Two-photon multi-mode imaging light path system and method for traditional Chinese medicine active ingredient target discovery and action mechanism analysis

Abstract

The invention provides a two-photon multi-mode imaging light path system and method for traditional Chinese medicine active ingredient target discovery and action mechanism analysis. The two-photon multi-mode imaging light path system comprises a laser emission module, a light path adjusting module, a confocal microscopic module, a space acquisition module and a high-resolution fluorescence imaging module which are sequentially connected along a propagation path of laser. Laser generated by the laser emission module is used for performing two-photon excitation on a traditional Chinese medicine active ingredient target in the confocal microscopic module. According to the system and the method, multi-mode imaging is carried out on the active ingredients of the traditional Chinese medicine through an efficient, accurate and safe two-photon affinity labeling method, so that target recognition and mechanism analysis and research of the active ingredients of the traditional Chinese medicine are promoted, and guidance and support are provided for solving the multi-target problem of the traditional Chinese medicine and promoting new medicine innovation and medicine discovery.

Description

Technical Field

[0001] The present invention belongs to the technical field of laser application, and designs a two-photon multimodal imaging optical path system and method for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine. Background Art

[0002] The research and development of innovative drugs derived from clinically effective active ingredients of traditional Chinese medicine is an advantageous field and effective approach for my country. The target identification and mechanism research of active ingredients of traditional Chinese medicine have always been a major scientific issue that has received much attention in the field of new drug research and development. The discovery of major drug targets such as morphine receptors, retinoid receptors, and dopamine receptors has promoted the discovery of more candidate drug molecules with excellent properties and has become an important driving force for the development of innovative drugs. However, active ingredients of traditional Chinese medicine have the characteristics of multiple targets and multiple mechanisms of action, and the targets and mechanisms of action of most active ingredients of traditional Chinese medicine have not yet been clarified.

[0003] Photoaffinity labeling is an effective target fishing technology with the advantages of efficient and rapid identification, simple operating conditions and applicability to in situ reactions. However, conventional photoaffinity labeling methods currently require the use of high-energy ultraviolet light to excite the photoaffinity labeling group, which not only causes certain damage to proteins and cells, but also single-photon ultraviolet light has no spatial localization, which can easily label non-specific proteins or miss some potential targets, reducing the specificity of labeling; in addition, ultraviolet light is difficult to penetrate deep into tissues, resulting in the current photoaffinity labeling method can only be used for in vitro target identification.

[0004] Cutting-edge technologies and measuring instruments such as two-photon excitation have made great progress and are widely used in pharmacological mechanism research, bringing new opportunities for exploring the targets and mechanisms of action of natural drugs. Two-photon excitation uses near-infrared light with twice the wavelength of the original excitation light for excitation. It has the advantages of deep penetration depth and precise excitation area, and is an important potential way to achieve accurate target identification. However, the lack of two-photon instruments targeted at active ingredients of traditional Chinese medicine has limited the research on two-photon use for target identification of traditional Chinese medicine.

[0005] The urgent problem to be solved for the active ingredients of traditional Chinese medicine is to find their targets and mechanisms of action. More precise target determination even requires real-time, in vivo measurements, which are difficult to obtain with existing detection methods. Existing cell imaging and spectral methods reflect the molecular characteristics of intracellular molecules and drug interactions in a certain aspect, but cannot highly sensitively and comprehensively characterize the interactions between intracellular macromolecules and drugs. In order to solve this problem, it is necessary to combine various spectral analysis and imaging methods and develop multimodal optical methods. By analyzing and fusing the data obtained by different modal optical methods, the spatial distribution, dynamic processes and drug-protein interactions of intracellular biomacromolecules can be sensitively and comprehensively characterized, and the cell structure changes caused by drug-protein interactions can be monitored. However, current optical instruments cannot meet the needs of multimodal imaging of the interactions between active ingredients of traditional Chinese medicine and proteins in cells. Therefore, the construction of a new multimodal imaging optical path system is essential for target discovery and mechanism research of active ingredients of traditional Chinese medicine. Summary of the invention

[0006] This application is written based on the priority of the prior application with application number 202410436127.1.

[0007] In order to solve the technical problems existing in the prior art, the present invention provides a two-photon multimodal imaging optical path system and method for target discovery and mechanism analysis of active ingredients of traditional Chinese medicine. The system and method perform multimodal imaging of active ingredients of traditional Chinese medicine through an efficient, accurate and safe two-photon affinity labeling method, thereby promoting target identification and mechanism analysis research of active ingredients of traditional Chinese medicine, and providing guidance and support for solving multi-target problems of traditional Chinese medicine, promoting new drug innovation and drug discovery.

