Fluorescent pathological section scanning system, method and device

Abstract

The invention discloses a fluorescent pathological section scanning system, method and device, and the system comprises a scanning control module which is used for enabling an imaging view field to scan a whole pathological section, synchronously triggering the fluorescence excitation light source to flash and the imaging module to photograph, and setting the flash duration of the excitation light source and the exposure duration of the imaging module based on the fluorescence lifetime of the used fluorescent dye; the flash duration, the exposure duration and the fluorescence lifetime of the fluorescence sample to be detected meet the following formula. According to the application, short-time pulse illumination light is used for exciting fluorescence, meanwhile, long-time exposure is used for collecting the fluorescence, fluorescence afterglow radiation energy is fully collected, and a high-brightness fluorescence microscopic image is obtained under low-energy excitation. According to the application, the to-be-detected fluorescent sample can be prevented from being damaged by light, and the fluorescence quenching or bleaching of the pathological section can be inhibited.

Description

Technical Field

[0001] This application relates to the field of fluorescence lifetime imaging technology, and more specifically, to a fluorescence pathological slide scanning system, method, and apparatus. Background Technology

[0002] Fluorescence pathology microscopy is a high-resolution imaging technique based on the fluorescence excitation-emission principle. It achieves visualization and analysis of the microscopic structure and molecular expression of pathological tissues by specifically labeling biomolecules. Its core technology relies on the specific binding or reaction of fluorescent dyes or proteins with target molecules, such as bacteria, tumor markers, and pathogen antigens. Under specific wavelength excitation light, these molecules emit longer-wavelength fluorescence signals, which are then separated by a filtering system to form a high-contrast image.

[0003] Fluorescence imaging technology, due to its high sensitivity, specificity, and real-time visualization capabilities, has become a core tool in life sciences, materials science, and medical diagnostics. However, fluorescence quenching, the irreversible or reversible decay of fluorescence signals under excitation light, severely limits the stability and quantitative reliability of imaging. Summary of the Invention

[0004] To address the aforementioned technical problem of poor imaging quality caused by fluorescence quenching, this application discloses a fluorescence pathological slide scanning system, method, and apparatus. By using short-time pulsed illumination to excite fluorescence and simultaneously using longer exposure time to collect fluorescence, the residual fluorescence radiation energy is fully collected, thereby achieving high-brightness fluorescence microscopic images under low-energy excitation.

[0005] Specifically, the technical solution of this application is as follows: In a first aspect, this application discloses a fluorescent pathological slide scanning system, comprising: a fluorescent excitation light source, an imaging module, and a scanning control module; The scanning control module is used to scan the entire pathological slide through the imaging field of view, synchronously trigger the fluorescence excitation light source flash and the imaging module to take pictures, and base the images on the fluorescence lifetime of the fluorescent dye used. Set the flash duration of the excitation light source. and the exposure time of the imaging module ; The flash duration, the exposure duration, and the fluorescence lifetime of the fluorescent sample to be tested satisfy the following formula: ; The scanning control module is also used to control the fluorescence excitation light source to continuously generate excitation light during the flash duration, irradiating the fluorescent sample to be tested; so that the fluorescent sample to be tested is excited and emits a fluorescence signal; The scanning control module is also used to simultaneously control the imaging module to continuously acquire the fluorescence signal within the exposure time, and to convert the fluorescence signal into a fluorescence image through exposure.

[0006] In some embodiments, the fluorescent pathological slide scanning system further includes: a scanning displacement stage; used to place the fluorescent sample to be tested and to move the fluorescent sample to be tested on the plane of the scanning displacement stage; The scanning control module is also used to acquire the position information of the fluorescent sample to be tested on the scanning displacement stage in real time, and to send trigger signals to the imaging module and the fluorescent excitation source synchronously according to the position information; The trigger signal is used to trigger the fluorescence excitation source and the imaging module to start performing corresponding operations.

[0007] In some embodiments, the scanning control module is further configured to determine the signal trigger duration of the trigger signal; and based on the signal trigger duration, determine the first moment when the fluorescence excitation source begins to generate the excitation light, and the second moment when the imaging module begins to expose.

[0008] In some implementations, the first time point is equal to the second time point.

[0009] In some embodiments, the fluorescent pathological slide scanning system further includes: a sample transport device for setting the sample transport speed so that the excitation light scans the fluorescent sample to be tested line by line.

