Multicolor pathology detection system, detection analysis method and controller

Abstract

The invention discloses a multicolor pathology detection system, a detection analysis method and a controller. The multicolor pathology detection system comprises a control module, and a light source module, a light splitting module, a microscopic module, a detection module and an imaging module which are electrically connected with the control module respectively. The light source module emits at least two kinds of exciting light with different wavelengths, the light splitting module guides the exciting light with the target wavelength to the microscopic module, light signals of various markers can be detected at the same time, and the detection efficiency is high. A to-be-processed electric signal is obtained after feedback light signals with different wavelengths are detected through the detection module, imaging is carried out through the imaging module, fluorescence signals of multiple different fluorescence markers and / or corresponding bright field signals can be detected at the same time, and therefore multi-dimensional detection is carried out, the application range is wide, complex molecular characteristics of a to-be-detected sample can be captured, and the detection accuracy is high. The invention can be widely applied to the optical technical field.

Description

Technical Field

[0001] The present invention relates to the technical field of pathology detection, and in particular to a multicolor pathology detection system, a detection and analysis method, and a controller. Background Art

[0002] Pathological testing is a medical examination method that diagnoses diseases and determines the nature and extent of lesions through microscopic observation and analysis of human tissues or cells. As the "gold standard" of medical diagnosis, pathological testing plays a key role in disease diagnosis, prognosis assessment and treatment plan formulation. With the rapid development of precision medicine, pathological testing technology is evolving towards multicolor, high throughput and intelligence. Multicolor pathological testing technology is an important development trend in the current field of pathology. This technology allows the simultaneous detection and visualization of multiple biomarkers in the same tissue sample, providing more comprehensive molecular information for the diagnosis and research of complex diseases. Multicolor pathological testing has shown great potential in cancer typing, immunotherapy response prediction, microenvironment analysis, etc., and is increasingly becoming an important supporting technology for precision medicine and personalized treatment.

[0003] However, the existing multi-color pathology detection systems mostly use a single wavelength excitation light source, which is difficult to effectively excite multiple fluorescent dyes, limiting the number of markers that can be detected simultaneously and resulting in low detection efficiency. In addition, the existing multi-color pathology detection systems are limited to a single detection dimension and have a small scope of application, which limits the diversity and comprehensiveness of the detection and makes it difficult to capture the complex molecular characteristics of the samples to be tested. Summary of the invention

[0004] In view of this, the purpose of the embodiments of the present invention is to provide a multicolor pathology detection system, a detection and analysis method and a controller, which can simultaneously detect multiple biomarkers, improve the detection efficiency of the samples to be tested, and the diversity and comprehensiveness of the detection.

[0005] In a first aspect, an embodiment of the present invention provides a multi-color pathology detection system, including a control module and a light source module, a spectroscopic module, a microscopic module, a detection module, and an imaging module electrically connected to the control module respectively;

[0006] The light source module is used to emit at least two excitation lights of different wavelengths, and guide the excitation light of the target wavelength to the light splitting module through the optical fiber, so that the light splitting module guides the excitation light of the target wavelength to the microscopy module;

[0007] The microscopy module is used to scan the sample to be tested using the excitation light of the target wavelength, so that the sample to be tested generates a feedback light signal under the action of the excitation light of the target wavelength, and transmits the feedback light signal to the spectroscopic module, and guides the feedback light signal to the detection module through the spectroscopic module, and the feedback light signal represents the light signal generated by the sample to be tested under the excitation of the excitation light of the target wavelength;

[0008] The detection module is used to detect the feedback optical signals of different wavelengths and convert the feedback optical signals into electrical signals to be processed;

[0009] The imaging module is used to process the electrical signal to be processed and generate image information.

[0010] In some optional embodiments, the light source module includes a laser integrator and a multi-wavelength laser source, the multi-wavelength laser source is used to emit at least two excitation lights of different wavelengths to the laser integrator, the laser integrator is used to simultaneously or sequentially select excitation lights of different wavelengths and integrate them to obtain the excitation light of the target wavelength, and the excitation light of the target wavelength is subsequently transmitted to the spectroscopic module.

[0011] In some optional embodiments, the multi-wavelength laser source includes a modulated nanosecond laser and an up-conversion laser, the modulated nanosecond laser is used to emit short-wave excitation light, the up-conversion laser is used to emit long-wave excitation light, the wavelength of the short-wave excitation light is between 300nm and 700nm, and the wavelength of the long-wave excitation light is between 700nm and 1000nm.

[0012] In some optional embodiments, the wavelength of the short-wave excitation light includes at least one of the following:

[0013] 375nm; 405nm; 485nm; 635nm; 680nm.

[0014] In some optional embodiments, the wavelength of the long-wave excitation light includes at least one of the following:

[0015] 730nm; 808nm; 980nm.

[0016] In some optional embodiments, the detection module includes at least one fluorescence detector, which is used to generate fluorescence image information after detecting a fluorescence signal in the feedback light signal, wherein the fluorescence signal is generated by a fluorescent marker under the excitation of the excitation light of the target wavelength.

[0017] In some optional embodiments, the detection module further includes at least one bright field detector, which is used to generate bright field image information after detecting a bright light signal in the feedback light signal, wherein the bright light signal represents the light signal generated by the sample to be tested under the excitation of the excitation light of the target wavelength.

[0018] In some optional embodiments, the detection module further includes a beam splitting unit, and the beam splitting unit is used to direct the fluorescence signal to the fluorescence detector and direct the bright light signal to the bright field detector.

[0019] In some optional embodiments, the fluorescence detector includes a fluorescence single photon detector, and the bright field detector includes a bright field transmission single photon detector.

[0020] In some optional embodiments, the modulated nanosecond laser includes a programmable broadband frequency laser modulator, which is used to continuously adjust the output of the excitation light in the sub-hertz to megahertz range.

[0021] In some optional embodiments, the broadband frequency laser modulator includes a digital frequency synthesizer, a high-speed optoelectronic modulation unit, a microprocessor control unit and a feedback closed-loop unit;

[0022] The digital frequency synthesizer is used to generate a digital frequency signal in the range of 1 Hz to 80 Hz;

[0023] The high-speed photoelectric modulation unit is used to adjust the intensity of the excitation light according to the digital frequency signal;

[0024] The microprocessor control unit is used to adjust and optimize the modulation parameters of the digital frequency synthesizer and the high-speed optoelectronic modulation unit in real time;

[0025] The feedback closed-loop unit is used to monitor and correct the modulated output excitation light.

[0026] In some optional embodiments, the light splitting module includes a first beam splitter, a second beam splitter, a third beam splitter, a fourth beam splitter and a fifth beam splitter;

[0027] The first beam splitter is used to reflect the excitation light of the target wavelength to the second beam splitter, the second beam splitter is used to reflect the excitation light of the target wavelength to the third beam splitter, the third beam splitter is used to reflect the excitation light of the target wavelength to the fourth beam splitter, the fourth beam splitter is used to reflect the excitation light of the target wavelength to the fifth beam splitter, and the fifth beam splitter is used to reflect the excitation light of the target wavelength to the microscopy module;

[0028] The fifth beam splitter is used to reflect the feedback light signal to the fourth beam splitter, the fourth beam splitter is used to reflect the feedback light signal to the third beam splitter, and the third beam splitter is used to transmit the feedback light signal to the beam splitting unit.

[0029] In some optional embodiments, the fluorescence detector includes a first detector and a second detector, the first detector is used to detect the fluorescence signal of the first wavelength, the second detector is used to detect the fluorescence signal of the second wavelength, and the bright field detector includes a third detector and a fourth detector, the third detector is used to detect the bright light signal of the third wavelength, and the fourth detector is used to detect the bright light signal of the fourth wavelength.

[0030] In some optional embodiments, the beam splitter unit includes a sixth beam splitter, a seventh beam splitter and an eighth beam splitter, the sixth beam splitter is used to transmit the fluorescence signal of the first wavelength and the second wavelength to the seventh beam splitter, and reflect the bright light signal of the third wavelength and the fourth wavelength to the eighth beam splitter, the seventh beam splitter is used to transmit the fluorescence signal of the first wavelength to the first detector, and reflect the fluorescence signal of the second wavelength to the second detector, and the eighth beam splitter is used to reflect the bright field signal of the third wavelength to the third detector, and transmit the bright field signal of the fourth wavelength to the fourth detector.

[0031] In some optional embodiments, the imaging module includes a data acquisition unit and an image generation unit, the data acquisition unit is used to convert the electrical signal to be processed into digital information, and the image generation unit is used to generate image information according to the digital information.

[0032] In some optional embodiments, the multi-color pathology detection system also includes an analysis module, which is used to generate pathology analysis information after processing the image information, and the pathology analysis information includes at least one of fluorescence lifetime image information, fluorescence lifetime phase information, energy conversion information, photon counting information and scanning fluorescence related spectrum information.

[0033] In some optional embodiments, the optical fiber includes a core and a cladding, the core has a diameter of 80 to 100 μm, the cladding has a diameter of 125 to 140 μm, the numerical aperture is 0.22 to 0.28, and the optical fiber is configured to transmit excitation light in the wavelength range of 300 to 1050 nm.

[0034] In some optional embodiments, the microscopy module includes a microscope unit and a laser confocal scanning head, the microscope unit is placed with the sample to be tested, the laser confocal scanning head uses the excitation light of the target wavelength to scan the sample to be tested, and transmits the feedback light signal to the spectroscopic module.

[0035] In some optional embodiments, the laser confocal scanning head includes a galvanometer unit, and the control module includes a galvanometer control subunit, and the galvanometer control subunit is communicatively connected to the galvanometer unit.

[0036] In some optional embodiments, the galvanometer unit includes a first high-speed galvanometer and a second high-speed galvanometer, the first high-speed galvanometer and the second high-speed galvanometer are arranged orthogonally, the first high-speed galvanometer is used to drive the X-axis scanning of the excitation light of the target wavelength, and the second high-speed galvanometer is used to drive the Y-axis scanning of the excitation light of the target wavelength.

[0037] In some optional embodiments, the first high-speed galvanometer and the second high-speed galvanometer both include a reflective mirror, a voice coil motor and a position sensor, the voice coil motor is used to adjust the reflective mirror after receiving command information from the galvanometer control subunit, and the position sensor is used to send the position information of the reflective mirror to the galvanometer control subunit.

[0038] In some optional embodiments, the microscope unit includes an inverted microscope, an objective lens turntable and a sample stage, the objective lens turntable is equipped with objective lenses of different magnifications, the sample stage is used to move the sample to be tested in the X-axis direction, the Y-axis direction and the Z-axis direction, and the inverted microscope is used to observe the sample to be tested through objective lenses of different magnifications and adjust the focal length of the laser confocal scanning head.

[0039] In some optional embodiments, the data acquisition mode of the data acquisition unit includes at least one of the following:

[0040] Fluorescence fluctuation spectrum counting mode; time-stamping mode; time-resolved mode of time-stamping.

