Cell fluorescence microscopic image scanning and target cell marking and screening device and application

Abstract

The invention belongs to the technical field of cell fluorescence detection methods and equipment, and particularly relates to a cell fluorescence microscopic image scanning and target cell marking and screening device and application. Comprising an optical imaging system, an image acquisition system and an image processing system, a to-be-detected sample is irradiated by exciting light with different wavelengths emitted by an excitation module, fluorescence images under multiple excitation conditions are acquired by an imaging camera, and fluorescence response acquisition of target cells is realized by combining natural light images acquired by a preview camera; the image processing system carries out interception, indexing and layer superposition on the fluorescence signal to form a multi-light-source superposed image; the multi-channel fluorescence characteristic of the target cell can be efficiently and accurately extracted, rapid screening of the target cell, the to-be-determined target cell and the non-target cell is realized, and the multi-channel fluorescence characteristic extraction method is suitable for the fields of tumor detection, immunolabeling and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell fluorescence detection methods and devices, and particularly relates to a cell fluorescence microscopic image scanning and target cell labeling and screening device and its application. Background Art

[0002] Cell fluorescence microscopy imaging technology is a cell analysis method based on the combination of fluorescence labeling and high-resolution optical imaging, and is widely used in fields such as life science research, drug screening, disease diagnosis and treatment mechanism exploration. By using specific fluorescent probes or antibodies to label specific cell structures or proteins, visual observation of biological characteristics such as cell morphology, distribution, and functional state can be achieved.

[0003] In the prior art, the acquisition of cell fluorescence microscopic images mostly relies on inverted fluorescence microscopes or laser confocal microscopes operated manually. Although their imaging quality is relatively high, there are problems such as low efficiency, poor repeatability, and strong manual intervention in large-scale sample analysis, continuous scanning, multi-channel fluorescence acquisition, etc., making it difficult to meet the requirements of high-throughput automated cell screening.

[0004] On the other hand, the identification and screening of target cells usually rely on manual inspection of images or analysis using image post-processing software. This process not only takes a long time but is also easily affected by subjective judgment. Especially in the case of multi-fluorescent labeling or weak signal backgrounds, the accuracy and robustness are relatively low. In addition, there is currently a lack of a device system that can integrate cell image acquisition, fluorescence label recognition, and automatic screening output. As a result, multiple devices need to be coordinated to complete the operation in practical applications, increasing the cost and operation complexity.

[0005] Therefore, how to design a comprehensive device system that integrates image scanning, fluorescence recognition, target cell screening, and automated processing to improve the efficiency and accuracy of cell image acquisition and analysis has become a key technical problem urgently needed to be solved in this field. The present invention is proposed under this background, aiming to achieve a high degree of integration and intelligence of cell fluorescence imaging and screening analysis. Summary of the Invention

[0006] In view of the above problems, the object of the present invention is to provide: a cell fluorescence microscopic image scanning and target cell labeling and screening device, the device comprising an optical imaging system, an image acquisition system, and an image processing system; The optical imaging system includes: An excitation module, the excitation module is used to emit a predetermined excitation light to irradiate the sample to be measured; A motion platform, the motion platform is used to place a slide carrying the sample to be measured, and the sample to be measured includes target cells and non-target cells; Objective lens, trinocular lens, eyepiece; the trinocular lens has three light output channels, including an eyepiece channel, a preview camera channel, and an imaging camera channel; The image acquisition system comprises: a preview camera and an imaging camera; The image processing system comprises: A labeling module, which is used to extract the fluorescence signal of the target cell in the cell fluorescence microscopy image labeled with multiple excitation lights; A multi-light source superposition module, which is used to superimpose the natural light image and the fluorescent image to obtain a multi-light source superposition image; A screening module is used to determine the type of target cells in the sample to be tested based on the extracted fluorescent signals of the target cells, and to classify the cells in the sample to be tested based on the type of the target cells.

[0007] In a preferred technical solution, the excitation module includes: An excitation unit, the excitation unit comprising a plurality of excitation light sources, the plurality of excitation light sources being used to respectively emit excitation light of different wavelengths to perform fluorescence excitation on the sample to be tested; An electric filter converter is used to output different excitation lights by switching filters.