[0010] In some implementations, the scanning control module is further configured to set the interval between two adjacent trigger signals based on the sample transport speed.

[0011] In some embodiments, the fluorescent pathological slide scanning system further includes: a fluorescent illumination lens group, a fluorescent spectral dispersive module, a focusing module, a microscope objective, and a sleeve lens; The fluorescent illumination lens group is used to shape and focus the excitation light to match the subsequent optical path; The fluorescence spectrometer module includes a dichroic mirror and a filter for reflecting the excitation light to separate the excitation light from the fluorescence signal, allowing only the fluorescence signal to pass through; The excitation light is shaped by the fluorescent illumination lens group and enters the fluorescent spectral module. It is reflected in the first direction and enters the microscope objective, and is finally focused on the surface of the fluorescent sample to be tested. The focusing module is used to assist focusing in order to maintain the stability of the imaging focal plane; The microscope objective is used to collect the fluorescence signal emitted by the fluorescent sample to be tested and form an intermediate image; The telescopic lens is used to further magnify the intermediate image, and the intermediate image is transmitted to the imaging module.

[0012] In some embodiments, the fluorescence excitation light source, the fluorescence illumination lens group, the fluorescence beam splitter module, the focusing module, the microscope objective, the scanning stage, the telescopic lens, and the imaging module are arranged sequentially along the optical path.

[0013] Secondly, this application also discloses a method for scanning fluorescent pathological sections, wherein the method is performed based on a fluorescent pathological section scanning system described in any of the above embodiments; the method includes the following steps: The imaging field of view is scanned across the entire pathological section, and the fluorescence excitation light source flashes synchronously with the imaging module to take a picture, based on the fluorescence lifetime of the fluorescent dye used. Set the flash duration of the excitation light source. and the exposure time of the imaging module ; The flash duration, the exposure duration, and the fluorescence lifetime of the fluorescent sample to be tested satisfy the following formula: ; The fluorescence excitation source is controlled to continuously generate excitation light during the flash duration, illuminating the fluorescent sample to be tested; thus exciting the fluorescent sample to be tested and causing it to emit a fluorescence signal. Simultaneously, the imaging module is controlled to continuously acquire the fluorescence signal within the exposure time, and the fluorescence signal is converted into a fluorescence image through exposure.

[0014] Thirdly, this application also discloses a fluorescent pathological slide scanning device, which includes at least one of the fluorescent pathological slide scanning systems described in any of the above embodiments.

[0015] Compared with the prior art, this application has at least one of the following beneficial effects: This application decouples the "excitation" and "camera exposure" processes in time by rationally setting the flash duration of the excitation light source and the exposure duration of the imaging module through a scanning control module. It uses short-pulse illumination to excite fluorescence while simultaneously using a longer exposure time to collect the fluorescence, fully capturing the afterglow radiation energy and achieving high-brightness fluorescence microscopic images under low-energy excitation. This application avoids photodamage to the fluorescent sample and inhibits quenching or bleaching of fluorescence in pathological sections. Attached Figure Description

[0016] The preferred embodiments will now be described in a clear and easy-to-understand manner, in conjunction with the accompanying drawings, to further explain the above-mentioned characteristics, technical features, advantages, and implementation methods of this application.

[0017] Figure 1 This is a schematic diagram of the structure of an embodiment of a fluorescent pathological slide scanning system according to this application; Figure 2 This is a schematic diagram of another embodiment of a fluorescent pathological slide scanning system according to this application; Figure 3 This is a schematic diagram illustrating the timing relationship between signal trigger duration, flash duration, and exposure duration in this embodiment of the present application; Figure 4 This is a schematic diagram of the sample transfer device in the embodiments of this application; Figure 5 This is a schematic diagram of another embodiment of a fluorescent pathological slide scanning system according to this application; Figure 6 This is a schematic diagram of another embodiment of a fluorescent pathological slide scanning system according to this application; Figure 7 This is a flowchart illustrating the steps of an embodiment of a fluorescent pathological section scanning method according to this application; Figure 8 This is a schematic diagram of the signal timing of another embodiment of a fluorescent pathological section scanning method according to this application; Figure 9 This is a signal timing diagram of another embodiment of a fluorescence pathological section scanning method according to this application. Detailed Implementation

[0018] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that this application can also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods have been omitted so as not to obscure the description of this application with unnecessary detail.

[0019] It should be understood that, when used in this specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or sets.