[0041] In some optional embodiments, the data acquisition unit includes a fluorescence lifetime imaging data acquisition subunit and a communication interface, the fluorescence lifetime imaging data acquisition subunit is configured to obtain and process fluorescence lifetime information, and the communication interface is configured to exchange data and transmit instructions with an external computing device through a universal serial bus.

[0042] In a second aspect, an embodiment of the present invention provides a method for detecting and analyzing pathological data, which is applied to a multicolor pathological detection system, wherein the multicolor pathological detection system includes a light source module, a spectroscopic module, a microscopy module, a detection module, an imaging module, and an analysis module; the method for detecting and analyzing pathological data includes:

[0043] Obtaining sample information of the sample to be tested;

[0044] Control the light source module to emit the excitation light of the target wavelength to the spectroscopic module according to the sample information, so that the spectroscopic module guides the excitation light of the target wavelength to the microscopic module;

[0045] Controlling the microscopy module to scan the sample to be tested by the excitation light of the target wavelength, so that the sample to be tested generates a feedback light signal;

[0046] Controlling the microscopy module to transmit the feedback light signal to the light splitting module, so as to guide the feedback light signal to the detection module through the light splitting module;

[0047] Controlling the detection module to detect the feedback optical signals of different wavelengths and converting the feedback optical signals into electrical signals to be processed;

[0048] Controlling the imaging module to process the electrical signal to be processed and generate image information;

[0049] The analysis module is controlled to analyze the image information to obtain pathological data.

[0050] In some optional embodiments, the light source module includes a laser integrator and a multi-wavelength laser source, the microscope module includes a microscope unit and a laser confocal scanner, the microscope unit is provided with the sample to be tested; the detection module includes a plurality of fluorescence detectors and at least one bright field detector; the detection module also includes a beam splitting unit, the beam splitting unit includes a sixth beam splitter, a seventh beam splitter and an eighth beam splitter, the light splitting module includes a first beam splitter, a second beam splitter, a third beam splitter, a fourth beam splitter and a fifth beam splitter, the fluorescence detector includes a first detector and a second detector, the bright field detector includes a third detector and a fourth detector, and the detection module? After obtaining the sample information of the sample to be tested, it further includes:

[0051] Controlling the multi-wavelength laser source to emit initial excitation light of at least one wavelength according to the sample information;

[0052] Controlling the laser integrator to integrate the initial excitation light to obtain the excitation light of the target wavelength;

[0053] The excitation light of the target wavelength is emitted to the first beam splitter through the laser integrator, so as to guide the excitation light of the target wavelength to the laser confocal scanning head through reflection actions of the first beam splitter, the second beam splitter, the third beam splitter, the fourth beam splitter and the fifth beam splitter in sequence;

[0054] Controlling the laser confocal scanning head to scan the sample to be tested, so that the sample to be tested generates a feedback light signal;

[0055] Controlling the laser confocal scanning head to transmit the feedback light signal to the fifth beam splitter, so as to transmit the feedback light signal to the sixth beam splitter through the reflection effect of the fifth beam splitter and the fourth beam splitter and the transmission effect of the third beam splitter;

[0056] The feedback light signals of different wavelengths are directed to the first detector, and / or the second detector, and / or the third detector, and / or the fourth detector through reflection and / or transmission of the sixth beam splitter, the seventh beam splitter, and the eighth beam splitter, so as to generate fluorescence image information through the first detector, and / or the second detector, and generate bright field image information through the third detector, and / or the fourth detector.

[0057] In some optional embodiments, the step of controlling the laser confocal scanning head to scan the sample to be tested so that the sample to be tested generates a feedback light signal comprises:

[0058] Obtaining the position information of the sample to be tested;

[0059] Controlling the scanning angle of the laser confocal scanning head according to the posture information of the sample to be measured, and controlling the microscope unit to move the sample to be measured;

[0060] The laser confocal scanning head is controlled to scan the sample to be tested at the scanning angle, so that the sample to be tested generates a feedback light signal.

[0061] In some optional embodiments, the imaging module includes a data acquisition unit and an image generation unit, and the controlling the imaging module to process the electrical signal to be processed and generate image information includes:

[0062] Controlling the data acquisition unit to acquire the fluorescent image information and / or the bright field image information and generate digital information;

[0063] The image generating unit generates image information from the digital information.

[0064] In some optional embodiments, the method further includes:

[0065] Acquire the time information of the excitation light of the target wavelength reaching the sample to be tested;

[0066] The wavelength and emission time of the next excitation light of the target wavelength emitted by the laser integrator are controlled according to the time information, so that the laser integrator emits the next excitation light of the target wavelength at the emission time.

[0067] In some optional embodiments, the laser confocal scanner includes a galvanometer unit, the galvanometer unit includes a first high-speed galvanometer and a second high-speed galvanometer, the first high-speed galvanometer and the second high-speed galvanometer are arranged orthogonally, the first high-speed galvanometer and the second high-speed galvanometer both include a reflective mirror, a voice coil motor and a position sensor, and the scanning angle of the laser confocal scanner is controlled according to the posture information of the sample to be measured, including:

[0068] Determining the scanning angle of the laser confocal scanning head according to the posture information of the sample to be tested;

[0069] Acquiring position information of the reflective mirror surface according to the position sensor;

[0070] The voice coil motor is controlled to adjust the reflective mirror according to the scanning angle and the posture information, so as to adjust the angle of the laser confocal scanning head to the scanning angle.

[0071] In some optional embodiments, the microscope unit further includes an inverted microscope, an objective lens turret and a sample stage, the objective lens turret is equipped with objective lenses of different magnifications, and before obtaining the sample information of the sample to be tested, the method further includes:

[0072] Selecting a target objective lens on the objective lens turret according to the sample to be tested on the sample stage;

[0073] When the focus adjustment process of the laser confocal scanning head is completed based on the target objective lens and the sample to be measured, the laser confocal scanning head is switched to a scanning mode.

[0074] In a third aspect, an embodiment of the present invention provides a controller, comprising: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the above method when executing the computer program.

[0075] In a fourth aspect, an embodiment of the present invention provides a computer storage medium, wherein the computer storage medium stores computer executable instructions, and the computer executable instructions are used to execute the above-mentioned method for detecting and analyzing pathological data.

[0076] The multicolor pathology detection system according to the embodiment of the present invention has at least the following beneficial effects: at least two different wavelengths of excitation light are emitted through the light source module, and the excitation light of the target wavelength is guided to the spectroscopic module through the optical fiber, so that the spectroscopic module guides the excitation light of the target wavelength to the microscopic module, thereby effectively exciting the multiple fluorescent markers of the sample to be tested on the microscopic module, and the fluorescent signals of multiple fluorescent markers can be detected at the same time, and the detection efficiency is high. The excitation light of the target wavelength can be guided to the microscopic module, and the feedback light signal of the sample to be tested can be guided to the detection module through the same spectroscopic module, with high detection efficiency and a simple detection optical path. After the feedback light signals of different wavelengths are detected by the detection module, the electrical signal to be processed is obtained, and imaging is performed through the imaging module, so that the fluorescent signals of multiple different fluorescent markers and / or the corresponding bright field signals can be detected simultaneously, thereby performing multi-dimensional detection, with a wide range of applications, and capable of capturing the complex molecular characteristics of the sample to be tested.

[0077] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is a structural schematic diagram of a multicolor pathology detection system provided by an embodiment of the present invention;

[0079] Figure 2 Schematic diagram of the optical path structure of a multi-color pathology detection system provided by an embodiment of the present invention;

[0080] Figure 3 Eu(tta) provided by the embodiment of the present invention 3 Schematic diagram of the absorption spectrum and lifetime decay curve of phen material;

[0081] Figure 4 It is a schematic diagram of the absorbance and fluorescence intensity of two materials, Nile Red and BTPEBTTD, provided in an embodiment of the present invention;

[0082] Figure 5 Schematic diagram of absorbance and fluorescence intensity of two materials, Cy5.5 and Cy780, provided in an embodiment of the present invention;

[0083] Figure 6 The present invention provides a flow chart for detecting and analyzing pathological data.

[0084] Reference numerals: base 100;

[0085] Microscope module 200, Z-axis automatic stage 210, inverted microscope 220, objective lens 230;

[0086] Light source module 300, laser integrator 310, modulated nanosecond laser 320, up-conversion laser 330, optical fiber 340;

[0087] A light splitting module 400, a first beam splitter 410, a second beam splitter 420, a third beam splitter 430, a fourth beam splitter 440, and a fifth beam splitter 450;

[0088] The sixth beam splitter 510 , the fifth filter 511 , the seventh beam splitter 520 , the first filter 521 , the second filter 522 , the second detector 523 , the first detector 524 , the eighth beam splitter 530 , the third filter 531 , the fourth filter 532 , the fourth detector 533 , and the third detector 534 . DETAILED DESCRIPTION

[0089] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0090] In the embodiments of the present invention, words such as "exemplary" or "for example" are used to indicate examples, illustrations or explanations. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present invention should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0091] In the following, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, unless otherwise specified, "plurality" means two or more.

[0092] First, refer to Figure 1-2The embodiment of the present invention provides a multi-color pathology detection system, including a control module and a light source module 300, a spectroscopic module 400, a microscope module 200, a detection module and an imaging module electrically connected to the control module respectively; the light source module 300 is used to emit at least two excitation lights of different wavelengths, and guide the excitation light of the target wavelength to the spectroscopic module 400 through the optical fiber 340, so that the spectroscopic module 400 guides the excitation light of the target wavelength to the microscope module 200; the microscope module 200 is used to scan the sample to be tested with the excitation light of the target wavelength, so that the sample to be tested generates a feedback light signal under the action of the excitation light of the target wavelength, and transmits the feedback light signal to the spectroscopic module 400, and guides the feedback light signal to the detection module through the spectroscopic module 400, and the feedback light signal represents the light signal generated by the sample to be tested under the excitation of the excitation light of the target wavelength; the detection module is used to detect the feedback light signals of different wavelengths and convert the feedback light signals into electrical signals to be processed; the imaging module is used to process the electrical signals to be processed and generate image information.

[0093] Specifically, the light source module 300, the spectroscopic module 400, the microscopic module 200 and the detection module are arranged on the base 100, and the imaging module is electrically connected to the detection module. The light source module 300 of the embodiment of the present application includes at least two light source exciters, each of which emits an excitation light of a specific wavelength, and the wavelengths of the excitation lights emitted by different light source exciters are different. According to the multiple different wavelengths of the required excitation light, multiple light source exciters can be configured accordingly. The light source exciter can be a target light source, a laser, etc., which is used to generate excitation light of a specific wavelength to excite the sample to be tested. In some scenarios, as needed, multiple light source exciters can also be replaced with tunable lasers, that is, the light source module 300 adjusts the parameters of the tunable laser so that the tunable laser emits excitation light of different wavelengths. The multicolor pathology detection system can use excitation light of different wavelengths simultaneously or selectively, so that the system can achieve multi-wavelength excitation, and then can simultaneously excite and detect multiple fluorescent markers or biomarkers.

