obtain... .

[0141] In one possible implementation, the preparation module 805, in the utilization The polydimethylsiloxane PDMS matrix and PDMS curing agent are used in the preparation of the oxygen-sensitive film for: According to the fourth preparation ratio, the fifth mass... The sixth mass of PDMS matrix was mixed with the fifth volume of toluene solution until it became transparent, thus obtaining the third solution; After ultrasonic treatment of the third solution for a sixth time, it is continuously magnetically stirred at a first preset speed for a seventh time at room temperature to obtain the initial composite material. The seventh mass of PDMS curing agent was added to the composite material and degassed in a vacuum environment for eight hours to obtain the target composite material; After rotating the target composite material continuously at the second preset rotation speed for a ninth time, it continues to rotate at the third preset rotation speed for a tenth time. The composite material, after being rotated for ten hours, is spin-coated onto a glass slide and cured at a second target temperature for a target time to obtain the oxygen-sensitive film.

[0142] In one possible implementation, the imaging path includes a Köhler illumination module and a polarization beam splitting module; The illumination module 802, when the imaging path alternately illuminates the tissue to be detected with multiple types of excitation light at target intervals, is used for: Multiple types of excitation light are flashed alternately at target intervals, and each type of excitation light passes through the Köhler illumination module and the polarization beam splitting module in sequence to irradiate the tissue to be detected.

[0143] In one possible implementation, the various types of excitation light include red light, infrared light, and blue light; the different cameras include at least one first camera 803-1 and one second camera 803-2, the first excitation light signal includes an oxygen phosphorescence signal excited by the blue light based on the oxygen quenching effect; the second excitation light signal includes a calcium fluorescence signal excited by the blue light based on the calcium fluorescence properties of the specific tissue cells; the reflected light signal includes reflected red light signal and reflected infrared light signal of blood oxygen in the tissue to be detected; The acquisition module 803, when continuously acquiring frames of first excitation light signals on the oxygen-sensitive film, frames of second excitation light signals of the specific tissue cells, and frames of reflected light signals of blood oxygen in the tissue to be detected using different cameras at the target interval, is used for: According to the target interval, the first camera 803-1 continuously acquires each frame of oxygen phosphorescence signal on the oxygen-sensitive film, the second camera 803-2 continuously acquires each frame of calcium fluorescence signal of the specific tissue cells, and the first camera 803-1 continuously acquires each frame of reflected red light signal or reflected infrared light signal of blood oxygen in the tissue to be detected.

[0144] In one possible implementation, the tissue to be tested includes at least brain tissue, gastrointestinal tissue, and tumor region tissue; the tissue to be tested has a preset pathological state.

[0145] For the system embodiments, since they basically correspond to the method embodiments, the relevant parts can be referred to in the description of the method embodiments. The system embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this application according to actual needs. Those skilled in the art can understand and implement this without creative effort.

[0146] like Figure 9 The diagram illustrates a specific application of the imaging method and system provided in this application. In this application example, a transgenic mouse model is used as the target, and its brain tissue is used as the target tissue. Electrodes are implanted in the hippocampus, and the aforementioned oxygen-sensitive membrane is installed. Using the imaging system described above, the field potential of the mouse brain, the phosphorescence signal of the oxygen-sensitive membrane (reflecting tissue oxygen partial pressure), the calcium fluorescence signal, and the blood oxygen reflection signal are recorded and acquired simultaneously. During this process, two interventions are applied: first, hippocampal electrical stimulation is performed (parameters: 60Hz, 1ms, 0.4mA, duration 1s), followed by an intraperitoneal injection of a specific chemical stimulant (2-carboxy-4-(1-methylvinyl)-3-pyrrolidine, dose 15mg / kg) at another time point. According to the real-time acquired data, the field potential immediately increases and aftermath discharge occurs after electrical stimulation. At the same time, the tissue oxygen partial pressure image shows a rapid decrease in tissue oxygen partial pressure, indicating brain tissue hypoxia; the calcium signal image shows a dramatic increase in neuronal activity; and the blood oxygen signal image reflects the corresponding hemodynamic changes. This embodiment demonstrates that the imaging method and imaging system provided in this application can successfully capture the complete and rapid multi-scale pathophysiological chain of "abnormal neuronal discharge - rapid tissue hypoxia - vascular response" in epilepsy cases, intuitively verifying its application value in revealing the mechanism of brain diseases.