[0020] To keep the drawings concise, each figure only schematically shows the parts relevant to the invention, and these do not represent the actual structure of the product. Furthermore, to facilitate understanding, in some figures, only one of components with the same structure or function is schematically depicted, or only one is labeled. In this document, "one" not only means "only one," but can also mean "more than one."

[0021] It should also be further understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0022] Furthermore, in the description of this application, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the specific implementation methods of this application will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings and other implementation methods can be obtained based on these drawings without creative effort.

[0024] Fluorescence imaging technology, due to its high sensitivity, specificity, and real-time visualization capabilities, has become a core tool in life sciences, materials science, and medical diagnostics. However, fluorescence quenching, the irreversible or reversible decay of fluorescence signals under excitation light, severely limits the stability and quantitative reliability of imaging.

[0025] When using a fluorescence pathology microscope, the quenching of fluorescent groups in pathological sections mainly occurs due to the compound formation quenching process. Specifically, when fluorescent molecules are under high-power-density excitation light, they absorb photons and transition to an excited state. Simultaneously, their chemical stability decreases, making them more susceptible to chemical reactions with oxygen or halogen molecules, generating non-fluorescent products and severely affecting fluorescence image acquisition. Furthermore, the thermal effect caused by high-power excitation light leads to an increase in local temperature in the pathological tissue, further intensifying molecular collisions and oxidation rates, accelerating the fluorescence quenching process. For example, fluorescein isothiocyanate (FIFC) and AlexaFluor 488, 4',6-diamidinyl-2-phenylindole (DAPI) are rapidly oxidized and bleached under strong blue-violet light. For heterochromatic dyes like acridine orange (which emits green light when bound to double-stranded DNA and orange-red light when bound to single-stranded DNA), high-intensity excitation light can also disrupt the generation of characteristic fluorescent signals by breaking the hydrogen bonds between the dye and DNA.

[0026] Fluorescent materials do not immediately stop emitting fluorescence after being de-excited; their fluorescence emission gradually decays over a period of time. In most cases, the intensity of fluorescence emission after de-excitation can be described by the following formula: ; Including fluorescence radiation intensity It decays exponentially over time. This is known as fluorescence lifetime.

[0027] In engineering, the time it takes for the fluorescence radiation intensity to decay to 10% after excitation is stopped is usually referred to as fluorescence afterglow. .

[0028] The range of fluorescence afterglow is extremely wide, from nanoseconds to tens of hours, and its length mainly depends on the material type, band structure, and doping control. Fluorescent dyes used for pathological sections typically use short-afterglow and medium-short-afterglow materials, i.e., dyes with an afterglow time of <1 ms.

[0029] For example, when acridine yellow dye is dispersed in polyvinyl alcohol (PVA) or epoxy resin, the fluorescence afterglow time reaches the second level. Platinum (Pt) complexes used as mitochondrial-targeting complexes have a fluorescence afterglow time of approximately 20 µs. Alexa Fluor 647, commonly used for protein staining, has an acid fluorescence afterglow of approximately 5 µs.

[0030] Existing techniques for fluorescence pathology microscopy systems typically employ a scintillation light source synchronized with the camera's exposure time to excite fluorescent pathology samples. The aim is to provide continuous excitation during exposure, but this also means the sample is exposed to light throughout the entire exposure period, and the total amount of photodamage and quenching remains proportional to the exposure time. Therefore, it is necessary to compensate for the quenching and attenuation of the fluorescence signal through camera control and image processing to obtain uniformly bright and sufficiently luminous fluorescent pathology slide micrographs.

[0031] This application differs from traditional synchronous pulse excitation schemes by decoupling the "excitation" and "camera exposure" processes in time. Excitation is completed in a very short time, while collection lasts for a longer period. Simultaneously, the flash duration of the excitation light source and the exposure duration of the imaging module are precisely controlled to ensure that the implementation of the scheme is not affected by the fluorescence lifetime of the sample being tested. This application avoids photodamage to the sample being tested and inhibits quenching or bleaching of fluorescence in pathological sections.

[0032] Reference manual attached Figure 1 As shown, one embodiment of a fluorescence pathological slide scanning system of this application specifically includes: a fluorescence excitation light source, an imaging module, and a scanning control module.

[0033] The scanning control module is used to scan the entire pathological slide through the imaging field of view, synchronously trigger the fluorescence excitation light source flash and the imaging module to take pictures, and base the images on the fluorescence lifetime of the fluorescent dye used. Set the flash duration of the excitation light source. and the exposure time of the imaging module .