[0008] In a preferred technical solution, the preview camera includes a natural light receiver, and the imaging camera includes a natural light receiver and a fluorescence receiver; The natural light receiver is used to receive the natural light image of the sample to be tested with the same coordinates on the slide under natural light; the fluorescence receiver is used to receive the fluorescence image of the sample to be tested with the same coordinates on the slide under different excitation lights.

[0009] In a preferred technical solution, the marking module includes: An interception unit, used for intercepting a fluorescence microscopic image generated under a single excitation light from a cell fluorescence microscopic image of multiple excitation lights input; An indexing unit, used to generate a plurality of single fluorescent labeling layers according to the intercepted fluorescent microscopic image produced under the single excitation light; The multi-light source superposition module is used to superimpose multiple single fluorescent marker layers on a natural light image to obtain a multi-light source superposition image.

[0010] In a preferred technical solution, intercepting a fluorescence microscopic image generated under a single excitation light specifically includes: T1. Analyze the distribution of various fluorescent signals in the fluorescence microscopy images under multiple excitation lights, and count the pixel proportions of each fluorescent signal in different single excitation light images; T2. When the proportion of the effective pixel number of a certain fluorescence signal in a single excitation light image to the total pixel number of the image reaches the maximum, then determine this fluorescence signal as the main fluorescence signal of the single excitation light; In a preferred technical solution, the generating of multiple single fluorescence marker layers specifically includes: T3. The indexing unit receives all the main fluorescence signals of any single excitation light, and generates a main fluorescence image for each main fluorescence signal; T4. Superimpose the main fluorescence images of the single excitation light to generate a single fluorescence marker layer of the single excitation light.

[0011] In a preferred technical solution, the screening module is based on the microscopic image under natural light at the same position, calls all the single fluorescence marker layers, compares with the known cell fluorescence reactions, and conducts cell classification; the cell classification includes known target cells, pending target cells, and non-target cells.

[0012] The present invention also provides a screening method based on the above-mentioned cell fluorescence microscopic image scanning and target cell marker screening device, including the following steps: S1. Sample preparation and platform positioning: Place the slide carrying the sample to be tested on the moving platform, and the sample to be tested includes target cells and non-target cells; S2. Multi-light source excitation and image acquisition: Control the excitation unit through the excitation module to emit excitation lights of different wavelengths, irradiate the sample to be tested in turn, and receive the fluorescence images generated by the sample under each excitation light through the imaging camera in the image acquisition system, and at the same time receive the natural light image collected by the preview camera to generate a cell fluorescence microscopic image of multiple excitation lights; S3. Fluorescence signal marking and layer generation: Extract the fluorescence signals of the target cells from the cell fluorescence microscopic image of multiple excitation lights through the marking module in the image processing system and conduct marking; S4. Cell classification and recognition based on layer comparison: Through the screening module, based on the microscopic image under natural light at the same position, call all the single fluorescence marker layers, compare with the known cell fluorescence reactions, and conduct cell classification; the cell classification includes known target cells, pending target cells, and non-target cells.

[0013] In a preferred technical solution, the step S3 includes: S31. Through the interception unit, extract the single fluorescence images under each single excitation light condition from the cell fluorescence microscopic image of multiple excitation lights; S32. The indexing unit indexes the fluorescence signals at each spatial position in the single fluorescence image according to the wavelength of the fluorescence signal; S33. Superimpose the indexed fluorescence images under all single excitation light conditions onto the natural light image at the same coordinate position to form a multi-source superimposed image with indexing information.

[0014] In a preferred technical solution, in step S4, the screening module, based on the microscopic image under natural light at the same position, calls all single fluorescence labeling layers, compares them with known cell fluorescence reactions, classifies the cells, and obtains a cell classification list; the cell classification includes known target cells, to-be-determined target cells, and non-target cells.

[0015] Beneficial effects: The present invention provides a cell fluorescence microscopic image scanning and target cell labeling and screening device, having the following beneficial effects: 1. Combining multi-band fluorescence excitation and natural light acquisition to improve cell recognition accuracy: By setting multiple excitation light sources with different wavelengths and combining the natural light channel to collect the background image of the sample, image information acquisition at the same position under different excitation conditions is realized, which helps to comprehensively compare and analyze the fluorescence response characteristics of target cells, and significantly improves the accuracy of cell labeling and recognition.