[0094] Between the light source module 300 and the microscopic module 200 where the sample to be tested is placed, a spectroscopic module 400 is provided. The spectroscopic module 400 includes an optical path structure composed of a plurality of spectroscopic elements. The spectroscopic elements may be semi-transparent and semi-reflective mirrors, prisms or diffraction gratings. The excitation light of the target wavelength from the light source module 300 is guided to the microscopic module 200 through the optical path structure, so that the microscopic module 200 scans the sample to be tested with the excitation light of the target wavelength, thereby exciting the sample to be tested, so that the sample to be tested generates a feedback optical signal. The feedback optical signal is guided to the detection module for detection through the spectroscopic module 400, that is, the optical path structure of the spectroscopic module 400 can also guide the feedback optical signal to the detection module, so that the guidance of different optical signals in different directions can be achieved through one optical path structure, the excitation light of the target wavelength and the feedback optical signal can share the same optical path, but can be separated at an appropriate position, and through a reasonably designed beam splitting ratio, the feedback optical signal can be collected to the maximum extent while ensuring sufficient excitation intensity of the excitation light of the target wavelength.

[0095] The detection module can detect feedback light signals of different wavelengths, thereby performing multi-dimensional detection on the sample to be tested, and can well capture the complex molecular characteristics of the sample to be tested. The detection module converts the feedback light signal into an electrical signal to be processed, generates corresponding image information through the imaging module, and then obtains image information of feedback light signals of different wavelengths. After analyzing and processing the image information, the required information can be obtained.

[0096] In some optional embodiments, the light source module 300 includes a laser integrator 310 and a multi-wavelength laser source, the multi-wavelength laser source is used to emit at least two excitation lights of different wavelengths to the laser integrator 310, the laser integrator 310 is used to simultaneously or sequentially select excitation lights of different wavelengths and integrate them to obtain excitation light of a target wavelength, and the excitation light of the target wavelength is subsequently transmitted to the spectroscopic module 400.

[0097] Specifically, the laser integrator 310 is connected to the spectroscopic module 400 via an optical fiber 340. The flexibility and bendability of the optical fiber 340 allow the laser source and the spectroscopic module 400 to be physically separated and not restricted by a strict straight optical path; this ensures efficient transmission of the excitation light, reduces optical path losses, and improves the flexibility and space utilization of the system. Through the collaborative work of the detection module and the imaging module, the system can simultaneously acquire and analyze multiple parameters. Since the system can simultaneously acquire multiple parameters (such as fluorescence signals of different wavelengths, fluorescence lifetime, etc.) and perform comprehensive analysis through the analysis module, it can provide more comprehensive sample information. This multi-dimensional, multi-parameter analysis method can more accurately identify and distinguish different pathological states, thereby reducing false positive and false negative results and improving the accuracy of pathological data. The laser integrator 310 of the embodiment of the present application can integrate at least two different wavelengths of excitation light emitted by the multi-wavelength laser source, so as to integrate multiple different wavelengths of excitation light together for simultaneous emission according to demand, or integrate one wavelength of excitation light at a time for emission, and sequentially integrate the next wavelength of excitation light for emission according to demand, so as to generate feedback light signals of different wavelengths by exciting the sample to be tested with the excitation light of different wavelengths. It can be known that the laser integrator 310 integrates multiple wavelengths of excitation light (i.e., the excitation light of the target wavelength) at a time and emits it to the spectroscopic module 400, and the spectroscopic module 400 guides the excitation light of the target wavelength to the microscope module 200 to scan the sample to be tested, and the sample to be tested generates multiple different wavelengths of feedback light signals accordingly; the laser integrator 310 integrates one wavelength of excitation light at a time and emits it to the spectroscopic module 400, and the spectroscopic module 400 guides the excitation light of the target wavelength to the microscope module 200 to scan the sample to be tested, and the sample to be tested generates a feedback light signal of one wavelength accordingly.

[0098] In some optional embodiments, the multi-wavelength laser source includes a modulated nanosecond laser 320 and an up-conversion laser 330, the modulated nanosecond laser 320 is used to emit short-wave excitation light, and the up-conversion laser 330 is used to emit long-wave excitation light, the wavelength of the short-wave excitation light is between 300nm and 700nm, and the wavelength of the long-wave excitation light is between 700nm and 1000nm.

[0099] Specifically, the combination of the modulated nanosecond laser 320 and the upconversion laser 330 of the embodiment of the present application significantly expands the excitation capability of the system. The modulated nanosecond laser 320 provides precise short pulse excitation (i.e., short-wave excitation light), while the upconversion laser 330 can emit long-wave excitation light with a longer wavelength. This diverse excitation source enables the system to adapt to the needs of various fluorescent markers and biological samples, greatly improving the flexibility and scope of imaging. Secondly, the nanosecond pulse excitation of the modulated nanosecond laser 320 brings time-resolved measurement capabilities to the system. This enables the system to not only obtain fluorescence intensity information, but also perform fluorescence lifetime imaging (FLIM), thereby providing dynamic information of the sample microenvironment, such as pH changes, intermolecular interactions, etc. The acquisition of this multi-dimensional information can be used to deeply understand biological processes and accurately obtain pathological information. Finally, the introduction of the upconversion laser 330 enables the system to have upconversion luminescence imaging capabilities, which can effectively reduce the interference of spontaneous fluorescence of biological tissues, improve the signal-to-noise ratio, and reduce light damage.

[0100] In some optional embodiments, the wavelength of the short-wave excitation light includes at least one of the following: 375nm; 405nm; 485nm; 635nm; 680nm.

[0101] Specifically, refer to Figure 2, the excitation light wavelength of the modulated nanosecond laser 320 includes 375nm, 405nm, 485nm, and 635nm. Specifically, four modulated nanosecond lasers 320 can be set, each modulated nanosecond laser 320 emits an excitation light of a wavelength, and the four modulated nanosecond lasers 320 are arranged in sequence in the first direction, and the excitation light is emitted or transmitted to the laser integrator 310 through four spectroscopic elements; specifically, the four spectroscopic elements are arranged in sequence in the first direction, and correspond to the modulated nanosecond laser 320 in the second direction, and the first direction and the second direction are perpendicular to each other, so that the excitation light emitted by the modulated nanosecond laser 320 can be reflected by the corresponding spectroscopic element, and transmitted by other spectroscopic elements located in the optical path, so as to introduce the excitation light into the laser integrator 310. For example, the first modulated nanosecond laser emits an excitation light of 375nm, the second modulated nanosecond laser emits an excitation light of 405nm, the third modulated nanosecond laser emits an excitation light of 485nm, and the fourth modulated nanosecond laser emits an excitation light of 635nm. The first beam splitter element corresponding to the first modulated nanosecond laser in the second direction reflects the 375nm excitation light to the second beam splitter element, and the second beam splitter element, the third beam splitter element and the fourth beam splitter element sequentially transmit the 375nm excitation light to the laser integrator 310; similarly, the second beam splitter element corresponding to the second modulated nanosecond laser in the second direction reflects the 405nm excitation light to the third beam splitter element, and the third beam splitter element and the fourth beam splitter element sequentially transmit the 405nm excitation light to the laser integrator 310; the third beam splitter element corresponding to the third modulated nanosecond laser in the second direction reflects the 485nm excitation light to the fourth beam splitter element, and the fourth beam splitter element transmits the 485nm excitation light to the laser integrator 310; the fourth beam splitter element corresponding to the fourth modulated nanosecond laser in the second direction reflects the 635nm excitation light to the laser integrator 310. The wavelength of the excitation light covers the excitation spectrum of most commonly used fluorescent dyes and markers, enabling the system to simultaneously or sequentially excite multiple specific markers, such as cell nuclei, cytoskeleton, specific proteins, etc. Multiple labeling can obtain rich pathological information in a single scan, greatly improving the accuracy and efficiency of subsequent data processing. In addition, the selection of these specific wavelengths provides new possibilities for the autofluorescence imaging of pathological tissues. For example, 375nm and 405nm wavelengths can effectively excite endogenous fluorescent substances such as NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) in tissues, revealing the metabolic state and morphological characteristics of tissues without additional staining.

[0102] In some optional embodiments, the wavelength of the long-wave excitation light includes at least one of the following: 730 nm; 808 nm; 980 nm.

[0103] Specifically, the wavelength of the excitation light of the up-conversion laser 330 includes any one or more of 730nm, 808nm, and 980nm. Taking 980nm as an example in this embodiment, the excitation light of 980nm significantly improves the ability of deep imaging of pathological samples (samples to be tested). This near-infrared excitation light has a strong tissue penetration ability and can penetrate into the tissue depth that is difficult to reach with conventional visible light. This feature enables the system to perform non-invasive deep detection of thicker pathological sections or biological tissues, providing new possibilities for three-dimensional pathological analysis and monitoring of living lesions. There is almost no spontaneous up-conversion luminescence under 980nm excitation, so the signal-to-noise ratio is greatly improved, allowing the system to detect trace amounts of pathological markers. In addition, the low phototoxicity of 980nm excitation light creates conditions for long-term observation and in vivo detection. Compared with short-wavelength lasers, near-infrared light causes significantly less photodamage to biological samples. This enables the system to perform long-term continuous observation of pathological samples or repeated detection of living tissues.

[0104] In some optional embodiments, the detection module includes at least one fluorescence detector, which is used to generate fluorescence image information after detecting a fluorescence signal in the feedback light signal, and the fluorescence signal is generated by the fluorescent marker under the excitation of excitation light of the target wavelength.

[0105] In some optional embodiments, the detection module also includes at least one bright field detector, which is used to generate bright field image information after detecting a bright light signal in the feedback light signal, and the bright light signal represents the light signal generated by the sample to be tested under the excitation of the excitation light of the target wavelength.

[0106] Specifically, the detection module of the present application includes at least one fluorescence detector and at least one bright field detector, so that fluorescence imaging and bright field imaging can be performed simultaneously or separately, thereby obtaining corresponding fluorescence image information and bright field image information. The bright field image information of bright field imaging provides the overall morphology and structural information of the sample to be detected, while the fluorescence image information of fluorescence imaging accurately locates specific molecular markers. This dual-modality imaging (fluorescence imaging and bright field imaging) method can accurately interpret changes at the molecular level in the context of tissue structure, greatly improving the accuracy and efficiency of pathological data analysis, especially in the diagnosis of complex lesions such as early cancer or inflammatory diseases.

[0107] In some optional embodiments, the fluorescence detector includes a fluorescence single photon detector, and the bright field detector includes a bright field transmission single photon detector.

[0108] Specifically, the fluorescent single-photon detector can detect extremely weak fluorescence signals, which is conducive to observing low-expression or fast-decaying fluorescent markers and supports fluorescence lifetime imaging (FLIM). The bright-field transmission single-photon detector can accurately capture the tiny structural changes and subtle optical density differences of the sample, providing high-resolution and high-contrast tissue morphology images; the cell morphology, tissue structure and lesion characteristics of the biological sample to be tested can be observed more clearly, which helps to improve the accuracy of pathological diagnosis. The transmission single-photon detector provides morphological information of the sample, and combined with the fluorescence signal of the fluorescent single-photon detector, the correlation analysis of structure and function can be realized.