[0034] The flash duration, the exposure duration, and the fluorescence lifetime of the fluorescent sample to be tested satisfy the following formula: .

[0035] The scanning control module is also used to control the fluorescence excitation source to continuously generate excitation light during the flash duration, illuminating the fluorescent sample to be tested. This excites the fluorescent sample to emit a fluorescence signal.

[0036] The scanning control module is also used to simultaneously control the imaging module to continuously acquire the fluorescence signal within the exposure time, and to convert the fluorescence signal into a fluorescence image through exposure.

[0037] Specifically, in the scanning process of fluorescent pathological sections, excitation and exposure are two key steps that work together to capture high-quality fluorescence images. Excitation refers to illuminating the sample with a fluorescent light source of a specific wavelength, causing the fluorescent dye labeled in the sample to absorb light energy and transition from the ground state to the excited state, thereby emitting fluorescence with a longer wavelength. This process is the basis for generating fluorescence signals and determines which specific target sites will be visualized. Exposure refers to the length of time the camera sensor receives the fluorescence signal. It controls the amount of light entering the camera and directly affects the brightness and signal-to-noise ratio of the image. Appropriate exposure ensures that weak fluorescence signals are clearly captured while avoiding overexposure and loss of detail in areas with strong signals. Excitation and exposure need to be precisely coordinated. This application optimizes the flash time of the excitation light and the exposure time of the camera to avoid light damage to the fluorescent sample being tested and to suppress quenching or bleaching of fluorescence in the pathological sections.

[0038] The fluorescent sample to be tested is a fluorescent pathological slide, which is a pathological slide labeled with a fluorophore. The quenching rate of the fluorophore is directly related to the excitation light intensity and time integral. After pulse excitation, the fluorescent molecules continue to emit fluorescence; this process is called fluorescence afterglow, which is the decay of fluorescence lifetime, and its intensity decays exponentially with time. Traditional synchronous pulse methods waste the fluorescence signal that has become very weak in the later stages of exposure. However, this application improves photon utilization efficiency by using a longer exposure time, collecting all of this afterglow energy, thereby achieving a higher signal-to-noise ratio at the same peak excitation light power. In other words, it achieves the lowest total excitation light energy while maintaining the same image signal-to-noise ratio. The fluorescence lifetime of the sample to be tested is usually determined experimentally, and the fluorescence lifetime varies for each material and even for different molecular configurations of the same material. For example, the lifetime of a common fluorophore is about 4 nanoseconds, while the lifetimes of some rare earth complexes or quantum dots can reach the microsecond or even millisecond range.

[0039] In this application, the scanning control module is connected to both the fluorescence excitation source and the imaging module. The flash duration of the excitation source is set accordingly. and the exposure time of the imaging module The following conditions must be met: First, the flash duration. Less than exposure time Secondly, exposure duration With flash duration The difference is greater than the fluorescence lifetime of the sample to be tested. .

[0040] The technical solution of this application sets the flash duration of the excitation light source and the exposure duration of the imaging module based on the fluorescence lifetime. This solution is applicable to both fluorescence samples with long inherent lifetimes and those with short fluorescence lifetimes.

[0041] Optionally, in another embodiment of this invention, those skilled in the art can further adjust the intensity of the excitation light to ensure the scanning system operates in a stable environment, and then base the exposure time on the aforementioned parameters. With flash duration By meeting certain conditions and selecting the most suitable target value within a specific time range, the quality of the acquired fluorescence images can be optimized. The acquired fluorescence images can be used to obtain the spatial distribution, morphological characteristics, and expression levels of specific target molecules, thereby revealing their functional information under physiological or pathological conditions and providing crucial evidence for pathological diagnosis.

[0042] Based on the above embodiments, this application discloses another embodiment of a fluorescence pathology slide scanning system. The fluorescence pathology slide scanning system is described in the appendix to the specification. Figure 2As shown, it also includes a scanning displacement stage. This stage is used to place the fluorescence sample to be tested and to move the sample on the plane of the scanning displacement stage.

[0043] The scanning control module is also used to acquire the position information of the fluorescent sample to be tested on the scanning displacement stage in real time, and synchronously send trigger signals to the imaging module and the fluorescence excitation source according to the position information. The trigger signals are used to trigger the fluorescence excitation source and the imaging module to start performing corresponding operations.