[0008] In order to achieve the above technical effects, the present invention adopts the following technical solutions:

[0009] One of the purposes of the present invention is to provide a two-photon multimodal imaging optical path system for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine, the two-photon multimodal imaging optical path system comprises a laser emission module, an optical path adjustment module, a confocal microscopy module, a spatial acquisition module and a high-resolution fluorescence imaging module which are sequentially connected along the propagation path of the laser;

[0010] The laser light generated by the laser emission module performs two-photon excitation on the active ingredient target of the traditional Chinese medicine in the confocal microscopy module.

[0011] As a preferred technical solution of the present invention, the laser emission module includes a femtosecond laser and an optical parametric oscillator.

[0012] As a preferred technical solution of the present invention, the repetition frequency of the second laser and the optical parametric oscillator is 80 to 100 MHz, such as 80 MHz, 82 MHz, 85 MHz, 88 MHz, 90 MHz, 92 MHz, 95 MHz, 98 MHz or 100 MHz, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0013] As a preferred technical solution of the present invention, the wavelength of the laser generated by the laser emission module is 300-2000nm, such as 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1100nm, 1200nm, 1300nm, 1400nm, 1500nm, 1600nm, 1700nm, 1800nm, 1900nm or 2000nm, the pulse width is not more than 100fs, and the maximum average power is not less than 1W, but it is not limited to the listed values, and other unlisted values ​​within the above numerical ranges are equally applicable.

[0014] As a preferred technical solution of the present invention, the optical path adjustment module includes a reflective chip and an attenuation lens.

[0015] As a preferred technical solution of the present invention, the lifetime imaging module is a platform for collecting and imaging the optical lifetime signal spectrum of the active ingredients of traditional Chinese medicine.

[0016] As a preferred technical solution of the present invention, the spatial acquisition module is a detection platform for the optical properties of the active ingredients of traditional Chinese medicine in a macroscopic state and a platform for collecting the interaction signals between the active ingredients of traditional Chinese medicine and related proteins in the solution.

[0017] As a preferred technical solution of the present invention, the high-resolution fluorescence imaging module includes a monochromator, a photomultiplier tube and a charge-coupled device which are sequentially connected along the optical propagation path.

[0018] As a preferred technical solution of the present invention, the two-photon multimodal imaging optical path system also includes a lifetime imaging module. The fluorescence signal generated by the sample in the confocal microscopy module after two-photon excitation by laser is divided into two branches, one passing through the lifetime imaging module, and the other passing through the spatial acquisition module and the high-resolution fluorescence imaging module.

[0019] The second object of the present invention is to provide a two-photon multimodal imaging method for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine. The two-photon multimodal imaging method uses the two-photon multimodal imaging system for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine provided in the first object. The two-photon multimodal imaging method comprises:

[0020] The laser generated by the laser emission module passes through the optical path adjustment module and is focused onto the sample using the confocal microscopy module;

[0021] The active ingredients in the traditional Chinese medicine or the fluorescent groups connected to the active ingredients of the traditional Chinese medicine generate fluorescent signals under the two-photon excitation of the laser;

[0022] The fluorescence signal enters the spatial acquisition module, detects the optical properties of the Chinese medicine active ingredient solution under a macroscopic state, collects the interaction signal between the Chinese medicine active ingredient and the related proteins in the solution, and then enters the high-resolution fluorescence imaging module to convert the collected weak light signal into an electrical signal and then convert it into a spectral value, finally obtaining the target optical spectrum or fluorescence imaging diagram.

[0023] As a preferred technical solution of the present invention, the fluorescence signal is divided into two branches, one branch enters the lifetime imaging module to collect and image the optical lifetime signal spectrum of the active ingredients of traditional Chinese medicine, and the other branch enters the space acquisition module.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] (1) The present invention provides a two-photon multimodal imaging optical path system and method for target discovery and mechanism analysis of active ingredients of traditional Chinese medicine. The system and method are based on the high resolution and multidimensional information extraction capabilities of two-photon excitation to accurately identify multiple potential targets of active ingredients of traditional Chinese medicine, and utilize the high spatial positioning capabilities of two-photon excitation to obtain a more accurate position of the target protein in the cell;