[0109] In some optional embodiments, the modulated nanosecond laser 320 includes a programmable broadband frequency laser modulator, which is used to continuously adjust the output of the excitation light in the sub-hertz to megahertz range.

[0110] In some optional embodiments, the broadband frequency laser modulator includes a digital frequency synthesizer, a high-speed photoelectric modulation unit, a microprocessor control unit and a feedback closed-loop unit; the digital frequency synthesizer is used to generate a digital frequency signal in the range of 1 Hz to 80 Hz; the high-speed photoelectric modulation unit is used to adjust the intensity of the excitation light according to the digital frequency signal; the microprocessor control unit is used to adjust and optimize the modulation parameters of the digital frequency synthesizer and the high-speed photoelectric modulation unit in real time; the feedback closed-loop unit is used to monitor and correct the modulated output excitation light.

[0111] Specifically, the programmable broadband frequency laser modulator can precisely control the output of the excitation light. A digital frequency signal with any modulation frequency in the range of 1 Hz to 80 Hz is generated by a high-precision digital frequency synthesizer, converted into an analog signal by a digital-to-analog converter, and then transmitted to a high-speed photoelectric modulation unit. The high-speed photoelectric modulation unit receives the analog signal and converts it into the corresponding light intensity modulation parameter, thereby adjusting the intensity of the excitation light according to the light intensity adjustment parameter. The high-speed photoelectric modulation unit directly acts on the excitation light path, and adjusts the intensity of the excitation light by changing the optical properties of the crystal or the acoustic diffraction effect, thereby achieving continuous modulation from sub-hertz to megahertz. The microprocessor control unit is connected to the main control computer through a high-speed digital interface, receives operating instructions and experimental parameters, and controls the digital frequency synthesizer and the high-speed photoelectric modulation unit at the same time, and adjusts and optimizes the modulation parameters in real time. The microprocessor control unit can dynamically adjust the modulation strategy according to specific needs, such as performing frequency scanning, generating complex pulse sequences, or achieving specific waveform output. The feedback closed-loop unit collects the modulated excitation light through a high-speed photodetector, converts the collected optical signal into an electrical signal to obtain feedback data, and then transmits the feedback data to the microprocessor control unit, which analyzes the feedback data and obtains the actual output data of the excitation light based on the feedback data. The actual data includes light intensity, frequency, wavelength, etc., which are not specifically limited; the microprocessor control unit compares the actual output data with the preset value to obtain a comparison result, and calculates the corresponding correction parameters based on the comparison result, thereby generating a correction instruction. The correction instruction is then sent to the digital frequency synthesizer and the high-speed photoelectric modulation unit to achieve real-time correction of the modulated output.

[0112] The microprocessor control unit sets the initial parameters according to the requirements and drives the digital frequency synthesizer to generate the required modulation signal. The modulation signal is converted into light intensity modulation parameters by the high-speed photoelectric modulation unit, which acts on the light intensity of the excitation light output. At the same time, the feedback closed-loop unit continuously monitors the actual output data and feeds the actual output data back to the microprocessor control unit. The microprocessor control unit analyzes the actual output data in real time, calculates the deviation from the preset value, and quickly adjusts the control parameters to achieve precise modulation of the excitation light output, thereby compensating for the influence of environmental changes, device drift and other factors, ensuring the long-term stable operation of the system and meeting the needs of high-precision imaging and detection.

[0113] In some optional embodiments, the optical fiber 340 includes a core and a cladding, the core has a diameter of 80 to 100 μm, the cladding has a diameter of 125 to 140 μm, and the numerical aperture is 0.22 to 0.28. The optical fiber 340 is configured to transmit excitation light in the wavelength range of 300 to 1050 nm.

[0114] Specifically, the fiber core is used to transmit optical signals; a total reflection interface is formed through the cladding to limit the propagation of light in the fiber core. A numerical aperture of 0.22 to 0.28 is conducive to improving the light coupling efficiency, that is, optimizing the light collection and transmission efficiency. In this way, a single optical fiber 340 can transmit multiple wavelengths of excitation light, which simplifies the system design and is also conducive to the rapid switching of different wavelengths of excitation light to improve the efficiency of multi-color imaging. In addition, it can also transmit 980nm excitation light to support the application of up-conversion nanomaterials. The optical fiber 340 of the embodiment of the present application is configured to transmit excitation light within the wavelength range of 300 to 1050 nm, and the wavelength transmission range of 300 to 1050 nm covers the spectrum from ultraviolet to near infrared, and is suitable for the excitation of a variety of fluorescent dyes, such as the existing fluorescent dye DAPI (excited by 375 nm excitation light), fluorescent dye GFP (excited by 488 nm excitation light), fluorescent dye Cy3 (excited by 550 nm excitation light) and fluorescent dye Cy5 (650 nm by 650 nm excitation light), etc., and also supports 980 nm excitation of up-conversion nanoparticles; and the numerical aperture in the range of 0.22 to 0.28 effectively controls the mode dispersion while ensuring sufficient light collection capability, so that the optical fiber 340 can maintain good transmission characteristics in the wide spectral range of 300 to 1050 nm. The core diameter of 80 to 100 μm provides a sufficiently large optical transmission channel, ensuring the effective transmission of high-power excitation light while maintaining the flexibility of the optical fiber 340. The cladding diameter of 125 to 140 μm is compatible with the standard optical fiber 340 interface and provides sufficient mechanical strength, which not only ensures a good match between the optical fiber 340 and conventional optical devices, but also enhances the durability of the optical fiber 340 in practical applications.

[0115] In some optional embodiments, the microscope module 200 includes a microscope unit and a laser confocal scanner. The microscope unit is placed with a sample to be tested. The laser confocal scanner uses excitation light of a target wavelength to scan the sample to be tested and transmits a feedback light signal to the spectroscopic module 400.

[0116] Specifically, after the laser light source is integrated by the laser integrator 310, the excitation light of the target wavelength is transmitted to the spectroscopic module 400 through the optical fiber 340, and the spectroscopic module 400 guides the excitation light of the target wavelength to the laser confocal scanning head. The scanning mirror in the laser confocal scanning head quickly scans the laser light of the target wavelength to the sample to be tested. The fluorescence signal and / or bright field signal in the feedback light signal generated by the sample to be tested is collected by the objective lens 230, separated by the spectrometer in the laser confocal scanning head, and then transmitted to the detection module through the spectroscopic module 400 for detection.

[0117] In some optional embodiments, the laser confocal scanning head includes a galvanometer unit, and the control module includes a galvanometer control subunit, and the galvanometer control subunit is communicatively connected to the galvanometer unit.

[0118] In some optional embodiments, the galvanometer unit includes a first high-speed galvanometer and a second high-speed galvanometer, the first high-speed galvanometer and the second high-speed galvanometer are arranged orthogonally, the first high-speed galvanometer is used to drive the X-axis scanning of the excitation light of the target wavelength, and the second high-speed galvanometer is used to drive the Y-axis scanning of the excitation light of the target wavelength.

[0119] In some optional embodiments, the first high-speed galvanometer and the second high-speed galvanometer both include a reflective mirror, a voice coil motor and a position sensor. The voice coil motor is used to adjust the reflective mirror after receiving command information from the galvanometer control subunit, and the position sensor is used to send the position information of the reflective mirror to the galvanometer control subunit.

[0120] Specifically, the galvanometer control subunit is integrated in the control module and connected to the central processing unit of the system through a high-speed digital interface. The galvanometer control subunit receives corresponding scanning parameters and control instructions. The galvanometer control subunit contains a dedicated digital signal processor and a high-precision digital-to-analog converter, which can generate accurate driving signals. The driving signal is transmitted to the galvanometer unit in the laser confocal scanning head through a well-shielded analog signal line. The galvanometer unit is located in the optical path of the laser confocal scanning head and is usually composed of two orthogonally arranged high-speed galvanometers, namely the first high-speed galvanometer and the second high-speed galvanometer, which are responsible for scanning the X-axis and Y-axis respectively, so as to scan the sample to be detected on the X-axis and Y-axis through the excitation light of the target wavelength. Each high-speed galvanometer is a precision electromechanical system, including a reflective mirror surface coated with a high-reflective coating, a high-response speed voice coil motor and a precision position sensor. The first high-speed galvanometer and / or the second high-speed galvanometer receive the driving signal from the galvanometer control subunit, and quickly and accurately change the angle of the reflective mirror surface through the voice coil motor, thereby realizing two-dimensional scanning of the laser beam on the sample plane (the plane formed by the X-axis and the Y-axis). The galvanometer control subunit generates an ideal scanning waveform according to the set scanning parameters (such as scanning range, frequency, mode, etc.). The scanning waveform is usually a sawtooth wave or a sine wave, corresponding to the linear scanning and resonant scanning modes, respectively. Then, the scanning waveform is converted into a precise voltage signal to drive the galvanometer unit. At the same time, the position sensors on the first high-speed galvanometer and the second high-speed galvanometer obtain the actual position information of the reflective mirror surface in real time. The galvanometer control subunit compares the actual position with the preset position, calculates the error, and adjusts the instruction information in real time, thereby driving the voice coil motor to adjust the reflective mirror surface through the instruction information to ensure the accuracy and repeatability of the scanning trajectory. When the excitation light is incident on the reflective mirror surface, a slight change in the angle of the reflective mirror surface will cause a significant change in the direction of the reflected light beam. By precisely controlling the angles of the two orthogonal galvanometers (the first high-speed galvanometer and the second high-speed galvanometer), the excitation light is precisely positioned to any point on the plane of the sample to be tested. Through high-speed scanning, the scanning imaging of the entire sample area to be tested can be completed in a very short time. The high bandwidth characteristics of the galvanometer unit allow rapid angle changes, thereby achieving high frame rate image acquisition.

[0121] In some optional embodiments, the microscope unit includes an inverted microscope 220, an objective lens turntable and a sample stage. The objective lens turntable is equipped with objective lenses 230 of different magnifications. The sample stage is used to move the sample to be tested in the X-axis direction, the Y-axis direction and the Z-axis direction. The inverted microscope 220 is used to observe the sample to be tested through the objective lenses 230 of different magnifications and adjust the focal length of the laser confocal scanning head.

