Experimental system and method for research on photodynamic therapy, photothermal therapy and combination of photodynamic therapy and photothermal therapy

Abstract

The invention relates to an experimental system and method for photodynamic therapy, photothermal therapy and combined research of photodynamic therapy and photothermal therapy. The experiment system comprises an optical irradiation module which comprises a plurality of laser light sources, each laser light source can output collimated light beams, the collimated light beams are combined to form a coaxial light path, and the coaxial light paths are projected to the surface of an observation object through a light beam adjusting assembly; the light path position adjusting module comprises an electric Z-axis displacement mechanism; the temperature monitoring module comprises an active temperature control substrate, an infrared thermal imager and an optical fiber temperature probe interface; the data acquisition and control module is configured to synchronously acquire laser output power, light spot area, electric Z-axis position, temperature of the active temperature control substrate, temperature sequences of a plurality of different areas and optical fiber temperature probe data, and associatively record the data with a unified timestamp; the power density can be calculated based on the real-time output power of the laser light source and the light spot area; and when the data of any temperature monitoring channel exceeds a preset safety threshold value, the laser light source is automatically controlled to close the output.

Description

Technical Field

[0001] This invention relates to the field of photodynamic therapy (PDT), photothermal therapy (PTT), and novel photodynamic / photothermal combination research technology. It relates to an experimental system and method for research on photodynamic therapy, photothermal therapy, and their combinations. In particular, it relates to a research device that can achieve comparability of different wavelength illumination conditions, controllable temperature field, transparent data, and is applicable to in vitro cell models and in vivo tumor models. Background Technology

[0002] In recent years, photodynamic therapy (PDT) and photothermal therapy (PTT), as novel tumor treatments with high spatiotemporal selectivity and minimally invasive advantages, have made significant progress in the biomedical field. To further improve the precision and efficiency of treatment, researchers have developed a variety of novel nanotherapeutic agents. Studies (such as Dong, Y., Xia, P., Xu, X. et al. Targeted delivery of organic small-molecule photothermal materials with engineered extracellular vesicles for imaging-guided tumor photothermaltherapy. J Nanobiotechnol 21, 442 (2023)) have reported the use of engineered extracellular vesicles (EVs) as carriers to load organic small-molecule photothermal agents (such as Croconaine dyes) or inorganic nanomaterials (such as quantum dots) to achieve tumor-targeted imaging-guided photothermal therapy. These studies typically use near-infrared lasers of a specific wavelength (such as 808nm) to irradiate the tumor site, converting light energy into heat energy through a photothermal agent to ablate the tumor, and rely on infrared thermal imaging technology to evaluate the photothermal conversion efficiency and treatment effect.

[0003] However, while photosensitive / photothermal materials used for therapy have achieved remarkable precision in chemical modification and biological targeting, the experimental setups and methods for evaluating their performance lag behind, lacking a standardized system. In most existing literature, including the aforementioned exosome studies, experimental platforms are typically cobbled together from separate lasers, handheld or stand-mounted infrared thermal imagers, and animal heating devices. This fragmented equipment architecture exposes a series of common technical problems in practical applications, severely impacting the physical consistency and reproducibility of experimental data.

[0004] First, the uniformity and comparability of illumination conditions are difficult to guarantee. Under existing experimental models, laser collimation and focusing typically lack a unified optical path adjustment mechanism, especially when involving multi-wavelength studies (such as visible light PDT and near-infrared PTT), where the spot morphology and focal spot position of different light sources are difficult to align. Furthermore, when the observation subject changes from a flat cell culture dish to a raised subcutaneous tumor model, the change in sample height leads to a change in the spot area. If researchers only calculate the power density based on the laser's nominal power (e.g., setting it to 0.8 W / cm²), the results are inconvenient. 2 Ignoring the decrease in actual energy density caused by light spot diffusion will directly lead to a misjudgment of the dose-effect relationship.

[0005] Secondly, the accuracy of temperature monitoring data is significantly affected by environmental background. In small animal experiments, heating pads or temperature-controlled stages are typically used to maintain the animal's body temperature. Existing general-purpose infrared thermal imagers often directly read the absolute temperature value within the field of view (e.g., recording a tumor surface temperature exceeding 50°C). This value is actually a mixture of laser-induced photothermal temperature rise, the animal's own body temperature, and background thermal radiation from the heating pad. Due to the lack of real-time background difference algorithms for regions of interest (ROIs), researchers struggle to distinguish the actual temperature rise contributed by the material from the environmental background heat, easily overestimating the material's photothermal conversion efficiency. Furthermore, infrared imaging can only characterize the temperature distribution on the tissue surface and cannot detect the actual temperature deep within the tumor. This "surface-to-inside" inference method has a significant blind spot when dealing with thick tissues or deep tumors.

[0006] Finally, the completeness and transparency of experimental data urgently need improvement. In traditional non-integrated experiments, laser control, image acquisition, and temperature recording are often carried out separately, lacking a unified timestamp synchronization mechanism. This discrete data recording method not only increases human error but also makes it difficult to fully trace the experimental process. In the absence of mandatory multi-channel synchronous recording, experimental results may face the risk of "data fragmentation" or "selective reporting" (e.g., only showing the optimal heating curve while ignoring fluctuation data), reducing the credibility of scientific conclusions. Therefore, developing a standardized experimental system that integrates optical path consistency control, background thermal interference elimination, and full-process data synchronous recording has become a key technical problem that urgently needs to be solved in this field.

[0007] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an experimental system and method for studying photodynamic therapy, photothermal therapy, and their combinations, thereby solving at least some of the aforementioned technical problems. One objective of this invention is to provide an experimental device that can truly solve the above-mentioned problems, making the illumination conditions comparable, the temperature field quantifiable, the data transparent and traceable, and the results difficult to manipulate in photodynamic / photothermal research.

[0009] This invention discloses an experimental system for research on photodynamic therapy, photothermal therapy, and their combinations, comprising: The optical illumination module includes at least two laser sources of different wavelengths, each laser source is equipped with an independent collimation component to output a collimated beam; the collimated beams are combined by a beam combiner to form a coaxial optical path, and then projected onto the surface of the object being observed by a beam adjustment component; The optical path position adjustment module includes an electric Z-axis displacement mechanism, which is used to adjust the distance between the optical illumination module and the surface of the observed object so that the area of ​​the light spot projected on the surface of the observed object is maintained at a preset value. The temperature monitoring module includes: an active temperature control substrate, positioned below the object being observed, for providing a uniform background temperature; an infrared thermal imager, positioned above or to the side of the object being observed, for simultaneously acquiring real-time temperature images including the region of interest and the background region; and a fiber optic temperature probe interface, for connecting a fiber optic temperature probe to measure the internal temperature of the object being observed. The data acquisition and control module is configured to: synchronously acquire laser output power, spot area, electric Z-axis position, temperature of active temperature control substrate, temperature sequences of two different regions, and fiber optic temperature probe data, and record the above data with a unified timestamp; calculate power density based on the real-time output power and spot area of ​​the laser source; and automatically control the laser source to shut down when the data of any temperature monitoring channel exceeds a preset safety threshold.