[0026] (2) The present invention provides a two-photon multimodal imaging optical path system and method for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine. The two-photon affinity labeling method proposed by the system and method has the advantage of simultaneously capturing information of multiple targets through a single experiment, which is conducive to more comprehensive disclosure of the interaction mechanism between active ingredients of traditional Chinese medicine and different target proteins;

[0027] (3) The present invention provides a two-photon multimodal imaging optical path system and method for target discovery and mechanism analysis of active ingredients of traditional Chinese medicine. The system and method realize multimodal optical imaging of traditional Chinese medicine and target proteins. By analyzing and fusing data obtained from different modal methods, sensitive and comprehensive characterization of drug-protein interaction information is achieved, providing a powerful technical platform support for multi-target treatment of active ingredients of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a structural schematic diagram of a laser emission module in a specific implementation manner;

[0029] Figure 2 It is a structural schematic diagram of an optical path adjustment module in a specific implementation manner;

[0030] Figure 3 It is a structural schematic diagram of a confocal microscopy module in a specific implementation manner;

[0031] Figure 4 It is a structural schematic diagram of a space acquisition module in a specific implementation mode;

[0032] Figure 5 It is a structural schematic diagram of a life imaging module in a specific implementation manner;

[0033] Figure 6 FIG. 4 is a schematic diagram of the structure of a high-resolution fluorescence imaging module in a specific implementation manner.

[0034] The present invention is further described in detail below. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. DETAILED DESCRIPTION

[0035] The technical solution of the present application is further described below through specific implementation methods.

[0036] A specific embodiment of the present invention provides a two-photon multimodal imaging optical path system for target discovery and mechanism analysis of active ingredients of traditional Chinese medicine. The two-photon multimodal imaging optical path system includes a laser emission module, an optical path adjustment module, a confocal microscopy module, a spatial acquisition module and a high-resolution fluorescence imaging module which are sequentially connected along the propagation path of the laser.

[0037] In the present invention, a confocal microscopy module is used to focus the laser beam onto the sample surface. After the active ingredients of traditional Chinese medicine in the sample are combined with the target protein, the photoaffinity probe connected to the active ingredients of traditional Chinese medicine is excited by the femtosecond laser, producing a two-photon absorption effect, and becomes a high-energy state to covalently bind to the amino acid sites on the protein, thereby enhancing the interaction between the small molecule and the target protein; at the same time, the active ingredients of traditional Chinese medicine themselves or the fluorescent groups connected to the active ingredients of traditional Chinese medicine generate fluorescent signals under laser excitation.

[0038] In a specific embodiment of the present invention, laser light of a certain frequency emitted by a femtosecond laser is passed through an optical parametric oscillator (OPO) to obtain a target laser light of a desired frequency.

[0039] In a specific embodiment of the present invention, an optical parametric oscillator (OPO) is a wavelength-tunable coherent light source that can convert a laser of one frequency into a coherent output of a signal and an idle frequency, and can be tuned within a wide frequency range.

[0040] In a specific embodiment of the present invention, the working principle of the reflective lens is based on the law of reflection of light. Its surface is specially processed so that the laser can be reflected along a predetermined path, thereby changing the propagation direction of the laser; the attenuation lens uses the absorption characteristics of the material to attenuate the laser to the required intensity. These two components together form an optical path adjustment module to achieve the purpose of changing the direction and intensity of the laser according to the experimental requirements.

[0041] In a specific embodiment of the present invention, the confocal microscopy module includes an XY piezoelectric displacement mechanical stage and a dichroic mirror. High-resolution fluorescence imaging of the sample can be achieved by adjusting the relative position between the sample and the excitation femtosecond laser through the XY piezoelectric displacement stage.

[0042] In a specific embodiment of the present invention, the confocal microscopy module introduces the outgoing laser in the optical path into the microscope, and utilizes the principles of light interference and diffraction to receive only the light at the focus through a small hole, thereby improving the depth resolution and achieving high-resolution image acquisition, thereby achieving the purpose of observing the localization and distribution of the active ingredients of traditional Chinese medicine in cells or tissues and their interaction with target proteins.

[0043] In a specific embodiment of the present invention, the confocal microscopy module also includes a collecting lens and a filtering assembly. The collecting lens collimates the output of the fluorescence signal, and then filters the excitation light component through the filtering assembly. The remaining fluorescence signal is focused again onto the incident slit of the monochromator to achieve the sorting of light wavelengths.