[0044] In some implementations, the scanning control module generates a trigger signal and synchronously sends the trigger signal to the fluorescence excitation source and the imaging module. Upon receiving the trigger signal, the fluorescence excitation source and the imaging module start simultaneously and perform flash / exposure operations, respectively.

[0045] In other implementations, during operation, the scanning control module generates a first trigger signal and sends it to the light source driver. Upon receiving the first trigger signal, the light source outputs a flash. The duration of the flash is determined by the width of the trigger signal or the internal settings of the light source. The scanning control module generates a second trigger signal and sends it to the imaging module. Upon receiving the second trigger signal, the imaging module begins acquiring signals.

[0046] This application provides another embodiment of a fluorescence pathological slide scanning system. Based on any of the embodiments described above, the scanning control module is further configured to determine the signal trigger duration of the trigger signal. Based on the signal trigger duration, a first moment when the fluorescence excitation source begins to generate the excitation light, and a second moment when the imaging module begins exposure, are determined.

[0047] Specifically, exposure time is the total time window during which the imaging module (camera) acquires signals. Flash duration is the actual time the fluorescent sample to be tested is irradiated by excitation light. Trigger signal is the electrical signal command that controls when the flash and exposure begin and continue.

[0048] The core function of the signal trigger duration is to provide a unified time reference and synchronization starting point for the entire flash exposure process, which is used to define the theoretical delay between the scanning control module issuing the trigger command and the relevant hardware actually starting to operate.

[0049] In some implementations, the first time point is equal to the second time point.

[0050] In its implementation, the scanning control module first sets a signal trigger duration, which represents the expected time required for the trigger signal to be emitted and responded to. Based on this common duration benchmark, the module can calculate when to emit the first trigger signal to the light source driver and the second trigger signal to the imaging module, respectively, to ensure that although the two trigger signals are sent separately, the generation of excitation light (first moment) and the start of exposure (second moment) occur precisely at the same time.

[0051] Reference manual attached Figure 3 As shown, Figure 3 This diagram illustrates the timing relationship between the signal trigger duration, flash duration, and exposure duration in this embodiment. The excitation light illumination time is synchronized with the camera exposure time.

[0052] This design achieves software-level synchronization compensation for hardware response delays by sending first and second trigger signals separately based on the signal trigger duration. This cancels out the possible response time differences between different hardware components, namely the lighting of the light source and the shutter of the camera. Ultimately, it aligns the two physically independent actions in time, ensuring that the strongest effective fluorescence signal can be captured within the exposure window.

[0053] Based on the above embodiments, this application discloses another embodiment of a fluorescence pathology slide scanning system. The fluorescence pathology slide scanning system is described in the appendix to the specification. Figure 4 As shown, it also includes: a sample transport device, used to set the sample transport speed so that the excitation light scans the fluorescent sample to be tested line by line.

[0054] This application provides another embodiment of a fluorescence pathological slide scanning system. Based on any of the above-described embodiments, the scanning control module is further configured to set the interval between two adjacent trigger signals based on the sample transport speed.

[0055] Specifically, the fluorescent sample to be tested, i.e., the fluorescent pathological section, is a pathological section labeled with a fluorophore. Because the preparation of a typical pathological section involves making consecutive ultrathin sections of the same tissue block and applying fluorescent labels, the resulting set of two-dimensional sections that are spatially adjacent is obtained.

[0056] The sample transport device controls the stage carrying the sample to move at a constant speed in one direction, allowing the excitation light to sequentially scan each row of the sample. The scanning control module dynamically sets the image acquisition frequency based on the uniform speed of the sample stage, ensuring that the final stitched image is free from distortion or overlap. The interval between two adjacent trigger signals is directly determined by the sample transport speed: the faster the speed, the faster the sample moves relative to the objective lens's field of view, i.e., the shorter the trigger interval; conversely, if the transport speed is slow, the trigger interval is extended, ensuring that each row of images is captured when the sample reaches the correct position.

[0057] Based on the above embodiments, this application discloses another embodiment of a fluorescence pathology slide scanning system. The fluorescence pathology slide scanning system is described in the appendix to the specification. Figure 5 As shown, it also includes: a fluorescent illumination lens group, a fluorescent beam splitter module, a focusing module, a microscope objective, and a telescopic lens.

[0058] The fluorescent illumination lens group is used to shape and focus the excitation light to match the subsequent optical path.