[0010] The experimental system of this invention systematically solves three core technical problems commonly found in existing technologies—"opaque illumination conditions," "temperature measurement contamination by background heat," and "selective data reporting"—through modular integration of light, heat, and control. First, the architecture, which independently collimates different wavelength light sources and coordinates with electric Z-axis adjustment, directly addresses the technical pain point of "incomparable power densities due to inconsistent light paths and light spots." By using Z-axis height adjustment to forcibly unify the light spot size of different wavelengths on the observation surface, the power density calculated by the data acquisition and control module is no longer a nominal value but a physically meaningful real value. Second, the linkage between the temperature monitoring module and the data acquisition and control module, through an active temperature-controlled substrate providing a stable background and combined with synchronous recording, solves the problem of "the inseparability of photothermal effects and ambient temperature," enabling net measurement of photothermal conversion efficiency. Finally, the mandatory multi-channel synchronous data acquisition mechanism cuts off the space for manually selecting the "optimal curve" from the hardware level, establishing an unalterable experimental evidence chain, thus providing a standard experimental platform that truly achieves comparable experimental conditions, quantifiable temperature fields, and transparent and traceable data.

[0011] In particular, to improve the temperature monitoring accuracy of multi-wavelength photothermal measurements and avoid "pseudo-temperature rise differences" introduced by the inconsistency between the position of the light spot of different wavelengths and the focal plane, this system first constructs a coaxial confocal optical path as the optical baseline for thermal measurements by the optical illumination module before the temperature control and differential algorithm links. The beam combining component uses a dichroic mirror, which utilizes its beam splitting characteristics for different wavelengths (e.g., high transmission at 660nm and high reflection at 808nm) to combine two spatially separated collimated beams into a coaxial output after calibration. This significantly reduces the difference in temperature rise between different wavelengths during wavelength switching or dual-band comparison. Lateral shift of the light spot on the sample surface; further, for the axial focal shift caused by material dispersion of the refractive focusing element, the focusing module is equipped with an achromatic lens to correct the focal difference between 660nm and 808nm, or a reflective parabolic mirror is equipped to take advantage of the color difference-free characteristic of reflective imaging, so that the focal planes of the two wavelengths overlap in the Z-axis direction and the focal position difference is controlled within a preset threshold, thereby ensuring that the region of interest ROI-1 has a consistent light-receiving position and focusing conditions under different wavelengths of illumination, so that the temperature change of ROI-1 is no longer mixed with geometric disturbances caused by light spot drift or defocus. Based on this, the active temperature control substrate clamps the background temperature of the corresponding background area to a constant value (e.g., 37°C) through a semiconductor cooler or liquid circulation, forming a thermodynamic steady-state boundary and suppressing environmental thermal fluctuations such as heat absorption by culture medium evaporation or animal body temperature drift, so that the background area ROI-2 captured by the infrared thermal imager remains stable. As a result, the differential temperature ΔT (ROI-1 minus ROI-2) executed by the data acquisition and control module obtains a higher signal-to-noise ratio under the dual constraints of coaxial confocal optical input and stable background thermal baseline. Its change can be more purely mapped to the real photothermal temperature rise induced by laser, thereby removing environmental interference and geometric errors at the algorithm level and realizing a comparable and repeatable "net value" measurement of the photothermal conversion efficiency of materials.

[0012] According to a preferred embodiment, the optical illumination module employs any of the following optical path configurations: The beam combining component uses a dichroic mirror for beam combining. The dichroic mirror has a transmittance of ≥95% for the first wavelength and a reflectance of ≥98% for the second wavelength. The beam combiner uses multiple dielectric film mirrors to sequentially introduce beams of different wavelengths into the coaxial optical path to eliminate chromatic aberration over a wide spectral range. An adjustable beam splitter is provided after beam combining to split a single coaxial beam into two parallel sub-spots, so as to form a parallel control experimental area on the same observation object.

[0013] To address the issue of data incomparability caused by the misalignment of different wavelength spot positions with the focal plane in multi-wavelength experiments, the optical illumination module of this invention constructs a coaxial confocal optical path: the beam combining component employs a dichroic mirror, utilizing its beam-splitting characteristics for different wavelengths (e.g., high transmission at 660nm and high reflection at 808nm). After calibration, it combines two spatially separated collimated beams into a coaxial output, thereby significantly reducing the lateral shift of different wavelength spots. Furthermore, to address the axial focal shift caused by material dispersion in the refractive focusing element, the focusing module is equipped with an achromatic lens to correct the focal difference between 660nm and 808nm; or a reflective parabolic mirror is configured to utilize the color-aberration-free characteristic of reflective imaging, achieving the overlap of the focal planes of different wavelengths in the Z-axis direction, so that the focal position difference between the two wavelengths can be controlled within a preset threshold.

[0014] This invention deepens the solution to the optical path consistency problem by further defining the specific configuration of the optical illumination module. When using a dichroic mirror beam combining scheme, the high transmission and high reflection of specific wavelengths solves the technical problem of the difficulty in perfectly aligning multiple wavelength light sources in physical space, achieving efficient transmission of lasers of different wavelengths in the coaxial optical path and ensuring that photodynamic excitation (e.g., 660nm) and photothermal triggering (e.g., 808nm) act on the exact same cell or tissue region. When using a dielectric film mirror combination, the physical characteristic of the reflective optical path being achromatic solves the problem of focal axis separation caused by lens dispersion over a wide spectral range, preventing energy density estimation errors caused by focal plane misalignment in multi-wavelength experiments. The introduction of a beam splitter creates a parallel control area with completely equal physical conditions on the same observation object, effectively eliminating time drift errors caused by sequential experiments and ensuring strict consistency of the comparative experiments from an optical physics perspective.

[0015] According to a preferred embodiment, the beam adjustment assembly adopts any of the following structures: Achromatic lenses or reflective parabolic mirrors are used to reduce the focal difference between beams of different wavelengths; A microlens array or speckle shaping plate is provided in front of the beam conditioning component used for focusing, so that the spot area presents a top-hat-shaped energy distribution; An electric scanning galvanometer is used to drive the light spot to scan and cover the surface of the observed object at a preset frequency.