[0147] like Figure 10The diagram illustrates a specific application embodiment two of the imaging method and system provided in this application. In this embodiment, a transient middle cerebral artery blood flow occlusion animal model is used as the subject of investigation, and its brain tissue is used as the tissue to be investigated. This animal model is typically established using the "suture occlusion method": a thin nylon suture is surgically inserted into the common carotid artery of the animal model and advanced to the origin of the middle cerebral artery to block its blood flow. The suture is retained for 60 minutes to induce focal cerebral ischemia, and then removed to restore blood flow, simulating the clinical "ischemia-reperfusion" process. First, the aforementioned oxygen-sensitive membrane is installed in a specific tissue area of ​​the subject of investigation. Subsequently, using the multimodal imaging system of this application, the tissue area to be investigated is simultaneously imaged at multiple consecutive time points: before blood flow occlusion (basal state), during occlusion (ischemic state), and after blood flow restoration (reperfusion state). The imaging system acquires and generates brain tissue oxygen partial pressure distribution maps, neuronal calcium activity images, and blood oxygen saturation change maps in real time. Experimental data show that during blood flow occlusion, the partial pressure of oxygen in the target brain region decreased significantly, calcium signal images showed abnormal activity, and blood oxygen saturation underwent characteristic changes simultaneously. After blood flow was restored, all of the above parameters showed varying degrees of recovery or dynamic evolution. This embodiment demonstrates that the imaging method and imaging system provided in this application can successfully achieve long-term, synchronous, and visualized observation of the spatiotemporal correlation between neural activity, energy metabolism disorders, and blood flow interruption during the dynamic process of cerebral ischemia and reperfusion, verifying its application capability in studying the pathophysiological mechanisms related to cerebrovascular diseases.

[0148] In addition, a specific application embodiment three based on the imaging method and system provided in this application is demonstrated. In this application embodiment three, an animal model object established by a specific cell model (GL261 glioma cell line (GL261) model) is used as the object to be detected, and the specific cell model is used as the tissue to be detected. First, specific cells are implanted into the target brain region of the animal model object through micromanipulation, and the aforementioned oxygen-sensitive membrane is installed. Subsequently, using the aforementioned multimodal imaging system, long-term, repetitive synchronous imaging of the tissue region to be detected is performed every 2 to 3 days. In each imaging, the imaging system can acquire and generate sequence data of tissue oxygen partial pressure distribution map, neuronal calcium activity image, and blood oxygen saturation change map in the region in real time. Through comparative analysis of continuous multi-time point image data, it was observed that with the physiological evolution of this specific region, the local tissue oxygen partial pressure showed a continuous downward trend, and the changes of the three parameters showed significant heterogeneity in spatiotemporal distribution, intuitively revealing the dynamic law of the synergistic evolution of neural activity, energy metabolism, and blood supply in the tissue region to be detected. This embodiment demonstrates that the imaging method and imaging system provided in this application can achieve long-term, synchronous, multi-parameter dynamic imaging and tracking of complex biological processes for up to several weeks, providing a powerful in-situ observation tool for studying their dynamic evolution mechanisms.

[0149] Based on the same technical concept, embodiments of this application also provide a computer device. (Refer to...) Figure 11 The diagram shown is a structural schematic of a computer device provided in an embodiment of this application, comprising: Processor 1101, memory 1102 and bus 1103. The memory 1102 stores machine-readable instructions executable by the processor 1101. The processor 1101 executes the machine-readable instructions stored in the memory 1102. When the machine-readable instructions are executed by the processor 1101, the processor 1101 performs the following steps: S101: Installing a pre-prepared oxygen-sensitive film with an oxygen-sensitive probe onto the tissue to be tested; the tissue to be tested contains specific tissue cells sensitive to calcium signals; S102: Alternatingly irradiating the tissue to be tested with various types of excitation light at target intervals through an imaging pathway; S103: At target intervals, continuously acquiring each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be tested using different cameras; and S104: Generating a tissue oxygen partial pressure image of the tissue to be tested based on each frame of the continuously acquired first excitation light signal, generating a calcium signal image of the tissue to be tested based on each frame of the continuously acquired second excitation light signal, and generating a blood oxygen signal image of the tissue to be tested based on each frame of the continuously acquired reflected light signal.

[0150] The aforementioned memory 1102 includes a main memory 11021 and an external memory 11022. The main memory 11021, also known as internal memory, is used to temporarily store the computational data in the processor 1101, as well as the data exchanged with external memory such as a hard disk 11022. The processor 1101 exchanges data with the external memory 11022 through the main memory 11021. When the computer device is running, the processor 1101 and the memory 1102 communicate through the bus 1103, so that the processor 1101 executes the execution instructions mentioned in the above method embodiments.