[0122] Specifically, the inverted microscope 220 is located at the bottom of the microscope unit, serving as the basic platform of the entire imaging system. The inverted microscope 220 is equipped with a replaceable objective turret, which is equipped with objective lenses 230 of different magnifications. The sample stage of the microscope unit can move in the X-axis direction, the Y-axis direction and the Z-axis direction. The specific sample stage is placed on the Z-axis automatic platform, and the sample stage can be moved in the Z-axis direction by the Z-axis automatic platform. The sample stage itself can move in the X-axis direction and the Y-axis direction, thereby adjusting the position of the sample to be measured. The laser confocal scanning head is installed on the side or rear of the inverted microscope 220, and is closely integrated with the optical path system of the microscope. The scanning head contains a high-speed scanning mirror (a first high-speed scanning mirror and a second high-speed scanning mirror) and a spectrometer, which are used to realize the rapid scanning of the excitation light and the separation of the fluorescence signal. The detection module is located on the light output path of the laser confocal scanning head, and includes a high-sensitivity detector such as a photomultiplier tube (PMT) or an avalanche photodiode (APD). The detection module is connected to the scanning head through an optical fiber 340 or a direct optical path. The control module is usually a high-performance computer placed near the system console. This module is connected to the laser light source, scanning head, detection module and other parts through data lines to achieve control of the entire system. The data acquisition module is integrated into the control module or placed near the system as an independent hardware unit. It is connected to the detection module through a high-speed data line and is used to collect and process image data in real time. The software system of the analysis module is installed on the computer of the control module and is used to post-process and analyze the collected data.

[0123] In some optional embodiments, the data acquisition unit includes a fluorescence lifetime imaging data acquisition subunit and a communication interface, the fluorescence lifetime imaging data acquisition subunit is configured to obtain and process fluorescence lifetime information, and the communication interface is configured to exchange data and transmit instructions with an external computing device through a universal serial bus.

[0124] In some optional embodiments, the data acquisition mode of the data acquisition unit includes at least one of the following: fluorescence fluctuation spectrum counting mode; time marking mode; time-resolved mode of time marking.

[0125] Specifically, the fluorescence fluctuation spectrum counting mode uses a multi-wavelength excitation light source and a multi-channel detector, and each channel corresponds to a fluorescence signal in a specific wavelength range. The fluorescence lifetime imaging data acquisition subunit simultaneously records the fluorescence intensity changes at different wavelengths. The time-stamping mode uses a high-speed photon counter and a precision time-to-digital converter to record the precise timestamp of the arrival of each fluorescent photon. The time-resolved mode of time-stamping combines time-stamping and time-correlated single-photon counting methods to simultaneously record the time delay between the photon arrival time and the excitation pulse. Through software control, multiple acquisition modes can be flexibly switched or used simultaneously to customize data acquisition strategies according to specific needs. Therefore, it can provide multi-dimensional and comprehensive sample information, including concentration, distribution, kinetics and microenvironment characteristics, and can adapt to different types of pathological samples and research needs, thereby improving the versatility and flexibility of the system.

[0126] In some optional embodiments, the light splitting module 400 includes a first beam splitter 410, a second beam splitter 420, a third beam splitter 430, a fourth beam splitter 440 and a fifth beam splitter 450; the first beam splitter 410 is used to reflect the excitation light of the target wavelength to the second beam splitter 420, the second beam splitter 420 is used to reflect the excitation light of the target wavelength to the third beam splitter 430, the third beam splitter 430 is used to reflect the excitation light of the target wavelength to the fourth beam splitter 440, the fourth beam splitter 440 is used to reflect the excitation light of the target wavelength to the fifth beam splitter 450, and the fifth beam splitter 450 is used to reflect the excitation light of the target wavelength to the microscopy module 200;

[0127] The fifth beam splitter 450 is used to reflect the feedback light signal to the fourth beam splitter 440 , the fourth beam splitter 440 is used to reflect the feedback light signal to the third beam splitter 430 , and the third beam splitter 430 is used to transmit the feedback light signal to the beam splitting unit.

[0128] Specifically, the first beam splitter 410 and the second beam splitter 420 are sequentially arranged in the first direction and are in the same plane and have the same tilt angle relative to the first direction. The excitation light of the target wavelength emitted by the laser integrator 310 reaches the second beam splitter 420 in the first direction after being reflected by the first beam splitter 410; the second beam splitter 420 and the third beam splitter 430 are arranged opposite to each other in the second direction, and the second beam splitter 420 and the third beam splitter 430 are symmetrical to each other about the central axis in the first direction, that is, the second beam splitter 420 is symmetrical to the third beam splitter 430 about the central axis in the first direction. At the same time, the third beam splitter 430 and the fourth beam splitter 440 are arranged opposite to each other in the first direction, and the third beam splitter 430 is symmetrical to the fourth beam splitter 440 about the central axis in the second direction; the fourth beam splitter 440 and the fifth beam splitter 450 are arranged opposite to each other in the second direction, and the fourth beam splitter 440 and the fifth beam splitter 450 have the same tilt angle in the first direction. The second beam splitter 420 reflects the excitation light of the target wavelength in the second direction to the third beam splitter 430, the third beam splitter 430 reflects the excitation light of the target wavelength in the first direction to the fourth beam splitter 440, the fourth beam splitter 440 reflects the excitation light of the target wavelength in the second direction to the fifth beam splitter 450, and the fifth beam splitter 450 reflects the excitation light of the target wavelength in the first direction to the microscopy module 200. The laser confocal scanning head of the microscopy module 200 scans the sample to be tested with the excitation light of the target wavelength, thereby exciting the sample to be tested to generate a feedback light signal.

[0129] The laser confocal scanning head sends the feedback light signal to the fifth beam splitter 450 through the original optical path. The fifth beam splitter 450 reflects the feedback light signal and reaches the fourth beam splitter 440 in the second direction. The fourth beam splitter 440 reflects the feedback light signal and reaches the third beam splitter 430 in the first direction. The third beam splitter 430 transmits the feedback light signal and reaches the beam splitting unit in the first direction. The feedback light signal and the excitation light of the target wavelength are transmitted through the same optical path, which saves the optical path structure and reduces the testing cost.

[0130] In some optional embodiments, the fluorescence detector includes a first detector 524 and a second detector 523, the first detector 524 is used to detect a fluorescence signal of a first wavelength, and the second detector 523 is used to detect a fluorescence signal of a second wavelength, and the bright field detector includes a third detector 534 and a fourth detector 533, the third detector 534 is used to detect a bright light signal of a third wavelength, and the fourth detector 533 is used to detect a bright light signal of a fourth wavelength.

[0131] Specifically, the first detector 524 and the second detector 523 of the fluorescence detector are used to detect fluorescence signals of different wavelengths, that is, the first detector 524 detects the fluorescence signal of the first wavelength, and the second detector 523 detects the fluorescence signal of the second wavelength; the fluorescence signals of different wavelengths can be detected simultaneously, so as to simultaneously obtain the fluorescence signals of different wavelengths emitted by different fluorescent markers on the sample to be tested, with high detection efficiency, and the pathological data can be obtained in a timely and comprehensive manner. At the same time, the third detector 534 and the fourth detector 533 of the bright field detector are used to detect bright field signals of different wavelengths, that is, the third detector 534 detects the bright field signal of the third wavelength, and the fourth detector 533 detects the bright field signal of the fourth wavelength; the bright field signals of different wavelengths can be detected simultaneously, and by simultaneously obtaining the bright field light signals of different wavelengths emitted by the sample to be tested, the overall morphology and structural information of the sample to be tested, such as cell morphology and tissue structure, can be obtained, thereby improving the accuracy and comprehensiveness of pathological data acquisition.

[0132] In some optional embodiments, the beam splitter unit includes a sixth beam splitter 510, a seventh beam splitter 520 and an eighth beam splitter 530. The sixth beam splitter 510 is used to transmit the fluorescence signals of the first wavelength and the second wavelength to the seventh beam splitter 520, and reflect the bright light signals of the third wavelength and the fourth wavelength to the eighth beam splitter 530. The seventh beam splitter 520 is used to transmit the fluorescence signal of the first wavelength to the first detector 524, and reflect the fluorescence signal of the second wavelength to the second detector 523. The eighth beam splitter 530 is used to reflect the bright field signal of the third wavelength to the third detector 534, and transmit the bright field signal of the fourth wavelength to the fourth detector 533.

[0133] Specifically, the third beam splitter 430 of the optical splitter module 400 transmits the feedback light signal to the sixth beam splitter 510, and the sixth beam splitter 510 transmits the fluorescent signal in the feedback light signal to the seventh beam splitter 520, and at the same time reflects the bright field signal in the feedback light signal to the eighth beam splitter 530, so that the fluorescent signal and the bright field signal are separated and detected at the same time, with high detection efficiency, avoiding mutual interference, and high detection accuracy. The seventh beam splitter 520 transmits the fluorescent signal of the first wavelength to the first detector 524, and at the same time reflects the fluorescent signal of the second wavelength to the second detector 523, so that the fluorescent signal of the first wavelength is detected by the first detector 524, and the fluorescent signal of the second wavelength is detected by the second detector 523, and fluorescent signals of different wavelengths are detected at the same time, so that different fluorescent markers are detected at the same time to obtain multiple pathological feature information, with high detection efficiency and more comprehensive detection. The eighth beam splitter 530 reflects the bright field signal of the third wavelength to the third detector 534, and transmits the bright field signal of the fourth wavelength to the fourth detector 533. The third detector 534 detects the bright field signal of the third wavelength, and the fourth detector 533 detects the bright field signal of the fourth wavelength. By detecting bright field signals of different wavelengths at the same time, a more comprehensive morphological image of the sample to be tested, such as cell morphology, tissue structure and lesion characteristics, is obtained, with high detection efficiency and more comprehensive detection. Combining the fluorescence signal and the bright field signal at the same time, the changes at the molecular level can be accurately interpreted in the context of the tissue structure, which greatly improves the accuracy and efficiency of pathological data analysis, especially in the pathological analysis of complex lesions such as early cancer or inflammatory diseases.

[0134] In some optional embodiments, a first filter 521 is provided between the seventh beam splitter and the first detector 524, and the feedback light signals of other wavelengths are filtered out by the first filter 521, so as to retain the fluorescence signal of the first wavelength; a second filter 522 is provided between the seventh beam splitter and the second detector 523, and the feedback light signals of other wavelengths are filtered out by the second filter 522, so as to retain the fluorescence signal of the second wavelength. A third filter 531 is provided between the eighth beam splitter 530 and the third detector 534, and the feedback light signals of other wavelengths are filtered out by the third filter 531, so as to retain the bright field signal of the third wavelength; a fourth filter 532 is provided between the eighth beam splitter 530 and the fourth detector 533, and the feedback light signals of other wavelengths are filtered out by the fourth filter 532, so as to retain the bright field signal of the fourth wavelength; a fifth filter 511 is provided between the sixth beam splitter 510 and the optical splitting module 400, and the light signals of other wavelengths are filtered out by the fifth filter 511, so as to retain the feedback light signal. Filters can also be used to suppress background, remove scattering and reflection of excitation light, and improve the signal-to-noise ratio of fluorescence signals and bright field signals.

[0135] In some optional embodiments, the imaging module includes a data acquisition unit and an image generation unit, the data acquisition unit is used to convert the electrical signal to be processed into digital information, and the image generation unit is used to generate image information according to the digital information.

[0136] In some optional embodiments, the multi-color pathology detection system also includes an analysis module, which is used to generate pathology analysis information after processing image information, and the pathology analysis information includes at least one of fluorescence lifetime image information, fluorescence lifetime phase information, energy conversion information, photon counting information and scanning fluorescence related spectrum information.