[0016] This invention addresses the dose assessment distortion caused by uneven energy distribution of the laser beam by optimizing the focusing and shaping components. By employing achromatic lenses or reflective parabolic mirrors, the refractive index differences of different wavelengths during transmission are specifically corrected, ensuring that the focal points of multi-wavelength lasers highly coincide along the Z-axis, avoiding energy density calculation errors caused by "one wavelength focusing while another is defocused." The introduction of microlens arrays or speckle shaping plates transforms the original Gaussian energy profile of the laser into a flat-topped, cap-shaped distribution, resolving the "central necrosis with peripheral survival" phenomenon where excessive energy at the center of a Gaussian beam easily leads to nonspecific thermal damage, while insufficient energy at the edges results in ineffective treatment, thus improving the uniformity of biological experimental results. The application of an electric scanning galvanometer transforms static irradiation into dynamic uniform scanning, providing a standardized excitation method for high-throughput material screening and avoiding human error caused by manual beam movement.

[0017] According to a preferred embodiment, the optical path position adjustment module is further configured with one or more of the following components: Universal joints or swivel brackets are used to adjust the incident angle of the optical illumination module, allowing it to tilt within a range of ±45°. A three-dimensional displacement module is used to replace a single Z-axis to realize the relative displacement of the optical illumination module or stage in the X, Y, and Z directions.

[0018] This invention resolves the technical contradiction of "mismatch between animal position and vertical optical path" in in vivo experiments by introducing a multi-degree-of-freedom mechanical adjustment mechanism. The combination of the universal joint and the three-dimensional displacement module breaks the limitation of traditional equipment that can only irradiate vertically, allowing the optical irradiation module to accurately align with irregular tumors located on the flank or axilla of mice at any normal angle. This design not only avoids the physiological stress caused by forcibly twisting the animal's position, but more importantly, it ensures that the laser always incident perpendicularly on the tumor surface, reducing Fresnel reflection loss caused by the tilt of the incident angle. This ensures that the actual light energy entering the tissue is consistent with the set value, further solidifying the physical basis of "comparable power density".

[0019] According to a preferred embodiment, the infrared thermal imager is mounted at an angle of 30° to 60° to the side of the optical path, and: a long-pass filter is provided in front of the optical path of the infrared thermal imager, the filter being configured to transmit 8 to 14 μm wavelengths while blocking visible and ultraviolet light; or, the infrared thermal imager is mounted on a movable rotating bracket, responding to a control signal generated by a data acquisition and control module, the control signal being generated based on image tracking technology to compensate for the displacement of the area identified by the image recognition module, thereby locking the corrected temperature measurement area in real time and measuring its temperature while maintaining a preset tilt angle to compensate for the displacement of the observed object.

[0020] This invention proposes targeted solutions to the problems of "optical artifact interference" and "dynamic position drift" in infrared thermometry. The addition of a long-pass filter completely blocks scattered light from the laser source and stray light from the environment by utilizing spectral cutoff characteristics, solving the problem of "false high-temperature readings" caused by infrared thermal imagers receiving laser reflection signals, and ensuring that the measured temperature originates entirely from thermal radiation. Furthermore, the image recognition-based tracking technology addresses the problem of tumor displacement caused by respiratory movements in live animals by establishing a real-time closed-loop correction mechanism. The system can dynamically lock onto the region of interest (ROI) to prevent the temperature measurement point from slipping into the background area, thereby ensuring the continuity and accuracy of temperature data during long-term monitoring and avoiding data breaks caused by ROI drift.

[0021] According to a preferred embodiment, the fiber optic temperature probe interface is configured as follows: connecting a single fiber optic probe for insertion into a subcutaneous tumor of an animal at a position of 1-3 mm; or connecting dual-channel or multi-channel fiber optic probes for insertion into different depths or positions of the observed object to construct an internal temperature gradient curve.

[0022] This invention directly addresses the critical technological gap of "lack of deep tissue temperature measurement and the absence of a gold standard for deep tissue temperature" by introducing an invasive fiber optic probe. Existing infrared imaging can only characterize surface temperature, often leading to an underestimation of the risk of thermal damage to deep tissues or misjudgment of treatment efficacy. This approach inserts a fiber optic probe 1 to 3 millimeters into the tumor to directly acquire the true temperature rise data of the core region, constructing a three-dimensional temperature measurement system combining internal and external measurement. This not only provides the only gold standard data for assessing the penetration capability of photothermal agents in deep tissues but also serves as a basis for safety interlocking, preventing unintended biological damage caused by internal overheating even when the surface temperature is within acceptable limits, significantly improving the safety and scientific rigor of the experiment.

[0023] According to a preferred embodiment, the active temperature control substrate is provided with a semiconductor cooler or a liquid circulation channel. The liquid circulation channel is located inside the tray of the object being observed. When the liquid circulation channel is used, the channel is connected to an external constant temperature water bath device to maintain the temperature stability of the bottom of the object being observed through the circulating liquid flow.

[0024] This invention utilizes an actively temperature-controlled substrate to solve the problem of "environmental thermal noise interference" from the perspective of thermodynamic boundary conditions. A constant-temperature platform (e.g., 37 degrees Celsius) constructed through a semiconductor cooler or liquid circulation channel provides a large, constant-temperature heat reservoir for the observed object, effectively shielding it from interference from environmental temperature fluctuations on the measurement of trace thermal effects. Particularly for open cell culture experiments, the uniform bottom heating provided by the liquid circulation channel is superior to traditional air baths, effectively preventing changes in optical properties and osmotic pressure fluctuations caused by culture medium evaporation. This active steady-state environment is the physical prerequisite for subsequent high-sensitivity differential temperature measurement, ensuring that any captured temperature rise signal primarily originates from photothermal conversion rather than environmental heat accumulation.

[0025] According to a preferred embodiment, the data acquisition and control module is configured to perform the following operations: calculate the differential temperature ΔT in real time, where ΔT is the difference between the average temperature and the highest temperature of the region of interest and the background region, in order to eliminate background thermal interference from the active temperature control substrate and the body temperature of the observed object itself.