[0151] This application also provides a computer-readable storage medium storing a computer program, which, when executed by a processor, performs the steps of the imaging method described in the above-described method embodiments. The storage medium can be a volatile or non-volatile computer-readable storage medium.

[0152] This application also provides a computer program product, which carries program code. The instructions included in the program code can be used to execute the steps of the imaging method described in the above method embodiments. For details, please refer to the above method embodiments, which will not be repeated here.

[0153] The computer program product can be implemented specifically through hardware, software, or a combination thereof. In one alternative embodiment, the computer program product is specifically embodied in a computer storage medium; in another alternative embodiment, the computer program product is specifically embodied in a software product, such as a software development kit (SDK), etc.

[0154] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems and devices described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here. In the several embodiments provided in this application, it should be understood that the disclosed systems, devices, and methods can be implemented in other ways. The device embodiments described above are merely illustrative. For example, the division of units is only a logical functional division; in actual implementation, there may be other division methods. Furthermore, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Another point is that the displayed or discussed mutual coupling or direct coupling or communication connection may be through some communication interfaces; the indirect coupling or communication connection of devices or units may be electrical, mechanical, or other forms.

[0155] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0156] In addition, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit.

[0157] If the aforementioned functions are implemented as software functional units and sold or used as independent products, they can be stored in a processor-executable, non-volatile, computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to existing technology, or a portion of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this application. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0158] If the technical solution of this application involves personal information, the product using this technical solution has clearly informed the user of the personal information processing rules and obtained the user's voluntary consent before processing the personal information. If the technical solution of this application involves sensitive personal information, the product using this technical solution has obtained the user's separate consent before processing the sensitive personal information, and also meets the requirement of "express consent". For example, at personal information collection devices such as cameras, clear and prominent signs are set up to inform users that they have entered the scope of personal information collection and that personal information will be collected. If an individual voluntarily enters the collection scope, it is deemed that they have agreed to the collection of their personal information; or on the personal information processing device, with clear signs / information informing users of the personal information processing rules, authorization is obtained from the user through pop-up information or by asking the user to upload their personal information; wherein, the personal information processing rules may include information such as the personal information processor, the purpose of personal information processing, the processing method, and the types of personal information processed.

[0159] Finally, it should be noted that the above-described embodiments are merely specific implementations of this application, used to illustrate the technical solutions of this application, and not to limit them. The scope of protection of this application is not limited thereto. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the scope of the technology disclosed in this application. Such modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. An imaging method, characterized in that, The method includes: An oxygen-sensitive film with a pre-prepared oxygen-sensitive probe is mounted on the tissue to be tested; the tissue to be tested contains specific tissue cells that are sensitive to calcium signals. Through the imaging pathway, multiple types of excitation light are alternately irradiated onto the tissue to be detected according to the target interval; According to the target interval, different cameras are used to continuously acquire each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected. Based on the first excitation light signal acquired in each consecutive frame, a tissue oxygen partial pressure image of the tissue to be detected is generated; based on the second excitation light signal acquired in each consecutive frame, a calcium signal image of the tissue to be detected is generated; and based on the reflected light signal acquired in each consecutive frame, a blood oxygen signal image of the tissue to be detected is generated.

2. The method according to claim 1, characterized in that, The oxygen-sensitive thin film is prepared by the following steps: Dendritic mesoporous silica nanoparticles (DMSN) were prepared using hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water. Dendritic mesoporous silica nanoparticles Ru(dpp)3Cl2@DMSN loaded with Ru(dpp)3Cl2 were prepared using a silica suspension prepared based on the aforementioned DMSN and an aqueous sodium hydroxide solution, and a mixed solution of tris(4,7-phenyl-1,10-o-diazaphenanthroline)ruthenium(II)di(ester)Ru(dpp)3Cl2 and anhydrous ethanol solution. The oxygen-sensitive film was prepared using Ru(dpp)3Cl2@DMSN, polydimethylsiloxane PDMS matrix, and PDMS curing agent.

3. The method according to claim 2, characterized in that, The preparation of dendritic mesoporous silica nanoparticles (DMSN) using hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water includes: According to the first preparation ratio, a first mass of hexadecyltrimethylammonium p-toluenesulfonate, a second mass of triethanolamine, and a first volume of deionized water are mixed and stirred at a first temperature for a first time to obtain a first solution; The second volume of tetraethyl silicate and the first solution were stirred and mixed at a second temperature for a second time to obtain the DMSN precursor; The pretreated DMSN precursor is placed into a sintering apparatus, and the temperature of the sintering apparatus is raised to a first target temperature at a preset heating rate. The first target temperature is maintained until the third duration is met, then heating is stopped and the mixture is cooled to room temperature to obtain the prepared DMSN.