[0059] The fluorescence spectrometer module includes a dichroic mirror and a filter for reflecting the excitation light to separate the excitation light from the fluorescence signal, allowing only the fluorescence signal to pass through.

[0060] The excitation light is shaped by the fluorescence illumination lens group and then enters the fluorescence spectrometer module. It is reflected in the first direction and enters the microscope objective, and is finally focused on the surface of the fluorescent sample to be tested.

[0061] The focusing module is used to assist focusing in order to maintain the stability of the imaging focal plane.

[0062] The microscope objective is used to collect the fluorescence signal emitted by the fluorescent sample to be tested and form an intermediate image.

[0063] The telescopic lens is used to further magnify the intermediate image, and the intermediate image is transmitted to the imaging module.

[0064] In some embodiments, the fluorescence excitation source, fluorescence illumination lens group, fluorescence spectrometer module, focusing module, microscope objective, scanning stage, telescopic lens, and imaging module are arranged sequentially along the optical path, as shown in the reference. Figure 5 As shown.

[0065] Figure 5The fluorescence pathology slide scanning system includes a fluorescence excitation source, a fluorescence illumination lens group, a fluorescence spectrometer module, a focusing module, a microscope objective, a pathology slide sample, a telescope lens, and a camera arranged sequentially along the optical path. The pathology slide sample is located on the scanning displacement stage and can be scanned in two dimensions on the pathology slide plane.

[0066] The fluorescence excitation source can be a laser source, LED source, gas discharge source, or similar type of light source, and a scanning control module controls the flash. The fluorescence spectrometer is typically a dichroic filter placed at an angle, which reflects the excitation light and is transparent to the fluorescence.

[0067] The excitation light source is shaped by the illumination lens group and enters the fluorescence spectrometer. It is then reflected downwards into the microscope objective lens and finally focused on the surface of the fluorescent pathological section sample, causing the fluorescent groups in the sample to be excited and emit fluorescence.

[0068] The focusing module can be a displacement stage that moves the microscope objective along the optical axis, or it can be a lens group with adjustable optical power to assist focusing.

[0069] Preferably, the microscope objective is aligned with the optical axis of the telescopic lens.

[0070] When the pathology slide scanning system is in fluorescence scanning mode, the scanning control module acquires the position information of the scanning stage in real time and synchronously sends trigger signals to the camera and fluorescence excitation source based on the position information of the scanning stage. The duration of the trigger signal is [duration missing]. Upon receiving a trigger signal, the excitation light flash control system immediately illuminates the excitation source, and the flash duration is [duration missing]. Simultaneously, the camera begins exposure immediately upon receiving the trigger signal, with an exposure time of [time missing]. ,in , The fluorescence lifetime of the fluorophore to be tested is given.

[0071] In other embodiments, the fluorescent pathological slide scanning system described herein is described in the appendix to the specification. Figure 6 As shown, it also includes: a second fluorescence excitation source, a second fluorescence illumination lens group, a second fluorescence spectral dispersive module, and a second microscope objective.

[0072] The fluorescence excitation light source, fluorescence illumination lens group, fluorescence beam splitter module, focusing module, microscope objective, scanning displacement stage, sleeve lens, and imaging module form the first optical path.

[0073] The system includes a second fluorescence excitation source, a second fluorescence illumination lens group, a second fluorescence beam splitter module, and a second microscope objective. The scanning stage, the telescopic lens, and the imaging module form the second optical path.

[0074] Figure 6 In this system, the fluorescence illumination system comprises two parts: top illumination and bottom illumination. The main advantage of the dual-light source design is that it can significantly improve the uniformity and intensity of the excitation light, thereby obtaining higher quality and more reliable fluorescence images. This design, by illuminating the sample from both the top and bottom, can effectively reduce shadow effects and blind spots caused by uneven sample thickness, internal structural obstruction, or differences in the distribution of fluorescent markers, ensuring that fluorophores throughout the entire field of view are fully and uniformly excited.

[0075] Especially for samples with a certain thickness or three-dimensional structure, a single-direction light source may not be able to effectively excite the bottom fluorophores due to light attenuation in the deep region. The supplementary illumination from the bottom light source makes up for this deficiency and enhances the excitation efficiency of deep signals.