[0026] This invention effectively addresses the technical challenge of separating the photothermal effect from the temperature field through a real-time differential temperature algorithm (ΔT calculation). Addressing the issue in existing technologies where heating pads are commonly used to maintain body temperature, resulting in infrared thermometry readings that are a mixture of photothermal temperature rise, body temperature, and heating pad background, this solution utilizes the differential principle to subtract temperature fluctuations in the background region in real time. This algorithm, analogous to "tare-weighing," maximally suppresses common-mode interference signals such as the animal's own metabolic heat production, the heating pad's heat conduction, and environmental temperature drift, extracting the differential-mode temperature rise signal primarily caused by laser excitation. This yields net data that truly reflects the material's photothermal conversion efficiency, eliminating experimental errors caused by background heat.

[0027] According to a preferred embodiment, the data acquisition and control module is further configured to: prohibit users from turning off any temperature or power recording channel during the recording process to ensure data integrity; support multi-wavelength sequence control, automatically switch the on / off state and power of different wavelength laser sources according to a preset time sequence, and record the switching events in a unified timestamp.

[0028] This invention addresses the integrity risk of selective data reporting in scientific research through a mandatory data association and recording mechanism. It prohibits closing recording channels and generating unmodifiable logs, forcing experimental records to cover the entire process from preheating and illumination to cooling. This eliminates the practice of simply capturing data from the "fastest heating phase" or "highest temperature moment," ensuring complete traceability of the experimental process. Furthermore, the multi-wavelength sequence control function automates the complex illumination process, eliminating time errors caused by manual light source switching. This ensures strict repeatability of the energy injection sequence for each experiment in multi-step cascade reaction studies, providing a transparent and verifiable data chain for peer review.

[0029] This invention also discloses an experimental method for studying photodynamic therapy, photothermal therapy, and their combinations, comprising the following steps: Optical path configuration steps: At least two lasers of different wavelengths are independently collimated and then combined through a beam combiner. The optical path is adjusted using an electric Z-axis displacement mechanism to form a preset light spot on the surface of the observed object. Temperature baseline construction steps: Activate the active temperature control substrate, stabilize its temperature at the set value, and obtain the temperature background of the observed object when it is not irradiated; Temperature monitoring steps: Use an infrared thermal imager to acquire the temperature of the region of interest and the background region, and calculate ΔT, where ΔT is the difference between the average temperature and the highest temperature of the region of interest and the background region; if internal temperature measurement is required, insert an optical fiber temperature probe to measure the internal temperature of the observed object. Irradiation and synchronous recording steps: Irradiate with a preset power density, and at the same time, the data acquisition module synchronously records the laser power, spot area, Z-axis position, substrate temperature, temperature sequence of two different regions and fiber probe temperature. Safety control procedures: When ΔT or internal temperature exceeds the safety threshold, the irradiation will be automatically terminated and the reason for termination will be recorded.

[0030] The experimental method of this invention solidifies the aforementioned hardware and algorithms into a standardized operating paradigm, systematically solving the data silo problem caused by "methodological inconsistency" in photodynamic / photothermal research. Through five rigorous logical steps—optical path configuration, baseline construction, differential monitoring, synchronous recording, and security control—this method mandates that experiments must be conducted under conditions of "consistent light spots," "background subtraction," and "full recording." This standardized workflow breaks away from the crude mode of arbitrarily adjusting distances and ignoring background thermal interference in traditional experiments, ensuring strict cross-comparability of data produced by different laboratories. This provides a scientific and rigorous evaluation system for objectively assessing the clinical translational potential of novel photosensitive drugs and photothermal materials. Attached Figure Description

[0031] Figure 1 This is a hardware connection diagram of an experimental system according to a preferred embodiment of the present invention, showing the data interaction logic between each sub-module and the control module; Figure 2 This is a three-dimensional structural diagram (view 1) of an experimental system according to a preferred embodiment of the present invention, showing the layout of each hardware component on the optical vibration isolation platform; Figure 3 This is a three-dimensional structural schematic diagram (view 2) of an experimental system according to a preferred embodiment of the present invention, which further shows the details of the optical fiber connection and mechanical adjustment mechanism; Figure 4 This is a schematic diagram of the optical path principle of a dual-wavelength optical illumination module according to a preferred embodiment of the present invention, showing the optical path of independent collimation and beam focusing; Figure 5 This is a flowchart of the experimental method according to a preferred embodiment of the present invention.

[0032] List of reference numerals 100: Optical illumination module; 110: Collimation component; 120: Beam combining component; 130: Focusing module; 140: Laser; 200: Optical path position adjustment module; 210: Electric Z-axis displacement mechanism; 300: Temperature monitoring module; 310: Active temperature control substrate; 320: Experimental tray; 330: Infrared thermal imager; 340: Contact fiber optic probe; 400: Data acquisition and control module; 500: Optical vibration isolation platform. Detailed Implementation

[0033] The following is a detailed explanation with reference to the accompanying drawings.

[0034] Example 1 This embodiment provides an experimental system for research on photodynamic therapy (PDT), photothermal therapy (PTT), and their combinations. For example... Figure 1 and Figure 2 As shown, the system may include an optical illumination module 100, an optical path position adjustment module 200, a temperature monitoring module 300, and a data acquisition and control module 400. All modules are integrated and mounted on a perforated optical vibration isolation platform 500 to ensure optical path stability and alignment accuracy.

[0035] Preferably, for the optical illumination module 100, this embodiment adopts a dual-wavelength independent collimation combined with a dichroic mirror beam combining architecture to solve the problems of inconsistent light spots and color difference between different wavelengths. Figure 4As shown, the system is configured with two independent light sources: one is a laser 140 with an output wavelength of 660nm (commonly used for photodynamic excitation), and the other is a laser 140 with an output wavelength of 808nm (commonly used for photothermal triggering). Exemplarily, both lasers 140 output through an FC / PC interface and are transmitted via multimode fiber to their respective independent collimation components 110. After collimation, the two laser beams form quasi-parallel beams with a diameter of approximately 10mm. These two parallel beams are incident on a dichroic mirror (beam combiner 120) in horizontal and vertical directions, respectively. This dichroic mirror undergoes a special coating treatment, achieving a transmittance of over 95% for 660nm wavelength light and a reflectance of over 98% for 808nm wavelength light, thereby achieving beam combining of the two beams on the same optical axis. The combined beam then enters the achromatic focusing module 130, which uses an achromatic lens design to effectively correct chromatic aberration and control the difference in the focal lengths of the two wavelengths, 660nm and 808nm, within 0.2mm in the axial direction, ensuring the physical consistency of the two wavelengths acting on the same sample area.