[0137] Specifically, the detection module converts the acquired light signal (feedback light signal) into an electrical signal, which is digitized by the data acquisition unit to obtain digital information. The image generation unit reconstructs the confocal image of the sample in real time based on the digital information to obtain the corresponding image information. The analysis module performs post-processing on the acquired image information, such as denoising, contrast enhancement, etc., and performs quantitative analysis. The control module manages the entire system and coordinates the work of each module, such as controlling the switch and intensity of the laser, adjusting scanning parameters, and synchronizing data acquisition. It can achieve high-resolution fluorescence imaging of pathological samples, meet the excitation requirements of a variety of fluorescent markers, and provide rich data analysis functions.

[0138] The analysis module includes at least one of the following functional units: a lifetime analysis unit, configured to generate a lifetime distribution histogram and / or a fluorescence lifetime decay curve (i.e., fluorescence lifetime image information) according to the fluorescence lifetime data in the image information; a Phasor analysis unit, configured to generate a phasor plots (phase diagram) data direct reading diagram (i.e., fluorescence lifetime phase information) in real time during the lifetime image acquisition process; a FRET ( The Foster resonance energy transfer (FRET) analysis unit is configured to calculate the energy transfer efficiency (energy conversion information) based on the FRET orbital data; the photon counting analysis unit is configured to perform photon counting bar graph analysis to obtain photon counting information; the scanning FCS (Fluorescence Correlation Spectroscopy) analysis unit is configured to parse the Scanning FCS (Scanning Fluorescence Correlation Spectroscopy) data to obtain scanning fluorescence correlation spectrum information.

[0139] In multi-color pathology detection, the lifetime analysis unit collects fluorescence lifetime data through time-correlated single photon counting technology, and generates lifetime distribution histograms and fluorescence lifetime decay curves, which can effectively distinguish fluorescent markers with similar emission spectra but different lifetimes, provide microenvironment information such as pH and ion concentration, and help distinguish normal and diseased tissues; at the same time, it can also reduce spontaneous fluorescence interference and improve the signal-to-noise ratio, thereby enhancing the accuracy and sensitivity of multi-color pathology detection. During the lifetime image acquisition process, the Phasor analysis unit performs Fourier transform on the fluorescence decay data of each pixel, and generates phasor plots data direct reading diagrams in real time. It can intuitively display multi-component fluorescence lifetime information without complex mathematical model fitting, quickly identify and distinguish different fluorescent markers and spontaneous fluorescence, help discover and analyze unknown fluorescent components in complex samples, and provide more comprehensive information for pathology diagnosis. The FRET analysis unit simultaneously detects the intensity and lifetime changes of donor and acceptor fluorescent molecules, calculates the energy transfer efficiency based on FRET orbital data, and can study molecular interactions and conformational changes, such as protein-protein interactions, and provide nanoscale spatial information. The photon counting analysis unit uses a high-sensitivity photomultiplier tube or a single-photon avalanche diode to perform photon counting histogram analysis. It has extremely high sensitivity and can detect extremely low concentrations of fluorescent markers. The scanning FCS analysis unit analyzes the Scanning FCS data, combining the advantages of fluorescence correlation spectroscopy and confocal microscopy; in multi-color pathology detection, scanning FCS can simultaneously analyze molecular dynamics at multiple spatial locations, providing spatial distribution information on molecular diffusion, concentration, and interaction. The combined use of multiple functional units of the analysis module provides multi-dimensional, high-sensitivity, and high-temporal and spatial resolution analysis capabilities for multi-color pathology detection, which can not only improve the accuracy and reliability of pathology data analysis, but also reveal subtle changes and dynamic processes that are difficult to capture with traditional methods.

[0140] In some optional embodiments, the specific process of fluorescence lifetime imaging includes: the excitation light of the target wavelength is scanned point by point on the sample through the laser confocal scanner to ensure high-resolution imaging. In order to achieve accurate time-resolved measurement, time-correlated single photon counting (TCSPC) is used, and a high-speed photodetector is equipped in the detection module. The TCSPC electronics module accurately records the arrival time of each detected photon relative to the excitation light, while the high-performance data acquisition unit is responsible for high-speed processing and storage of large amounts of time-resolved data. Subsequently, the analysis module uses a specific algorithm (such as least squares fitting or maximum likelihood estimation) to calculate the fluorescence lifetime of each pixel, and finally maps these lifetime values ​​to the corresponding spatial positions to form a complete fluorescence lifetime image.

[0141] First, the sample is precisely placed on the Z-axis automatic stage 210 to ensure the best imaging position. Then the system is set up, including selecting a suitable excitation light source and wavelength, adjusting the laser power and scanning parameters, and setting the appropriate filter combination. In the scanning stage, the excitation light scans the sample finely point by point through the laser confocal scanner, and each scanning point receives multiple excitation pulses to collect enough fluorescence photons. During the data acquisition process, the time-correlated single photon counting (TCSPC) unit accurately records the arrival time of each detected photon relative to the excitation pulse, and repeats this process for each pixel point to construct a detailed time-resolved fluorescence decay curve. Next, in the data processing stage, the analysis module fits the fluorescence decay curve of each pixel and calculates the fluorescence lifetime value. Finally, the calculated fluorescence lifetime value is mapped to the corresponding spatial position to generate a fluorescence lifetime image, and pseudo-color is usually used to represent different lifetime values ​​to intuitively present the results. In addition, fluorescence intensity images and fluorescence lifetime images can be displayed simultaneously, including multi-dimensional data such as fluorescence lifetime distribution and component analysis, providing comprehensive and in-depth sample information.

[0142] In some optional embodiments, the nanosecond pulses (excitation light) emitted by the modulated nanosecond laser 320 provide a clear and precise excitation start time. This precise time reference point is the basis for time-resolved measurement. Each pulse can be regarded as a "time marker", and the system starts timing based on this. The duration of the nanosecond pulse is extremely short, much shorter than the lifetime of most fluorescence processes. This ensures that the excitation process can be approximated as an instantaneous event and will not interfere with the subsequent measurement of the fluorescence decay process. Combining spatial and temporal information, this technology also realizes multi-parameter imaging, simultaneously acquiring fluorescence intensity, lifetime and spectral information, enhancing the system's information acquisition capabilities and application range. Finally, it provides a basis for time-resolved super-resolution imaging techniques (such as STED), which helps to break through the resolution limit of traditional optical microscopes.

[0143] In some optional embodiments, the FRET track data represents the time series data obtained in the single molecule FRET experiment. The time series data records the donor and acceptor fluorescence intensities that change over time. Specifically, it includes: the change of the donor fluorescence intensity over time; the change of the acceptor fluorescence intensity over time; the change of the FRET efficiency over time.

[0144] The energy transfer efficiency is calculated as follows:

[0145] 1. Method based on intensity ratio:

[0146] E=I A / (I A +γI D )

[0147] Where E is the FRET efficiency, I A is the fluorescence intensity of the receptor, I D is the fluorescence intensity of the donor and γ is the correction factor.

[0148] 2. Donor quenching-based method:

[0149] E=1-(F DA / F D )

[0150] Among them, F DA is the fluorescence intensity of the donor in the presence of the acceptor, F D is the fluorescence intensity of the donor in the absence of the acceptor.

[0151] 3. Lifespan-based approach:

[0152] E=1-(τ DA / τ D )

[0153] Among them, τ DA is the fluorescence lifetime of the donor in the presence of the acceptor, τ D is the fluorescence lifetime of the donor in the absence of the acceptor.

[0154] 4. Single-molecule FRET data analysis:

[0155] The FRET trajectories were analyzed using a hidden Markov model (HMM) to identify different FRET states.

[0156] The average FRET efficiency was calculated for each state.

[0157] Analyze state transition dynamics.

[0158] 5. Time-resolved FRET:

[0159] The FRET efficiency was calculated by analyzing the fluorescence decay curve using time-correlated single photon counting (TCSPC) technology.

[0160] 6. Spectral FRET:

[0161] The FRET efficiency is calculated by analyzing the emission spectra changes of the donor and acceptor.

[0162] use The equation converts FRET efficiency to intermolecular distance:

[0163]

[0164] Where R is the actual distance, R 0 yes distance.

[0165] In some optional embodiments, Scanning FCS (Scanning Fluorescence Correlation Spectroscopy) is a variant of Fluorescence Correlation Spectroscopy (FCS) that acquires data by scanning the laser focus in the sample, thereby providing more spatial information.

[0166] Scanning FCS data consists of: time series fluorescence intensity data; corresponding spatial position information; scanning parameters (such as scanning speed, scanning mode, etc.).

[0167] Data characteristics: Contains information in two dimensions: time and space; usually has a high time resolution (microsecond to millisecond level); spatial resolution depends on the scanning step size and laser focus size.

[0168] Data acquisition methods: 1) Linear scanning: scanning repeatedly along a straight line; 2) Circular scanning: scanning along a circular path; 3) Grid scanning: raster scanning on a two-dimensional plane.

[0169] After parsing the Scanning FCS data, the following information can be obtained:

[0170] 1) Molecular diffusion coefficient: The diffusion time of the molecule is obtained by fitting the autocorrelation function; the diffusion coefficient is calculated using the known confocal volume.

[0171] 2) Molecular concentration: The number of molecules in the sample is estimated from the amplitude of the autocorrelation function; the local concentration is calculated based on the observed volume.

[0172] 3) Spatial inhomogeneity: Analyze the diffusion coefficient and concentration changes at different spatial locations; draw two-dimensional or three-dimensional diffusion coefficient / concentration distribution diagrams.

[0173] 4) In cell membrane research, analyze the lateral diffusion of proteins on the membrane; identify membrane microdomain structures (such as lipid rafts).

[0174] 5) Molecular interactions: Detect the formation of molecular complexes by analyzing changes in diffusion time; evaluate the strength and kinetics of the interaction.

[0175] 6) Anomalous diffusion behavior: Identify non-Brownian diffusion, such as confined diffusion or directed motion; analyze the spatial dependence of diffusion behavior.

[0176] 7) Time-resolved kinetics: study of rapid molecular processes such as conformational changes or chemical reactions; analysis of the temporal correlation of fluorescence intensity fluctuations.

[0177] 8) Flow velocity: In a system with flow, measure the local flow velocity; distinguish the contributions of flow and diffusion to molecular motion.

[0178] 9) Multicomponent analysis: Identify and quantify multiple components with different diffusion characteristics; analyze the proportions of each component in a complex mixture.

[0179] 10) Spatial correlation: Analyze the correlation between different spatial positions; study the spatial coupling of molecular motion.

[0180] 11) Membrane topology: In cell membrane research, reconstruct the three-dimensional structure of the membrane; analyze the effect of membrane curvature on molecular diffusion.

[0181] 12) Spectral information: If combined with spectral detection, the dynamics of different fluorescent species can be analyzed.

[0182] 13) Long-term dynamics: Study slow processes such as changes in cell morphology through long-term scanning.