[0076] Based on the same concept, this application also discloses a method for scanning fluorescent pathological sections. The implementation of this method is based on an embodiment of any of the aforementioned fluorescent pathological section scanning systems. Specifically, an embodiment of a fluorescent pathological section scanning method of this application is provided in the appendix to the specification. Figure 7 Specifically, it includes: S100, the imaging field of view is scanned across the entire pathological section, simultaneously triggering the fluorescence excitation light source flash and the imaging module to take an image, and based on the fluorescence lifetime of the fluorescent dye used. Set the flash duration of the excitation light source. and the exposure time of the imaging module .

[0077] S200, the flash duration, the exposure duration, and the fluorescence lifetime of the fluorescent sample to be tested satisfy the following formula: .

[0078] S300: The fluorescence excitation source is controlled to continuously generate excitation light during the flash duration, illuminating the fluorescent sample to be tested. This excites the fluorescent sample to emit a fluorescence signal.

[0079] S400, simultaneously controls the imaging module to continuously acquire the fluorescence signal within the exposure time, and converts the fluorescence signal into a fluorescence image through exposure.

[0080] Based on the above embodiments, this application discloses an embodiment of a fluorescent pathological section scanning method, specifically including: A fluorescence pathology scanning system was used to observe glycosylated protein pathological sections stained with pyrene-polyethylene glycol-hydroxylamine dye, which has a fluorescence lifetime of approximately 0.1 μs. A 338 nm ultraviolet LED was used as the excitation source. The light source passed through a collimating lens, a 350 nm cutoff short-pass filter, a homogenizing compound eye lens, and an integrating lens to produce a uniformly collimated beam. This beam was then reflected by a 370 nm long-wavelength dichroic filter and projected onto a 40x microscope objective, where it converged onto the pathological section sample, emitting violet fluorescence with a center wavelength of 384 nm. The fluorescence was collected by the microscope objective, passed through the dichroic filter, and imaged onto the target surface of a CMOS camera via a telescopic lens. (System structure reference...) Figure 5 This fluorescent pathology slide scanning system also includes a sample delivery system to enable automated, high-throughput detection. (See reference for sample delivery system.) Figure 6 .

[0081] The scanning stage moves the excitation light across the pathological slide sample line by line at a speed of 20 mm / s. During intra-line scanning, whenever the scanning stage travels 0.5 mm, the scanning control module sends a length signal to the camera and LED driver control circuit. The trigger signal is set to a rising edge trigger mode. The LED driver control circuit detects the rising edge of the trigger signal and starts lighting the LED, with a blinking duration set to... Simultaneously, the camera begins exposure after detecting the rising edge of the trigger signal, with the exposure duration set to... The signal timing is referenced in the attached instruction manual. Figure 8 As shown.

[0082] This application discloses another embodiment of a fluorescence pathological section scanning method, with the system structure referenced. Figure 6 The excitation source is a laser with a wavelength of 680 nm. The fluorescent pathological sample to be tested contains europium ions. The labeled antibody protein has a fluorescence lifetime of approximately 1.1 μs.

[0083] The method in this embodiment specifically includes: the scanning stage moves the excitation light across the pathological slide sample line by line at a speed of 5 mm / s. During the subsequent intra-line scanning, whenever the scanning stage travels 0.3 mm, the scanning control module sends a length signal to the camera and laser drive control circuit. The trigger signal is set to a falling edge trigger mode. Upon detecting the falling edge of the trigger signal, the laser drive control circuit simultaneously illuminates both the top and bottom laser sets, with the flashing duration set to... Simultaneously, the camera begins exposure after detecting the falling edge of the trigger signal, with the exposure duration set to... The signal timing is referenced in the attached instruction manual. Figure 9 As shown.

[0084] Based on the same concept, this application also discloses a fluorescent pathological slide scanning device, which includes at least one of the fluorescent pathological slide scanning systems described in any of the above embodiments. The fluorescent pathological slide scanning system, method, and device of this application share the same technical concept, and the technical details of the embodiments of the three are interchangeable. To reduce repetition, they will not be repeated here.

[0085] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the above-described division of program modules is merely an example. In practical applications, the above functions can be assigned to different program modules as needed, that is, the internal structure of the device can be divided into different program units or modules to complete all or part of the functions described above. The program modules in the embodiments can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one processing unit. The integrated unit can be implemented in hardware or as a software program unit. Furthermore, the specific names of the program modules are only for easy differentiation and are not intended to limit the scope of protection of this application.