[0036] Preferably, the aforementioned optical components (including the collimation component 110, the dichroic mirror, and the focusing module 130) can be integrally mounted on the electric Z-axis displacement mechanism 210 of the optical path position adjustment module 200. For example, the electric Z-axis displacement mechanism 210 has a stroke of 80 mm and a step resolution of up to 5 μm, enabling the optical head to move up and down with high precision relative to the surface of the observed object. This design allows the operator to flexibly adjust the focal length according to the height difference of the observed object (such as a cell culture dish or mouse tumor) until a light spot of a preset diameter is obtained on the sample surface. More importantly, the data acquisition and control module 400 can automatically calculate and display the actual power density (unit: mW / cm²) in real time based on the current light spot area (calculated from the Z-axis height) and the output power of the laser 140. 2 Furthermore, this module is equipped with a constant power density closed-loop control algorithm: when the researcher adjusts the Z-axis position, causing a change in the spot area, the system automatically adjusts the output power of laser 140 in the opposite direction to maintain the preset power density value. Conversely, if the laser power experiences thermal decay, the system can also fine-tune the Z-axis position to reduce the spot size to compensate for the density. This two-way closed-loop feedback mechanism eliminates the problem of inaccurate dosing caused by errors in spot size estimation in traditional experiments.

[0037] Preferably, for the optical illumination module 100, this embodiment adopts a dual-wavelength independent collimation combined with dichroic mirror beam combining architecture. The beam combining component 120 is a dichroic mirror beam combining structure disposed downstream of each independent collimation component 110. When the first wavelength is 660nm and the second wavelength is 808nm, it is stably positioned at a preset incident angle for beam splitting and beam combining, and has a transmittance of ≥95% for the first wavelength and a reflectance of ≥98% for the second wavelength. This allows the two spatially separated collimated beams output from each collimation component 110 to be coaxially combined at the beam combining component 120 and form a coaxial output optical path with a substantially consistent lateral position on the surface of the observed object.

[0038] Preferably, the focusing module 130, as a downstream focusing component of the coaxial output optical path, is in a confocal correction position in the form of an achromatic lens or a reflective parabolic mirror when the coaxial beam combining relationship is established. This ensures that the 660nm beam and the 808nm beam have a focal plane overlap in the Z-axis direction and limits the difference between the focal positions of the two wavelengths to a preset threshold (e.g., ≤0.2mm). This allows the electric Z-axis displacement mechanism 210 to continuously and repeatably adjust the distance between the optical illumination module 100 and the surface of the observed object, so that the distance change corresponds to the same focusing state for different wavelengths.

[0039] Preferably, for the temperature monitoring module 300, this embodiment features an active temperature control substrate 310 and a detachable experimental tray 320 positioned directly below the optical path. This aims to provide a stable and controllable thermal background for the observed object, thereby eliminating interference from ambient temperature fluctuations and animal body temperature on the experimental data. Exemplarily, the active temperature control substrate 310 embeds a TEC (thermal condenser) module and an NTC temperature sensor. Through a PID closed-loop control algorithm, the temperature of the substrate surface is precisely maintained at 37℃ ± 0.2℃ (simulating physiological temperature). The experimental tray 320 is placed on this constant-temperature substrate to support cell culture dishes or experimental animals. This design avoids the uneven temperature distribution and resulting infrared temperature measurement background noise associated with traditional heating pads.

[0040] Preferably, the temperature monitoring module 300 can use a combination of a non-contact infrared thermal imager 330 and a contact fiber optic probe 340 for temperature monitoring. For example... Figure 2 and Figure 3 As shown, the infrared thermal imager 330 is mounted to the side of the optical path, at an angle of approximately 45° to the surface of the observed object, ensuring that its field of view simultaneously covers the illuminated area (defined as ROI-1) and the adjacent unilluminated background area (defined as ROI-2). The system uses image algorithms to obtain the average and maximum temperatures of ROI-1 and ROI-2 in real time and performs differential calculations, outputting ΔT=T. ROI-1 –T ROI-2 , among which, T ROI-1T represents the average or highest temperature of ROI-1. ROI-2 The average or maximum temperature of ROI-2 is used, and both are taken when calculating ΔT. This difference value can automatically subtract the background temperature rise caused by substrate heating or animal body temperature, retaining only the true photothermal temperature rise data caused by laser irradiation. In addition, for mouse tumor and other thick tissue samples, this embodiment is also equipped with an optical fiber temperature probe that can be inserted from the side of the experimental tray 320. The probe tip can penetrate 1-3 mm into the center of the tumor for direct measurement of the real-time temperature of deep tissues, making up for the limitation of the infrared thermal imager 330, which can only measure the surface temperature.

[0041] Preferably, the system can perform forced synchronous acquisition and recording of all experimental parameters through the data acquisition and control module 400. The system integrates all channel data, including laser 140 power, current, Z-axis position (and corresponding spot area), ROI-1 temperature sequence, ROI-2 temperature sequence, substrate temperature, and fiber optic probe temperature, into a complete data chain using a unified timestamp, and writes it to a log file with an unmodifiable format. Specifically, to achieve data tamper-proofing and integrity verification, the data acquisition and control module 400 uses an encrypted frame structure for storage. Each data frame contains: [frame header identifier - millisecond-level absolute timestamp - unique hardware ID of the device - original data of each channel - hash value of the previous frame - CRC32 checksum]. The system runs a daemon thread independent of the user interface in the underlying firmware. This daemon thread is configured to forcibly lock the data writing channel and block any "stop recording" or deletion commands from the user interface as soon as current output from the laser 140 is detected or the temperature monitoring module 300 is active. Only after the laser output returns to zero and the temperature returns to baseline, and after a preset cooling delay, does the daemon thread release its recording privileges. This full-channel forced recording mechanism, based on underlying thread locking and chained hash verification, ensures the transparency and traceability of the experimental process and prevents selective data reporting. Simultaneously, the module also features a safety interlock function: if the internal temperature measured by the fiber optic probe exceeds 45°C (or other user-defined thresholds), or the surface temperature of ROI-1 is too high, the system will respond in milliseconds and automatically cut off the laser output. Simultaneously, it will automatically mark a "safety trigger" event in the log and force the data recording of each temperature monitoring channel to continue until the temperature drops back to a safe range. This "recording continues even when light is cut off" control logic prevents unexpected excessive thermal damage to the experimental object and fully preserves the thermal relaxation data after the incident for subsequent analysis.

[0042] Under the constraint of coaxial beam combining relationship and focal plane coincidence relationship that "the same absolute Z-axis position corresponds to the same optical focusing geometry", the data acquisition and control module 400 performs a power / area calculation relationship between the real-time acquired laser output power and the actual spot area to output power density. This ensures that the power density remains comparable and physically accurate under multi-wavelength switching conditions, thereby reducing dose-effect misjudgment caused by lateral spot drift, axial defocusing and uneven energy distribution.