[0044] In a specific embodiment of the present invention, the confocal microscopy module further includes a high-sensitivity detection component connected to a single-photon counting controller for recording the intensity of the sorted monochromatic light and the dynamic process of the intensity changing with time.

[0045] In a specific embodiment of the present invention, the sample in the confocal microscopy module can be a traditional Chinese medicine chip, a cell or a model animal.

[0046] In a specific embodiment of the present invention, the spatial acquisition module can make the research on the target of the active ingredient of traditional Chinese medicine more in-depth by mutual confirmation or contradiction of macroscopic and microscopic analysis results.

[0047] In a specific embodiment of the present invention, the high-resolution fluorescence imaging module is electrically connected to the computing module.

[0048] In a specific embodiment of the present invention, the monochromator in the high-resolution fluorescence imaging module selectively transmits light of a specific wavelength by adjusting the angle of the grating, realizing spectrophotometry for further spectrum collection; the photomultiplier tube (PMT) is an electronic vacuum device based on the external photoelectric effect and the secondary electron emission effect, which can convert weak light signals into electrical signals; the charge-coupled device (CCD) can convert light into electric charges, which are converted into digital signals through an analog-to-digital converter chip, and then transmitted to the computing module, and the image is obtained by the processing means of the computer in the computing module. The module composed of the above three components can convert the collected weak light signals into electrical signals and then convert them into spectral values, and finally obtain the target optical spectrum or fluorescence imaging diagram.

[0049] In a specific embodiment of the present invention, dynamic and imaging analysis of fluorescence signals of different wavelengths from a sample can be achieved by changing the emission wavelength of the monochromator.

[0050] Fluorescence lifetime imaging is a microscopic imaging technology based on fluorescence lifetime. Its imaging results provide lifetime information of pixel sites, so that in addition to fluorescence intensity imaging, it can perform more in-depth functional measurements of samples without being affected by factors such as fluorescent substance concentration, photobleaching, and excitation light intensity. In a specific embodiment of the present invention, the lifetime imaging module can collect and image the optical lifetime signal spectra of different active ingredients of traditional Chinese medicines to achieve the purpose of multimodal optical analysis.

[0051] To better illustrate the present invention and facilitate understanding of the technical solution of the present invention, typical but non-limiting embodiments of the present invention are as follows:

[0052] Example 1

[0053] This embodiment provides a two-photon multimodal imaging optical path system for target discovery and mechanism analysis of active ingredients in traditional Chinese medicine. Figure 1-5 As shown, the two-photon multimodal imaging optical path system includes a laser emission module, an optical path adjustment module, a confocal microscopy module, a spatial acquisition module and a high-resolution fluorescence imaging module which are sequentially connected along the propagation path of the laser;

[0054] The two-photon multimodal imaging optical path system also includes a lifetime imaging module. The fluorescence signal generated by the sample in the confocal microscopy module after two-photon excitation by laser is divided into two branches, one passing through the lifetime imaging module, and the other passing through the space acquisition module and the high-resolution fluorescence imaging module;

[0055] The laser emission module includes a femtosecond laser and an optical parametric oscillator. The repetition frequency of the femtosecond laser is 80MHz. The femtosecond laser and the optical parametric oscillator are used to generate laser pulses with a wavelength of 300 to 2000nm and a pulse width of about 100fs, and a maximum average power of 1W.

[0056] The optical path adjustment module includes a reflection chip and an attenuation lens;

[0057] The high-resolution fluorescence imaging module includes a monochromator, a photomultiplier tube and a charge-coupled device connected in sequence along the optical propagation path;

[0058] The high-resolution fluorescence imaging module is electrically connected to the computing module.

[0059] Example 2

[0060] This application example provides a two-photon multimodal imaging optical path method for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine. The method uses the two-photon multimodal imaging system for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicine provided in Example 1. The two-photon multimodal imaging method includes:

[0061] The laser generated by the laser emission module is attenuated to the required experimental power through the reflection chip and attenuation lens of the optical path adjustment module, and is focused on the cell surface using the confocal microscopy module;

[0062] After the active ingredients of traditional Chinese medicine bind to the target protein in the cell, the photoaffinity probe connected to the active ingredients of traditional Chinese medicine is excited by the femtosecond laser, producing a two-photon absorption effect, turning into a high-energy state and covalently binding to the amino acid sites on the protein, thereby enhancing the interaction between the small molecule and the target protein; at the same time, the active ingredients of traditional Chinese medicine themselves or the fluorescent groups connected to the active ingredients of traditional Chinese medicine generate fluorescent signals under laser excitation;