[0086] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

Claims

1. A fluorescent pathological slide scanning system, characterized in that, include: Fluorescent excitation source, imaging module, and scanning control module; The scanning control module is used to scan the entire pathological slide through the imaging field of view, synchronously trigger the fluorescence excitation light source flash and the imaging module to take pictures, and base the images on the fluorescence lifetime of the fluorescent dye used. Set the flash duration of the excitation light source. and the exposure time of the imaging module ; The flash duration, the exposure duration, and the fluorescence lifetime of the fluorescent sample to be tested satisfy the following formula: ; The scanning control module is also used to control the fluorescence excitation light source to continuously generate excitation light during the flash duration, irradiating the fluorescent sample to be tested; so that the fluorescent sample to be tested is excited and emits a fluorescence signal; The scanning control module is also used to simultaneously control the imaging module to continuously acquire the fluorescence signal within the exposure time, and to convert the fluorescence signal into a fluorescence image through exposure.

2. The fluorescence pathological slide scanning system as described in claim 1, characterized in that, Also includes: A scanning displacement stage; used to place the fluorescence sample to be tested and to move the fluorescence sample to be tested on the plane of the scanning displacement stage; The scanning control module is also used to acquire the position information of the fluorescent sample to be tested on the scanning displacement stage in real time, and to send trigger signals to the imaging module and the fluorescent excitation source synchronously according to the position information; The trigger signal is used to trigger the fluorescence excitation source and the imaging module to start performing corresponding operations.

3. The fluorescence pathological slide scanning system as described in claim 2, characterized in that, The scanning control module is further configured to determine the signal trigger duration of the trigger signal; and based on the signal trigger duration, determine the first moment when the fluorescence excitation source begins to generate the excitation light, and the second moment when the imaging module begins to expose.

4. The fluorescence pathological slide scanning system as described in claim 3, characterized in that, The first time point is equal to the second time point.

5. The fluorescence pathological slide scanning system as described in claim 2, characterized in that, Also includes: A sample transport device is used to set the sample transport speed so that the excitation light scans the fluorescent sample to be tested line by line.

6. The fluorescence pathological slide scanning system as described in claim 5, characterized in that, The scanning control module is also used to set the interval between two adjacent trigger signals based on the sample transport speed.

7. The fluorescent pathological slide scanning system as described in claim 2, characterized in that, Also includes: Fluorescent illumination lens assembly, fluorescent beam splitter module, focusing module, microscope objectives, and telescopic lens; The fluorescent illumination lens group is used to shape and focus the excitation light to match the subsequent optical path; The fluorescence spectrometer module includes a dichroic mirror and a filter for reflecting the excitation light to separate the excitation light from the fluorescence signal, allowing only the fluorescence signal to pass through; The excitation light is shaped by the fluorescent illumination lens group and enters the fluorescent spectral module. It is reflected in the first direction and enters the microscope objective, and is finally focused on the surface of the fluorescent sample to be tested. The focusing module is used to assist focusing in order to maintain the stability of the imaging focal plane; The microscope objective is used to collect the fluorescence signal emitted by the fluorescent sample to be tested and form an intermediate image; The telescopic lens is used to further magnify the intermediate image, and the intermediate image is transmitted to the imaging module.

8. The fluorescence pathological slide scanning system as described in claim 7, characterized in that, The fluorescence excitation light source, fluorescence illumination lens group, fluorescence beam splitter module, focusing module, microscope objective, scanning stage, telescopic lens, and imaging module are arranged sequentially along the optical path.

9. A method for scanning fluorescent pathological sections, characterized in that, The method is performed based on a fluorescent pathological slide scanning system according to any one of claims 1-8; and includes the following steps: The imaging field of view is scanned across the entire pathological section, and the fluorescence excitation light source flashes synchronously with the imaging module to take a picture, based on the fluorescence lifetime of the fluorescent dye used. Set the flash duration of the excitation light source. and the exposure time of the imaging module ; The flash duration, the exposure duration, and the fluorescence lifetime of the fluorescent sample to be tested satisfy the following formula: ; The fluorescence excitation source is controlled to continuously generate excitation light during the flash duration, illuminating the fluorescent sample to be tested; thus exciting the fluorescent sample to be tested and causing it to emit a fluorescence signal. Simultaneously, the imaging module is controlled to continuously acquire the fluorescence signal within the exposure time, and the fluorescence signal is converted into a fluorescence image through exposure.

10. A fluorescent pathological slide scanning device, characterized in that, It includes at least one fluorescent pathological slide scanning system as described in any one of claims 1-8.

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