[0043] Example 2 This embodiment is a further improvement on embodiment 1, and repeated content will not be described again.

[0044] This embodiment is an optimized design based on Embodiment 1, specifically addressing the unique requirements of photodynamic experiments on adherent cells in multi-well plates. In this scenario, the observed objects are typically shallowly adherent cells cultured in 6-well, 12-well, or 96-well plates, with a monolayer thickness usually less than 15 μm, making them extremely sensitive to the uniformity of light spot energy distribution.

[0045] Preferably, for the optical illumination module 100, in order to overcome the differences in cell phototoxicity caused by excessively high energy at the center of the Gaussian beam, this embodiment performs specific shaping optimization on the optical path. The beam adjustment component, as a shaping element disposed on the focusing path, while the actual spot area is calculated in real time, uses a microlens array or speckle shaping plate arranged at the front end of the beam adjustment component for focusing to reshape the energy distribution of the coaxial combined beam, so that the spot area changes from a Gaussian energy distribution to a top-hat energy distribution and obtains an approximately uniform energy meaning within the plane. For example, the optical illumination module 100 can use a precision achromatic lens with a focal length of 60mm, whose wavefront distortion is controlled below λ / 10 to ensure the basic quality of the spot. More importantly, a speckle shaping plate or microlens array is added to the optical path at the front end of the achromatic lens. After reshaping and optimization, the original Gaussian-distributed laser beam can be transformed into a "top-hat" laser spot with a flat energy distribution, thus ensuring uniform irradiation of cells at the bottom of the culture dish. In addition, the position of the electric Z-axis displacement mechanism 210 is set and fixed at about 60 mm above the bottom of the culture dish, so that the optical transfer function of the laser spot remains in the linear region, effectively avoiding energy attenuation of the edge spot due to slight changes in the depth of focus.

[0046] Preferably, to address the issues of easy evaporation and low heat capacity of the culture medium in multi-well plates, this embodiment improves the design of the experimental tray 320 of the temperature monitoring module 300. The experimental tray 320 has a shallow liquid circulation channel inside, connected to an external constant-temperature water bath system, allowing for large-area heat exchange directly with the bottom of the multi-well plate via a 37°C circulating water flow. This design maintains the temperature stability of the culture medium better than an air bath or solid heating block, reducing osmotic pressure changes caused by evaporation.

[0047] Preferably, the infrared thermal imager 330 of the temperature monitoring module 300 is installed at a 45° viewing angle. Since the cell layer is extremely thin, its surface temperature can be regarded as the true temperature; therefore, this embodiment does not use an invasive fiber optic probe.

[0048] Preferably, when processing thermal imaging data, the data acquisition and control module 400 can set ROI-1 as the light spot area within the irradiated well, and ROI-2 as the non-irradiated edge area within the same well or an adjacent non-irradiated well area. The system automatically calculates the temperature difference ΔT between the two, which can effectively correct for slight temperature shifts caused by differences in the height of the culture medium or the different reflectivity of the substrate plastic material, thereby ensuring that the experimental data between different well locations are strictly comparable.

[0049] Example 3 This embodiment is a further improvement on Embodiment 1 and / or 2, and the repeated content will not be described again.

[0050] This embodiment demonstrates a configuration scheme for multi-wavelength (UV / blue / infrared) combined photodynamic / photothermal research, particularly suitable for studying "excitation-catalysis-thermal triggering" cascade reactions. Such experiments often involve wavelength combinations spanning a wide range, such as 405nm (generating reactive oxygen species ROS), 660nm (photodynamic photothermal ...

[0051] Preferably, for the optical illumination module 100, this embodiment abandons the lens refraction optical path and instead uses a total internal reflection optical path combination. Three lasers 140 of different wavelengths are first collimated into beams with a diameter of approximately 6 mm via independent optical fibers, and then sequentially introduced into the coaxial optical path through three 45° high-reflectivity dielectric film mirrors. The focusing component at the end uses a reflective parabolic focusing mirror, utilizing the physical characteristic of the reflective optical path being achromatic, successfully controlling the axial position difference of the focal points for the ultraviolet, visible, and infrared wavelengths within 0.05 mm. This high-precision confocal design ensures that during photosensitizer "pre-excitation" or "co-excitation" experiments, light energy of different wavelengths always acts on the same physical space.

[0052] Preferably, the data acquisition and control module 400 includes a multi-wavelength sequence control function in its software. Users can set complex time sequences, such as "first irradiate with 405nm for 30 seconds, then irradiate with 660nm for 2 minutes." The system can record the on / off status, power parameters, and switching times of all light sources in the same timestamp chain, ensuring the time logic of the cascade reaction is traceable.

[0053] Preferably, considering that ultraviolet wavelengths may interfere with or damage the sensor of the infrared thermal imager 330, this embodiment adds a long-pass filter in front of the lens of the infrared thermal imager 330 in the temperature monitoring module 300. This filter only allows thermal radiation of 8~14μm to pass through, while completely blocking visible light and ultraviolet light, ensuring the purity of the temperature measurement data. Through the above design, this embodiment solves the problem of spot misalignment caused by the combination of multiple devices, providing physical consistency assurance for the study of complex photochemical cascade reactions.

[0054] Example 4 This embodiment is a further improvement on the foregoing embodiment, and repeated content will not be described again.

[0055] This embodiment incorporates mechanical structural modifications for experiments on large animals (such as rabbits) or mice with variable body positions (such as abdominal tumors, lateral subcutaneous tumors, intramuscular injection tumors, etc.).

[0056] Preferably, in terms of mechanical adjustment, the optical irradiation module 100 is no longer fixed vertically downward, but adopts a universal joint optical head design, allowing the terminal optical path to tilt freely within a range of ±45° to adapt to the tumor angle on the animal's side or abdomen. Correspondingly, the electric Z-axis displacement mechanism 210 can be upgraded to a three-dimensional displacement mechanism, with X, Y, and Z-axis adjustment capabilities (each axis step distance is 10μm). Combined with multi-point restraint devices on the animal support plate (such as abdominal fixation straps and lateral abdominal supports), it can achieve precise focusing and irradiation of tumors in irregular locations.