[0183] 14) Statistical fluctuation analysis: Study the statistical characteristics of fluorescence intensity fluctuations; identify non-equilibrium processes or energy metabolism fluctuations.

[0184] The specific applications are as follows:

[0185] Abnormal cell membrane fluidity detection: Scanning FCS can reveal changes in membrane fluidity by analyzing the diffusion coefficients of molecules on the cell membrane. In certain disease states, such as cancer or neurodegenerative diseases, the fluidity of the cell membrane often changes significantly. This change may reflect abnormalities in the composition of membrane lipids or changes in the distribution of membrane proteins. By comparing the membrane fluidity of healthy cells and diseased cells, it can provide important clues for early disease diagnosis.

[0186] Detection and characterization of protein aggregates: In many neurodegenerative diseases, such as Alzheimer's or Parkinson's, abnormal protein aggregation is a key pathological feature. Scanning FCS is able to detect the formation process of these aggregates. By analyzing the changes in diffusion time and spatial distribution, the early stages of protein aggregation can be identified, even before they are detectable by traditional pathological methods. This provides a valuable time window for early diagnosis and therapeutic intervention.

[0187] Dynamic monitoring of abnormal cell signaling pathways: Abnormal activation of cell signaling pathways is a common phenomenon in cancer and other diseases. Scanning FCS can monitor the dynamic changes of signaling molecules in real time, including their concentration, spatial distribution, and interactions. By analyzing these parameters, abnormal activation or inhibition of signaling pathways can be identified, providing a basis for the formulation of personalized treatment strategies.

[0188] Real-time evaluation of drug response: In drug development and personalized treatment, it is crucial to understand the immediate effects of drugs on cells. Scanning FCS can monitor the interaction between drug molecules and targets in real time, as well as the resulting changes in intracellular molecular dynamics. This dynamic information can help evaluate drug effects, optimize dosing strategies, and predict possible side effects.

[0189] Assessment of cellular metabolic state: Certain fluorescent markers can reflect the metabolic state of cells. By analyzing the kinetic characteristics of these markers, Scanning FCS can provide information on cellular energy metabolism. In diseases such as cancer, cellular metabolism often changes significantly. This method can help identify metabolic abnormalities and provide new perspectives for cancer diagnosis and grading.

[0190] Characterization of microenvironment heterogeneity: The heterogeneity of the tumor microenvironment is an important factor affecting the therapeutic effect. Scanning FCS can depict the spatial heterogeneity of the microenvironment at the subcellular level, including the microscale distribution of parameters such as pH and oxygen concentration. This information is of great significance for understanding the growth mechanism of tumors and designing targeted treatment strategies.

[0191] Dynamic monitoring of apoptosis: apoptosis is a key event in many diseases and treatments. Scanning FCS can capture molecular events in the apoptosis process, such as membrane phospholipid turnover, protease activation, etc. By monitoring these changes in real time, the therapeutic effect can be evaluated and the treatment plan can be optimized.

[0192] In some optional embodiments, the multicolor pathology detection system is used to achieve at least one of the following quantitative imaging measurements: laser confocal fluorescence intensity imaging; fluorescence lifetime imaging; phosphorescence lifetime imaging; upconversion fluorescence imaging or upconversion fluorescence lifetime imaging; rare earth luminescence imaging or rare earth luminescence lifetime imaging; delayed fluorescence imaging or delayed fluorescence lifetime imaging; single quantum dot luminescence imaging or single quantum dot luminescence lifetime imaging; single molecule imaging; single molecule fluorescence resonance transfer imaging, including alternating excitation imaging.

[0193] The application scope of multicolor pathology detection system has been greatly expanded through upconversion fluorescence imaging and rare earth luminescence imaging. Upconversion fluorescence imaging and rare earth luminescence imaging use special optical materials to convert long-wavelength light into short-wavelength light, or use the unique optical properties of rare earth elements to achieve high-sensitivity, low-background noise imaging. It is suitable for deep tissue imaging and long-term in vivo observation, and also provides the possibility for the development of new biomarkers. The integration of delayed fluorescence imaging and single quantum dot luminescence imaging further improves the sensitivity and time resolution of multicolor pathology detection system. The addition of single molecule imaging and single molecule fluorescence resonance transfer imaging has pushed the analytical capability of multicolor pathology detection system to the molecular level.

[0194] In some optional embodiments, the Eu(europium) complex Eu(tta) 3 The absorption of phen is before 400nm, and adding a 375nm laser can meet the testing requirements of most Eu complexes. Figure 3 ,in Figure 3 (a) is the absorption spectrum, Figure 3 (b) is the life decay curve.

[0195] In some optional embodiments, see Figure 4 Organic small molecule dyes are common materials with luminescence lifetimes in the nanosecond range. However, the Stokes shift of general organic small molecule dyes is relatively small. Taking the common Nile Red fluorescent dye as an example, the maximum absorption peak is at 520nm. Therefore, a 485nm laser is added to meet the testing needs of such materials. In addition, for Nile Red and the AIE molecule BTPEBTTD, both can detect luminescence signals under 485nm excitation. Under 405nm excitation, the signal of the AIE molecule BTPEBTTD can be measured, while the signal of Nile Red is almost undetectable. The dimension of the excitation light can be used to expand the encoding and decoding methods.

[0196] See also, for example, Figure 5 Since the wavelength detection range of the equipped APD detector is 400-1050nm, it can be used to detect the signal of near-infrared materials. Taking the common near-infrared cyanine dye as an example, the absorption is usually after 600nm, so a 635nm laser is added to meet the testing needs of near-infrared luminescent materials.

[0197] In addition, Yb 3+ Rare earth upconversion nanoparticles used as sensitizers usually use an excitation wavelength of 980 nm, so a 980 nm upconversion laser 330 is added to meet the testing requirements of such materials.

[0198] The multicolor pathology detection system according to the embodiment of the present invention has at least the following beneficial effects: at least two different wavelengths of excitation light are emitted by the light source module 300, and the excitation light of the target wavelength is guided to the spectroscopic module 400 through the optical fiber 340, so that the spectroscopic module 400 guides the excitation light of the target wavelength to the microscopic module 200, thereby effectively exciting multiple fluorescent markers of the sample to be tested on the microscopic module 200, and the fluorescent signals of multiple fluorescent markers can be detected simultaneously, and the detection efficiency is high. The excitation light of the target wavelength can be guided to the microscopic module 200, and the feedback light signal of the sample to be tested can be guided to the detection module by the same spectroscopic module 400, and the detection efficiency is high, and the detection optical path is simple. After the feedback light signals of different wavelengths are detected by the detection module, the electrical signal to be processed is obtained, and imaging is performed by the imaging module, and the fluorescent signals of multiple different fluorescent markers and / or the corresponding bright field signals can be detected simultaneously, so as to perform multi-dimensional detection, with a wide range of applications, and the complex molecular characteristics of the sample to be tested can be captured.

[0199] Second, refer to Figure 6 The present invention provides a method for detecting and analyzing pathological data, which is applied to a multi-color pathological detection system. Figure 1-2 The multicolor pathology detection system includes a light source module 300, a spectroscopic module 400, a microscope module 200, a detection module, an imaging module and an analysis module; the detection and analysis method of pathology data includes:

[0200] S601, obtaining sample information of the sample to be tested;

[0201] S602, controlling the light source module 300 to emit the excitation light of the target wavelength to the spectroscopic module 400 according to the sample information, so that the spectroscopic module 400 guides the excitation light of the target wavelength to the microscope module 200;

[0202] S603, controlling the microscope module 200 to scan the sample to be tested with the excitation light of the target wavelength, so that the sample to be tested generates a feedback light signal;

[0203] S604, controlling the microscope module 200 to transmit the feedback light signal to the spectroscopic module 400, so as to guide the feedback light signal to the detection module through the spectroscopic module 400;

[0204] S605, controlling the detection module to detect feedback optical signals of different wavelengths, and converting the feedback optical signals into electrical signals to be processed;

[0205] S606, controlling the imaging module to process the electrical signal to be processed and generate image information;

[0206] S607, controlling the analysis module to analyze the image information to obtain pathological data.

[0207] In some optional embodiments, the light source module 300 includes a laser integrator 310 and a multi-wavelength laser source, the microscope module 200 includes a microscope unit and a laser confocal scanner, and the microscope unit is placed with a sample to be tested; the detection module includes a plurality of fluorescence detectors and at least one bright field detector; the detection module also includes a beam splitting unit, the beam splitting unit includes a sixth beam splitter 510, a seventh beam splitter 520 and an eighth beam splitter 530, the light splitting module 400 includes a first beam splitter 410, a second beam splitter 420, a third beam splitter 430, a fourth beam splitter 440 and a fifth beam splitter 450, the fluorescence detector includes a first detector 524 and a second detector 523, and the bright field detector includes a third detector 534 and a fourth detector 533. After obtaining the sample information of the sample to be tested, it also includes:

[0208] Controlling the multi-wavelength laser source to emit initial excitation light of at least one wavelength according to sample information;

[0209] Controlling the laser integrator 310 to integrate the initial excitation light to obtain excitation light of a target wavelength;

[0210] The laser integrator 310 emits the excitation light of the target wavelength to the first beam splitter 410, so as to guide the excitation light of the target wavelength to the laser confocal scanning head through the reflection of the first beam splitter 410, the second beam splitter 420, the third beam splitter 430, the fourth beam splitter 440 and the fifth beam splitter 450 in sequence;

[0211] Controlling the laser confocal scanning head to scan the sample to be tested, so that the sample to be tested generates a feedback light signal;

[0212] Control the laser confocal scanning head to transmit the feedback light signal to the fifth beam splitter 450, so as to transmit the feedback light signal to the sixth beam splitter 510 through the reflection effect of the fifth beam splitter 450 and the fourth beam splitter 440 and the transmission effect of the third beam splitter 430;

[0213] Feedback light signals of different wavelengths are directed to the first detector 524, and / or the second detector 523, and / or the third detector 534, and / or the fourth detector 533 through the reflection and / or transmission of the sixth beam splitter 510, the seventh beam splitter 520, and the eighth beam splitter 530, so as to generate fluorescence image information through the first detector 524, and / or the second detector 523, and generate bright field image information through the third detector 534, and / or the fourth detector 533.

[0214] In some optional embodiments, controlling the laser confocal scanning head to scan the sample to be tested so that the sample to be tested generates a feedback light signal includes:

[0215] Obtaining the posture information of the sample to be tested;

[0216] Control the scanning angle of the laser confocal scanning head according to the position information of the sample to be tested, and control the microscope unit to move the sample to be tested;

[0217] The laser confocal scanning head is controlled to scan the sample to be tested at a scanning angle so that the sample to be tested generates a feedback light signal.

[0218] In some optional embodiments, the imaging module includes a data acquisition unit and an image generation unit, and controlling the imaging module to process the electrical signal to be processed and generate image information includes:

[0219] Controlling the data acquisition unit to acquire fluorescence image information and / or bright field image information and generate digital information;

[0220] The digital information is converted into image information by the image generating unit.