[0016] 2. Adopting the main fluorescence signal determination and layer superposition mechanism to achieve multi-source information fusion expression: By extracting the main fluorescence signal through an analysis algorithm based on clustering or pixel distribution, and superimposing the main fluorescence images under each excitation condition onto the natural light image to form a multi-source superimposed image, the intuitiveness and visualization expression effect of the image are enhanced, which is beneficial for subsequent screening and recognition processing by operators or algorithms.

[0017] 3. Introducing a triple eyepiece structure and a dual-camera acquisition path to achieve parallel processing of observation and imaging: The triple eyepiece structure includes an eyepiece observation channel, a preview camera channel, and an imaging camera channel, enabling the operator to complete high-definition image acquisition while observing in real time, effectively improving the performance of the device in terms of operation convenience and data acquisition efficiency.

[0018] 4. The modular image processing process improves the screening efficiency and adaptability: The present invention divides the image processing process into multiple modules such as interception, indexing, superposition, and classification, having good system scalability, facilitating the integration of different algorithm strategies to adapt to the screening requirements of various types of target cells.

[0019] 5. The cell classification dimension is more refined and the result has more medical reference value: By performing fitting analysis on the fluorescence responses of each target cell under multiple excitation channels, precise classification of known target cells, to-be-determined target cells, and non-target cells is realized, which helps to construct a high-throughput and highly specific cell screening system, applicable to scenarios such as circulating tumor cell (CTC) recognition, immune typing, and drug sensitivity screening. Brief Description of the Drawings

[0020] Figure 1 is a schematic structural diagram of the system of the present invention; Figure 2 is a schematic external view diagram of the system of the present invention; Figure 3 is a schematic flowchart of the method of the present invention. Detailed Embodiments

[0021] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0022] Embodiment 1 According to Figure 1 and Figure 2 shown, this embodiment provides a device for scanning cell fluorescence microscopic images and screening target cell markers. The device includes an optical imaging system, an image acquisition system, and an image processing system.

[0023] Among them, the optical imaging system includes the following components: An excitation module for emitting a predetermined excitation light to irradiate a sample to be measured; A moving platform for placing a slide carrying the sample to be measured, and the sample to be measured includes target cells and non-target cells; An objective lens, a trinocular lens, and an eyepiece. The trinocular lens has three light output channels, which are respectively connected to the eyepiece channel, the preview camera channel, and the imaging camera channel, for realizing the parallel operation of observation and image acquisition.

[0024] The excitation module includes an excitation unit and an electric filter converter. The excitation unit is provided with a plurality of excitation light sources for respectively emitting excitation lights of different wavelengths to realize multi-channel fluorescence excitation. The electric filter converter is configured with a plurality of filter plates, and can adjust the filter path by means of electric switching to realize precise control of the excitation light wavelength, and improve the specificity and sensitivity of fluorescence excitation.

[0025] The image acquisition system includes a preview camera and an imaging camera. Preferably, the preview camera integrates a natural light receiver, and the imaging camera is simultaneously configured with a natural light receiver and a fluorescence receiver. The natural light receiver is used to collect the bright-field microscopic image of the sample to be measured at the same position coordinate on the slide under natural light irradiation; the fluorescence receiver is used to respectively collect the fluorescence images generated at this position under different excitation light irradiations, so as to realize the full coverage of the fluorescence information of the same target area under multiple excitation conditions.

[0026] The image processing system includes a marking module, a multi-light source superposition module, and a screening module.

[0027] Among them, the marking module is used to extract and mark the target cell fluorescence signals in the cell fluorescence images collected under multiple excitation light conditions.

[0028] The marking module includes a cropping unit and an indexing unit: The cropping unit is responsible for extracting the fluorescence microscopic images formed under a single excitation light condition from the input multiple excitation light images. The specific process includes the following steps: T1. Analyze the fluorescence signals frame by frame in the images under multiple excitation light conditions, and count the number of effective pixels of each fluorescence signal in different images; T2. Judge in which excitation light image the pixel ratio of a certain fluorescence signal is the largest, and take the corresponding image as the main fluorescence image of this signal.