[0057] Preferably, to address the minute displacement of the tumor location caused by the animal's respiratory movements, this embodiment introduces real-time image tracking technology. The infrared thermal imager 330 is mounted on a servo-responsive, multi-degree-of-freedom rotating support. This support is configured to respond to feedback from the image recognition module, maintaining a constant angle (i.e., tilt angle, such as 45°) with the surface normal of the observed object, and to perform translational or rotational compensation within the XY plane following the tumor location. Alternatively, when the support only performs simple gimbal rotation, the data acquisition and control module 400 incorporates a geometric correction algorithm that can calculate and compensate for emissivity directional errors caused by changes in viewing angle in real time, ensuring the accuracy of the temperature measurement data. The software of the data acquisition and control module 400 incorporates a GPU-accelerated image recognition module, capable of identifying and locking the tumor region (ROI-1) in real time. When the tumor location undergoes periodic displacement due to the animal's respiration, the system can automatically correct the coordinate reading range of ROI-1, ensuring that the temperature measurement data always originates from the tumor surface, not the surrounding skin.

[0058] Furthermore, the fiber optic temperature probe in this embodiment can be designed as a multi-channel array (such as a pin array structure), allowing it to be inserted into different depths of the tumor at once (e.g., 1mm, 3mm, and 5mm subcutaneously). The system simultaneously records temperature data at these three depths, thereby constructing a three-dimensional temperature gradient curve within the tissue, which facilitates researchers in analyzing the attenuation of photothermal effects in deep tissues.

[0059] Example 5 This embodiment is a further improvement on the foregoing embodiment, and repeated content will not be described again.

[0060] This embodiment is applicable to the study of deep tissue models or 3D spheroidomas. For 3D spheroidomas with a thickness of 200~2000μm, due to their poor light transmittance and slow internal heat diffusion, significant internal and external temperature differences are easily formed, and simple surface temperature measurement cannot assess the risk of internal necrosis.

[0061] In this embodiment, the observed object (globular nodule) is fixed in a transparent gel matrix (such as Matrigel) and placed in a glass dish. Preferably, the optical illumination module 100 still maintains vertical illumination, but the focal length of the focusing lens is shortened to 30-45 mm to obtain a finer light spot (approximately 4-6 mm in diameter) to match the small size of the globular nodule.

[0062] Preferably, for the temperature monitoring module 300, given that the surface temperature rise (ΔT) detected by the infrared thermal imager 330 only represents the surface thermal effect, this embodiment specifically configures a dual-channel microfiber temperature probe system. The first probe is placed in the gel approximately 0.5 mm directly above the nodule to monitor the ambient thermal field; the second probe is precisely inserted into the geometric center of the nodule (approximately 2 mm deep). The data acquisition and control module 400 can synchronously record and compare the internal temperatures at these two points with the surface temperatures measured by the infrared thermal imager 330. This combined internal and external monitoring method allows researchers to clearly reconstruct the deposition and conduction model of photothermal energy in three-dimensional tissues, which is an important verification method for evaluating the penetration depth and photothermal conversion efficiency of novel photosensitizers.

[0063] Example 6 This embodiment is a further improvement on the foregoing embodiment, and repeated content will not be described again.

[0064] This embodiment aims to realize a multi-spot parallel control experiment, that is, to test the experimental group and the control group simultaneously on the same animal or the same cell plate, so as to eliminate the systematic error caused by individual differences to the greatest extent.

[0065] Preferably, in this embodiment, an adjustable beam splitter is added after the beam combining component 120 for the optical illumination module 100. This component splits the single combined laser beam into two parallel sub-spots, and the center-to-center distance between the two spots is adjustable, typically set to 15-20 mm. To achieve independent control, the system is equipped with a dual-channel power attenuation module, allowing the power density of each spot to be adjusted independently (e.g., the left spot is the high-power treatment group, and the right spot is the low-power control group).

[0066] Preferably, in terms of temperature monitoring and data analysis, the field of view of the infrared thermal imager 330 of the temperature monitoring module 300 is expanded to simultaneously cover two spot regions; the software of the data acquisition and control module 400 can define ROI-1 as spot region A and ROI-2 as spot region B, and calculate ΔT(A) and ΔT(B) respectively. This design allows comparative experiments of "material concentration A vs. material concentration B" or "treatment parameter A vs. treatment parameter B" to be conducted under completely identical physiological environments and time backgrounds, greatly improving the statistical reliability of the experimental results.

[0067] Example 7 This embodiment is a further improvement on the foregoing embodiment, and repeated content will not be described again.

[0068] This embodiment is used for high-throughput screening of novel photothermal materials, aiming to rapidly evaluate the photothermal conversion performance of various materials in in vitro solutions.

[0069] Preferably, this embodiment fundamentally adjusts the optical path structure of the optical irradiation module 100, abandoning the use of a static focusing lens and introducing a high-speed motorized scanning galvanometer system. The control unit of this system can drive the galvanometer to rapidly scan the laser spot on the sample surface at a frequency of 20~200Hz, thereby forming a uniform excitation field covering the entire porous plate area macroscopically, or performing jump-point irradiation on multiple specific well positions.

[0070] Preferably, in terms of temperature monitoring, the temperature monitoring module 300 can increase the acquisition frame rate of the infrared thermal imager 330 to match the scanning speed, wherein the acquisition frame rate of the infrared thermal imager 330 can be increased, for example, to 100Hz. The image processing algorithm has an automatic well location recognition function, which can automatically locate each sample well in the 96-well plate and independently record the temperature rise curve (ΔT) and maximum temperature rise value of each well.

[0071] This embodiment allows researchers to simultaneously test the photothermal properties of 24 to 96 different materials or samples of different concentrations in a single experiment. The automatically generated screening report can greatly accelerate the screening process in the early stages of new material development, providing a highly efficient verification platform.

[0072] Example 8 This embodiment is a further improvement on the foregoing embodiment, and repeated content will not be described again.

[0073] like Figure 5 As shown, the present invention also discloses an experimental method for research on photodynamic therapy, photothermal therapy, and their combinations, comprising the following steps: Optical path configuration steps: At least two lasers of different wavelengths are independently collimated and then combined through the beam combiner 120. The optical path is adjusted by the electric Z-axis displacement mechanism 210 so that a preset light spot is formed on the surface of the observed object. Temperature baseline construction steps: Activate the active temperature control board 310 to stabilize its temperature at the set value, and obtain the temperature background of the observed object when it is not irradiated; Temperature monitoring steps: Use an infrared thermal imager 330 to acquire the temperatures of two different areas and calculate ΔT, where ΔT is the difference between the average temperature or the difference between the highest temperature of the two different areas; if internal temperature measurement is required, insert an optical fiber temperature probe to measure the internal temperature of the observed object. Irradiation and synchronous recording steps: Irradiate with a preset power density, and at the same time, the data acquisition module synchronously records the laser power, spot area, Z-axis position, substrate temperature, temperature sequence of two different regions and fiber probe temperature. Safety control procedures: When ΔT or internal temperature exceeds the safety threshold, the irradiation will be automatically terminated and the reason for termination will be recorded.