[0221] In some optional embodiments, the method further comprises:

[0222] Obtain the time information of the excitation light of the target wavelength reaching the sample to be tested;

[0223] The wavelength and emission time of the next target wavelength of the excitation light emitted by the laser integrator 310 are controlled according to the time information, so that the laser integrator 310 emits the next target wavelength of the excitation light at the emission time.

[0224] It should be noted that the time information indicates the time point when the excitation light of the target wavelength reaches the sample to be tested. The wavelength of the excitation light of the next target wavelength is determined according to the sample to be tested or the test requirements, and is not specifically limited. By controlling the emission time of the excitation light of the next target wavelength, the excitation light of the next target wavelength is prevented from interfering with the generation and detection of the feedback light signal.

[0225] In some optional embodiments, the laser confocal scanning head includes a galvanometer unit, the galvanometer unit includes a first high-speed galvanometer and a second high-speed galvanometer, the first high-speed galvanometer and the second high-speed galvanometer are arranged orthogonally, the first high-speed galvanometer and the second high-speed galvanometer both include a reflective mirror, a voice coil motor and a position sensor, and the scanning angle of the laser confocal scanning head is controlled according to the posture information of the sample to be measured, including:

[0226] Determine the scanning angle of the laser confocal scanning head according to the posture information of the sample to be measured;

[0227] Acquire the position information of the reflective mirror surface according to the position sensor;

[0228] The voice coil motor is controlled to adjust the reflective mirror according to the scanning angle and posture information, so as to adjust the angle of the laser confocal scanning head to the scanning angle.

[0229] In some optional embodiments, the microscope unit further includes an inverted microscope 220, an objective lens turret and a sample stage, and the objective lens turret is equipped with objective lenses 230 of different magnifications. Before obtaining the sample information of the sample to be tested, the method further includes:

[0230] Select a target objective lens 230 on the objective lens turret according to the sample to be tested on the sample stage;

[0231] When the focus adjustment process of the laser confocal scanning head is completed based on the target objective lens 230 and the sample to be measured, the laser confocal scanning head is switched to the scanning mode.

[0232] It can be seen that the contents of the above-mentioned multi-color pathology detection system embodiment are all applicable to the present method embodiment, the functions specifically implemented by the present method embodiment are the same as those in the above-mentioned multi-color pathology detection system embodiment, and the beneficial effects achieved are also the same as those achieved by the above-mentioned multi-color pathology detection system embodiment.

[0233] In a third aspect, an embodiment of the present invention provides a controller, comprising: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the above method is implemented when the processor executes the computer program.

[0234] It can be seen that the contents of the above-mentioned pathological data detection and analysis method embodiments are all applicable to the present controller embodiment, the functions specifically implemented by the present controller embodiment are the same as those in the above-mentioned pathological data detection and analysis method embodiments, and the beneficial effects achieved are also the same as those achieved by the above-mentioned pathological data detection and analysis method embodiments.

[0235] In a fourth aspect, an embodiment of the present invention provides a computer storage medium, wherein the computer storage medium stores computer executable instructions, and the computer executable instructions are used to execute the above-mentioned method for detecting and analyzing pathological data.

[0236] It can be seen that the contents of the above-mentioned pathological data detection and analysis method embodiments are all applicable to the present storage medium embodiments, the functions specifically implemented by the present storage medium embodiments are the same as those of the above-mentioned pathological data detection and analysis method embodiments, and the beneficial effects achieved are also the same as those achieved by the above-mentioned pathological data detection and analysis method embodiments.

[0237] It is understood that all or some steps and systems in the disclosed method above can be implemented as software, firmware, hardware and appropriate combinations thereof. Some physical components or all physical components can be implemented as processors, such as software executed by a central processing unit, a digital information processor or a microprocessor, or implemented as hardware, or implemented as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, and the computer-readable medium can include a computer storage medium (or a non-transitory medium) and a communication medium (or a temporary medium). As known to those of ordinary skill in the art, the term computer storage medium includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information (such as computer-readable instructions, data structures, program modules or other data). Computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tapes, disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and can be accessed by a computer. Furthermore, it is well known to those skilled in the art that communication media generally contain computer readable instructions, data structures, program modules, or other data in modulated data information such as carrier waves or other transport mechanisms, and may include any information delivery media.

[0238] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the technical field without departing from the purpose of the present invention.

Claims

1. A multicolor pathology detection system, characterized in that: It includes a control module and a light source module, a spectroscopic module, a microscopic module, a detection module and an imaging module which are electrically connected to the control module respectively; The light source module is used to emit at least two excitation lights of different wavelengths, and guide the excitation light of the target wavelength to the light splitting module through the optical fiber, so that the light splitting module guides the excitation light of the target wavelength to the microscopy module; The microscopy module is used to scan the sample to be tested using the excitation light of the target wavelength, so that the sample to be tested generates a feedback light signal under the action of the excitation light of the target wavelength, and transmits the feedback light signal to the spectroscopic module, and guides the feedback light signal to the detection module through the spectroscopic module, and the feedback light signal represents the light signal generated by the sample to be tested under the excitation of the excitation light of the target wavelength; The detection module is used to detect the feedback optical signals of different wavelengths and convert the feedback optical signals into electrical signals to be processed; The imaging module is used to process the electrical signal to be processed and generate image information.

2. The multicolor pathology detection system according to claim 1, characterized in that: The light source module includes a laser integrator and a multi-wavelength laser source. The multi-wavelength laser source is used to emit at least two excitation lights of different wavelengths to the laser integrator. The laser integrator is used to simultaneously or sequentially select excitation lights of different wavelengths and integrate them to obtain the excitation light of the target wavelength, and then transmit the excitation light of the target wavelength to the spectroscopic module.

3. The multicolor pathology detection system according to claim 2, characterized in that: The multi-wavelength laser source includes a modulated nanosecond laser and an up-conversion laser. The modulated nanosecond laser is used to emit short-wave excitation light, and the up-conversion laser is used to emit long-wave excitation light. The wavelength of the short-wave excitation light is between 300nm and 700nm, and the wavelength of the long-wave excitation light is between 700nm and 1000nm.

4. The multicolor pathology detection system according to claim 3, characterized in that: The wavelength of the short-wave excitation light includes at least one of the following: 375nm; 405nm; 485nm; 635nm; 680nm.

5. The multicolor pathology detection system according to claim 3, characterized in that: The wavelength of the long-wave excitation light includes at least one of the following: 730nm; 808nm; 980nm.

6. The multicolor pathology detection system according to claim 1, characterized in that: The light splitting module includes a first beam splitter, a second beam splitter, a third beam splitter, a fourth beam splitter and a fifth beam splitter; The first beam splitter is used to reflect the excitation light of the target wavelength to the second beam splitter, the second beam splitter is used to reflect the excitation light of the target wavelength to the third beam splitter, the third beam splitter is used to reflect the excitation light of the target wavelength to the fourth beam splitter, the fourth beam splitter is used to reflect the excitation light of the target wavelength to the fifth beam splitter, and the fifth beam splitter is used to reflect the excitation light of the target wavelength to the microscopy module; The fifth beam splitter is used to reflect the feedback light signal to the fourth beam splitter, the fourth beam splitter is used to reflect the feedback light signal to the third beam splitter, and the third beam splitter is used to transmit the feedback light signal to the detection module.

7. The multicolor pathology detection system according to claim 1, characterized in that: The detection module includes a fluorescence detector and a bright field detector, the fluorescence detector includes a first detector and a second detector, the first detector is used to detect a fluorescence signal of a first wavelength, the second detector is used to detect a fluorescence signal of a second wavelength, the bright field detector includes a third detector and a fourth detector, the third detector is used to detect a bright light signal of a third wavelength, the fourth detector is used to detect a bright light signal of a fourth wavelength, and the feedback light signal includes a fluorescence signal and a bright field signal.

8. A method for detecting and analyzing pathological data, characterized in that: Applicable to a multicolor pathology detection system, the multicolor pathology detection system comprising a light source module, a spectroscopic module, a microscopic module, a detection module, an imaging module and an analysis module; The detection and analysis method of the pathological data comprises: Obtaining sample information of the sample to be tested; Control the light source module to emit the excitation light of the target wavelength to the spectroscopic module according to the sample information, so that the spectroscopic module guides the excitation light of the target wavelength to the microscopic module; Controlling the microscopy module to scan the sample to be tested by the excitation light of the target wavelength, so that the sample to be tested generates a feedback light signal; Controlling the microscopy module to transmit the feedback light signal to the light splitting module, so as to guide the feedback light signal to the detection module through the light splitting module; Controlling the detection module to detect the feedback optical signals of different wavelengths and converting the feedback optical signals into electrical signals to be processed; Controlling the imaging module to process the electrical signal to be processed and generate image information; The analysis module is controlled to analyze the image information to obtain pathological data.

9. The method for detecting and analyzing pathological data according to claim 8, characterized in that: The light source module includes a laser integrator and a multi-wavelength laser source, the microscopy module includes a microscope unit and a laser confocal scanning head, the microscope unit is provided with the sample to be tested; the detection module includes a plurality of fluorescence detectors and at least one bright field detector; the detection module also includes a beam splitting unit, the beam splitting unit includes a sixth beam splitter, a seventh beam splitter and an eighth beam splitter, the light splitting module includes a first beam splitter, a second beam splitter, a third beam splitter, a fourth beam splitter and a fifth beam splitter, the fluorescence detector includes a first detector and a second detector, the bright field detector includes a third detector and a fourth detector, and after obtaining the sample information of the sample to be tested, the method further includes: Controlling the multi-wavelength laser source to emit initial excitation light of at least one wavelength according to the sample information; Controlling the laser integrator to integrate the initial excitation light to obtain the excitation light of the target wavelength; The excitation light of the target wavelength is emitted to the first beam splitter through the laser integrator, so as to guide the excitation light of the target wavelength to the laser confocal scanning head through reflection actions of the first beam splitter, the second beam splitter, the third beam splitter, the fourth beam splitter and the fifth beam splitter in sequence; Controlling the laser confocal scanning head to scan the sample to be tested, so that the sample to be tested generates a feedback light signal; Controlling the laser confocal scanning head to transmit the feedback light signal to the fifth beam splitter, so as to transmit the feedback light signal to the sixth beam splitter through the reflection effect of the fifth beam splitter and the fourth beam splitter and the transmission effect of the third beam splitter; The feedback light signals of different wavelengths are directed to the first detector, and / or the second detector, and / or the third detector, and / or the fourth detector through reflection and / or transmission of the sixth beam splitter, the seventh beam splitter, and the eighth beam splitter, so as to generate fluorescence image information through the first detector, and / or the second detector, and generate bright field image information through the third detector, and / or the fourth detector.

10. A controller, characterized in that: include: A memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the method according to any one of claims 8 to 9 when executing the computer program.

11. A computer storage medium, characterized in that: The computer storage medium stores computer executable instructions, and the computer executable instructions are used to execute the pathology data detection and analysis method described in any one of claims 8-9.

Images