[0029] The indexing unit is used to process the cropped single excitation light images to generate a main fluorescence layer. Its processing flow is as follows: T3. The indexing unit receives all the main fluorescence signals under each single excitation light, and generates corresponding main fluorescence images for each main fluorescence signal; T4. Perform spatial alignment and gray-scale normalization processing on multiple main fluorescence images belonging to the same excitation light channel, and finally superimpose them to form a single fluorescence marking layer corresponding to this excitation light channel.

[0030] The multi-light source superimposing module superimposes the above-mentioned multiple single fluorescence marking layers onto the natural light image respectively, so as to form a multi-light source superimposed image containing multi-channel fluorescence information and bright-field structure information. This image retains the structural and morphological characteristics of the sample, and at the same time enhances the fluorescence localization effect, which is convenient for subsequent screening and identification.

[0031] The screening module is based on the natural light image, calls all the single fluorescence marking layers, and compares and matches them with a preset known cell fluorescence response feature library to perform the identification and classification task of target cells. Preferably, this module determines whether each cell is: A known target cell; A to-be-determined target cell (with potential biological characteristics but not yet matched to the database); A non-target cell (not conforming to any known response pattern).

[0032] Through the description of the above embodiments, the device provided by the present invention can not only realize the integrated acquisition of multi-band fluorescence excitation and imaging, but also achieve efficient and accurate cell classification and recognition through a modular processing flow, and is applicable to various biomedical application scenarios such as tumor cell screening, immunolabeling analysis, and live cell tracking.

[0033] Embodiment 2 Such as Figure 3As shown in the figure, this embodiment provides a screening method based on the above-mentioned cell fluorescence microscopy image scanning and target cell labeling and screening device. This method combines multi-band excitation imaging technology and image processing algorithms to achieve automatic recognition and classification of target cells in a sample. The specific steps are as follows: S1. Sample preparation and platform positioning: Place the slide carrying the sample to be tested steadily on the moving platform to ensure that the slide is within the focal plane of the imaging system. The sample to be tested may contain target cells and non-target cells of unknown types. This step aims to provide a stable basis for subsequent fluorescence excitation and image acquisition.

[0034] S2. Multi-light source excitation and image acquisition: Control multiple excitation light sources to work through the excitation module in the optical imaging system, and sequentially emit excitation lights of different wavelengths to the sample to be tested. After each excitation light acts, the imaging camera collects the corresponding fluorescence image and records the fluorescence response characteristics of the cells at a specific wavelength band. At the same time, the preview camera synchronously collects the bright-field microscopy image of the sample in the natural light channel to form the corresponding background structure information. Through this process, a multi-channel and multi-spectral cell fluorescence microscopy image sequence is obtained as the basic data for subsequent image processing.

[0035] S3. Fluorescent signal labeling and layer generation: The image processing system starts the labeling module, analyzes the obtained multiple excitation light images, extracts the fluorescent signals of various target cells, and generates layer information for recognition. This step includes the following preferred sub-steps: S31. Fluorescent image interception: The interception unit analyzes the multiple excitation light images in sequence, separates the single fluorescent image generated under each excitation light, and ensures that the images under different wavelength conditions are processed independently; S32. Fluorescent signal indexing: The indexing unit performs tagging processing on the target signals according to the wavelengths and position coordinates of the fluorescent signals in different fluorescent images, that is, marks each pixel position with the corresponding fluorescent channel identifier; S33. Layer generation and superposition: Superimpose the image layers indexed under each single excitation light onto the structural image in natural light to form a multi-light source superposition image with both the comprehensive structure and fluorescent signals, significantly improving the contrast and interpretability of the image.

[0036] S4. Cell classification and recognition based on layer comparison: The image processing system further calls the screening module, uses the natural light image as the coordinate benchmark, superimposes and analyzes each single fluorescently labeled layer, and compares it with the preset cell fluorescence feature library. Through matching analysis of the fluorescence response combinations of target cells in multiple channels, automatic cell classification is achieved.