[0074] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. An experimental system for research on photodynamic therapy, photothermal therapy, and their combinations, characterized in that, It includes: The optical illumination module (100) includes at least two laser sources of different wavelengths, each of which is equipped with an independent collimation component (110) to output a collimated beam; the collimated beam is combined by a beam combiner (120) to form a coaxial optical path, and then projected onto the surface of the object being observed by a beam adjustment component with focusing, guiding or beam guiding functions. The optical path position adjustment module (200) includes an electric Z-axis displacement mechanism (210) for adjusting the distance between the optical illumination module (100) and the surface of the observed object, so that the area of ​​the light spot projected on the surface of the observed object is maintained at a preset value; Temperature monitoring module (300) includes: active temperature control board (310), disposed below the object being observed, for providing uniform background temperature; An infrared thermal imager (330) is positioned above or to the side of the object being observed to simultaneously acquire real-time temperature images including the region of interest and the background region; a fiber optic temperature probe interface is used to connect a fiber optic temperature probe to measure the internal temperature of the object being observed. The data acquisition and control module (400) is configured to: synchronously acquire laser output power, spot area, electric Z-axis position, temperature of active temperature control substrate (310), temperature sequences of at least two different regions, and fiber optic temperature probe data, and record the above data with a unified timestamp; calculate power density based on the real-time output power of the laser source and the spot area; and automatically control the laser source to shut down when the data of any temperature monitoring channel exceeds a preset safety threshold.

2. The system according to claim 1, characterized in that, The optical illumination module (100) adopts any of the following optical path configurations: The beam combining component (120) uses a dichroic mirror for beam combining, wherein the dichroic mirror has a transmittance of ≥95% for the first wavelength and a reflectance of ≥98% for the second wavelength; The beam combining component (120) employs multiple dielectric film mirrors to sequentially introduce beams of different wavelengths into the coaxial optical path, thereby eliminating chromatic aberration over a wide spectral range. An adjustable beam splitter is provided after beam combining to split a single coaxial beam into two parallel sub-spots, so as to form a parallel control experimental area on the same observation object.

3. The system according to claim 1 or 2, characterized in that, The beam adjustment component adopts any of the following structures: Achromatic lenses or reflective parabolic mirrors are used to reduce the focal difference between beams of different wavelengths; A microlens array or speckle shaping plate is provided in front of the beam conditioning component used for focusing, so that the spot area presents a top-hat-shaped energy distribution; An electric scanning galvanometer is used to drive the light spot to scan and cover the surface of the observed object at a preset frequency.

4. The system according to any one of claims 1 to 3, characterized in that, The optical path position adjustment module (200) is also configured with one or more of the following components: Universal joints or rotating brackets are used to adjust the incident angle of the optical illumination module (100) so that it can tilt within a range of ±45°; A three-dimensional displacement module is used to replace a single Z-axis to realize the relative displacement of the optical illumination module (100) or the stage in the X, Y, and Z directions.

5. The system according to any one of claims 1 to 4, characterized in that, The infrared thermal imager (330) is mounted at an angle of 30° to 60° to the side of the optical path, and: a long-pass filter is provided in front of the optical path of the infrared thermal imager (330), the filter is configured to transmit 8 to 14 μm wavelength and block visible light and ultraviolet light; or, the infrared thermal imager (330) is mounted on a movable rotating bracket, responding to the control signal generated by the data acquisition and control module (400), the control signal is generated based on image tracking technology to compensate for the displacement of the area identified by the image recognition module, in order to lock the corrected temperature measurement area in real time and measure its temperature in order to compensate for the displacement of the observed object.

6. The system according to any one of claims 1 to 5, characterized in that, The fiber optic temperature probe interface is configured to: connect a single fiber optic probe for insertion into a subcutaneous tumor in an animal at a depth of 1-3 mm; or connect dual-channel or multi-channel fiber optic probes for insertion into different depths or locations of the observed object to construct an internal temperature gradient curve.

7. The system according to any one of claims 1 to 6, characterized in that, The active temperature control substrate (310) is provided with a semiconductor cooler or a liquid circulation channel inside. The liquid circulation channel is located inside the tray of the object being observed. When the liquid circulation channel is used, the channel is connected to an external constant temperature water bath device to maintain the temperature stability of the bottom of the object being observed through the circulating liquid flow.

8. The system according to any one of claims 1 to 7, characterized in that, The data acquisition and control module (400) is configured to perform the following operations: calculate the differential temperature ΔT in real time, where ΔT is the difference between the average temperature of the region of interest and the background region or the difference between the highest temperature, in order to eliminate background thermal interference from the active temperature control substrate (310) and the body temperature of the observed object.

9. The system according to any one of claims 1 to 8, characterized in that, The data acquisition and control module (400) is also configured to: prohibit users from closing any temperature or power recording channel during the recording process to ensure data integrity; support multi-wavelength sequence control, automatically switch the on / off state and power of different wavelength laser sources according to a preset time sequence, and record the switching event in the unified timestamp.

10. An experimental method for studying photodynamic therapy, photothermal therapy, and their combinations, characterized in that, Includes the following steps: Optical path configuration steps: At least two lasers of different wavelengths are independently collimated and then combined through a beam combiner (120), and the optical path is adjusted by an electric Z-axis displacement mechanism (210) to form a preset light spot on the surface of the observed object; Temperature baseline construction steps: Activate the active temperature control board (310) to stabilize its temperature at the set value and obtain the temperature background of the observed object when it is not irradiated; Temperature monitoring steps: Use an infrared thermal imager (330) to acquire the temperature of the region of interest and the background region, and calculate ΔT, where ΔT is the difference between the average temperature of the region of interest and the background region or the difference between the highest temperature; if internal temperature measurement is required, insert an optical fiber temperature probe to measure the internal temperature of the observed object. Irradiation and synchronous recording steps: Irradiate with a preset power density, and at the same time, the data acquisition module synchronously records the laser power, spot area, Z-axis position, substrate temperature, temperature sequence of two different regions and fiber probe temperature. Safety control procedures: When ΔT or internal temperature exceeds the safety threshold, the irradiation will be automatically terminated and the reason for termination will be recorded.

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