4. The method according to claim 2, characterized in that, The preparation of uniformly loaded Ru(dpp)3Cl2 dendritic mesoporous silica nanoparticles Ru(dpp)3Cl2@DMSN using a silica suspension prepared based on DMSN and sodium hydroxide aqueous solution, and a mixed solution of Ru(dpp)3Cl2 and anhydrous ethanol, and washing with ethanol, includes: According to the second preparation ratio, the third mass of DMSN and the third volume of sodium hydroxide aqueous solution are mixed and stirred continuously for a fourth time to obtain a silica suspension. According to the third preparation ratio, the fourth mass of Ru(dpp)3Cl2 and the fourth volume of anhydrous ethanol solution are mixed to obtain the second solution; The second solution was added to the silica suspension, and after continuous stirring for five hours, vacuum filtration was performed to obtain Ru(dpp)3Cl2@DMSN.

5. The method according to claim 2, characterized in that, The oxygen-sensitive film is prepared using Ru(dpp)3Cl2@DMSN, polydimethylsiloxane PDMS matrix, and PDMS curing agent, comprising: According to the fourth preparation ratio, the fifth mass of Ru(dpp)3Cl2@DMSN and the sixth mass of PDMS matrix were mixed in the fifth volume of toluene solution until transparent to obtain the third solution; After ultrasonic treatment of the third solution for a sixth time, it is continuously magnetically stirred at a first preset speed for a seventh time at room temperature to obtain the initial composite material. The seventh mass of PDMS curing agent was added to the composite material and degassed in a vacuum environment for eight hours to obtain the target composite material; After rotating the target composite material continuously at the second preset rotation speed for a ninth time, it continues to rotate at the third preset rotation speed for a tenth time. The composite material, after being rotated for ten hours, is spin-coated onto a glass slide and cured at a second target temperature for a target time to obtain the oxygen-sensitive film.

6. The method according to claim 1, characterized in that, The imaging pathway includes a Köhler illumination module and a polarization beam splitting module; The process of alternately irradiating the tissue to be detected with multiple types of excitation light at target intervals through an imaging pathway includes: Multiple types of excitation light are flashed alternately at target intervals, and each type of excitation light passes through the Köhler illumination module and the polarization beam splitting module in sequence to irradiate the tissue to be detected.

7. The method according to claim 1, characterized in that, The various types of excitation light include red light, infrared light, and blue light; the different cameras include at least one first camera and one second camera, the first excitation light signal includes the oxygen phosphorescence signal excited by the blue light based on the oxygen quenching effect; the second excitation light signal includes the calcium fluorescence signal excited by the blue light based on the calcium fluorescence characteristics of the specific tissue cells; the reflected light signal includes the reflected red light signal and the reflected infrared light signal of blood oxygen in the tissue to be detected; The step of continuously acquiring, according to the target interval, each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected using different cameras includes: According to the target interval, the first camera continuously acquires each frame of oxygen phosphorescence signal on the oxygen-sensitive film, the second camera continuously acquires each frame of calcium fluorescence signal of the specific tissue cells, and the first camera continuously acquires each frame of reflected red light signal or reflected infrared light signal of blood oxygen in the tissue to be detected.

8. The method according to claim 1, characterized in that, The tissue to be tested includes at least brain tissue, gastrointestinal tissue, and tumor area tissue; the tissue to be tested has a preset pathological state.

9. An imaging system, characterized in that, The system includes an oxygen-sensitive thin film and an imaging unit, the imaging unit including an illumination module, an acquisition module and a generation module; The oxygen-sensitive membrane is used to be installed on the tissue to be tested; the tissue to be tested has specific tissue cells that are sensitive to calcium signals. The illumination module is used to alternately irradiate the tissue to be detected with multiple types of excitation light at target intervals through the imaging path; The acquisition module is used to continuously acquire, according to the target interval, each frame of the first excitation light signal on the oxygen-sensitive film, each frame of the second excitation light signal of the specific tissue cells, and each frame of reflected light signal of blood oxygen in the tissue to be detected using different cameras. The generation module is used to generate a tissue oxygen partial pressure image of the tissue to be detected based on each frame of the first excitation light signal acquired continuously, to generate a calcium signal image of the tissue to be detected based on each frame of the second excitation light signal acquired continuously, and to generate a blood oxygen signal image of the tissue to be detected based on each frame of the reflected light signal acquired continuously.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the program is executed by a processor, it implements the steps of the method as described in any one of claims 1 to 8.

11. A computer device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that, When the processor executes the program, it implements the steps of the method as described in any one of claims 1 to 8.

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