[0063] The fluorescence signal is divided into two branches, one of which enters the lifetime imaging module to collect the optical lifetime signal spectrum of the active ingredients of traditional Chinese medicine and form an image, and the other enters the space acquisition module;

[0064] The fluorescence signal enters the spatial acquisition module, and the optical properties of the active ingredients of traditional Chinese medicine are detected under the macroscopic state. The interaction signals between the active ingredients of traditional Chinese medicine and related proteins in the solution are collected, and then enters the high-resolution fluorescence imaging module to convert the collected weak light signals into electrical signals and then convert them into spectral values, finally obtaining the target optical spectrum or fluorescence imaging diagram.

[0065] The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed structural features, that is, it does not mean that the present invention must rely on the above-mentioned detailed structural features to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the components selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0066] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0067] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0068] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A two-photon multimodal imaging optical path system for target discovery and action mechanism analysis of active ingredients in traditional Chinese medicine, characterized in that: The two-photon multimodal imaging optical path system comprises a laser emission module, an optical path adjustment module, a confocal microscopy module, a spatial acquisition module and a high-resolution fluorescence imaging module which are sequentially connected along the propagation path of the laser; The laser light generated by the laser emission module performs two-photon excitation on the active ingredient target of the traditional Chinese medicine in the confocal microscopy module.

2. The two-photon multimodal imaging optical path system according to claim 1, characterized in that: The laser emission module includes a femtosecond laser and an OPO optical parametric oscillator.

3. The two-photon multi-modal imaging optical path system according to claim 1, characterized in that: The repetition frequency of the femtosecond laser and the OPO optical parametric oscillator is 80-100 MHz.

4. The two-photon multimodal imaging optical path system according to claim 1, characterized in that: The wavelength of the laser generated by the laser emission module is 300-2000nm, the pulse width is not greater than 100fs, and the maximum average power is not less than 1W.

5. The two-photon multi-modal imaging optical path system according to claim 1, characterized in that: The optical path adjustment module includes a reflective chip and an attenuation lens.

6. The two-photon multi-modal imaging optical path system according to claim 1, characterized in that: The spatial acquisition module is a detection platform for the optical properties of the active ingredient liquid of traditional Chinese medicine in a macroscopic state and a platform for collecting the interaction signals between the active ingredient of traditional Chinese medicine and related proteins in the solution.

7. The two-photon multi-modal imaging optical path system according to claim 1, characterized in that: The high-resolution fluorescence imaging module comprises a monochromator, a photomultiplier tube and a charge-coupled device which are sequentially connected along the optical propagation path.

8. The two-photon multi-modal imaging optical path system according to claim 1, characterized in that: The two-photon multimodal imaging optical path system also includes a lifetime imaging module. The fluorescence signal generated by the sample in the confocal microscopy module after two-photon excitation by laser is divided into two branches, one passing through the lifetime imaging module, and the other passing through the spatial acquisition module and the high-resolution fluorescence imaging module.

9. A two-photon multimodal imaging method for target discovery and mechanism analysis of active ingredients in traditional Chinese medicine, characterized in that: The method uses the two-photon multimodal imaging system for target discovery and action mechanism analysis of active ingredients of traditional Chinese medicines according to any one of claims 1 to 8, and the two-photon multimodal imaging method comprises: The laser generated by the laser emission module passes through the optical path adjustment module and is focused onto the sample using the confocal microscopy module; The active ingredients in the traditional Chinese medicine or the fluorescent groups connected to the active ingredients of the traditional Chinese medicine generate fluorescent signals under the two-photon excitation of the laser; The fluorescence signal enters the spatial acquisition module, detects the optical properties of the Chinese medicine active ingredient solution under a macroscopic state, collects the interaction signal between the Chinese medicine active ingredient and the related proteins in the solution, and then enters the high-resolution fluorescence imaging module to convert the collected weak light signal into an electrical signal and then convert it into a spectral value, finally obtaining the target optical spectrum or fluorescence imaging diagram.

10. The two-photon multimodal imaging method according to claim 9, characterized in that: The fluorescent signal is divided into two branches, one branch enters the lifetime imaging module to collect the optical lifetime signal spectrum of the active ingredients of traditional Chinese medicine and form an image, and the other branch enters the space acquisition module.

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