[0037] Based on the above comparison results, the screening module generates a cell classification list. The list can be divided into three categories: Known target cells: Typical fluorescent feature cells with successful matching; Pending target cells: Partial feature matching, with possible expression of new markers; Non-target cells: Background cells with no obvious fluorescence reaction or inconsistent features.

[0038] The above method can achieve rapid screening and classification recognition of multiple types of cells in high-throughput samples without relying on external manual interpretation, and is applicable to various application scenarios such as tumor cell screening, immunomarker analysis, live cell recognition, and drug sensitivity detection.

[0039] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A cell fluorescence microscopic image scanning and target cell marker screening device, characterized in that, The device includes an optical imaging system, an image acquisition system, and an image processing system; The optical imaging system includes: An excitation module for emitting a predetermined excitation light to irradiate the sample to be tested; A moving platform for placing a slide carrying the sample to be tested, where the sample to be tested includes target cells and non-target cells; An objective lens, a trinocular lens, and an eyepiece; the trinocular lens has three light output channels, including an eyepiece channel, a preview camera channel, and an imaging camera channel; The image acquisition system includes a preview camera and an imaging camera; The image processing system includes: A marking module for extracting the fluorescence signal of target cells in the cell fluorescence microscopic image marked with multiple excitation lights; A multi-light source superposition module for superposing a natural light image and a fluorescence image to obtain a multi-light source superposition image; A screening module for determining the type of target cells in the sample to be tested according to the extracted fluorescence signal of the target cells, and classifying the cells in the sample to be tested according to the type of the target cells.

2. The cell fluorescence microscopic image scanning and target cell labeling and screening device according to claim 1, wherein The excitation module includes: An excitation unit including a plurality of excitation light sources for respectively emitting excitation lights of different wavelengths to perform fluorescence excitation on the sample to be tested; An electric filter converter for outputting different excitation lights by switching filters.

3. The cell fluorescence microscopic image scanning and target cell labeling and screening device according to claim 2, wherein The preview camera includes a natural light receiver, and the imaging camera includes a natural light receiver and a fluorescence receiver; The natural light receiver is used to receive the natural light image of the sample to be tested at the same position coordinates on the slide under natural light; the fluorescence receiver is used to receive the fluorescence image of the sample to be tested at the same position coordinates on the slide under different excitation lights.

4. The cell fluorescence microscopic image scanning and target cell marking and screening device according to any one of claims 1-3, characterized in that The marking module includes: An intercepting unit for intercepting the fluorescence microscopic image generated under a single excitation light from the input cell fluorescence microscopic image marked with multiple excitation lights; An indexing unit for generating a plurality of single fluorescence marking layers according to the intercepted fluorescence microscopic image generated under a single excitation light; The multi-light source superposition module is used to superpose a plurality of single fluorescence marking layers on the natural light image to obtain a multi-light source superposition image.

5. The cell fluorescence microscopic image scanning and target cell marker screening device according to claim 4, characterized in that, Intercepting the fluorescence microscopic image generated under a single excitation light specifically includes: T1. Analyze the distribution of various fluorescence signals in the fluorescence microscopic image under multiple excitation lights in sequence, and count the pixel proportion of each fluorescence signal in different single excitation light images; T2. When the proportion of the effective pixel number of a certain fluorescence signal in a certain single excitation light image to the total pixel number of the image reaches the maximum, then determine the certain fluorescence signal as the main fluorescence signal of the single excitation light.

6. The cell fluorescence microscopic image scanning and target cell labeling and screening device according to claim 5, wherein Generating a plurality of single fluorescence marking layers specifically includes: T3. The indexing unit receives all the main fluorescence signals of any single excitation light, and generates a main fluorescence image for each main fluorescence signal; T4. Superpose the main fluorescence images of a single excitation light to generate a single fluorescence marking layer of the single excitation light.

7. The cell fluorescence microscopic image scanning and target cell labeling and screening device according to claim 6, characterized in that, Based on the microscopic images under natural light at the same position, the screening module calls all single fluorescence-labeled layers, compares them with known cell fluorescence reactions, and classifies cells; the cell classification includes known target cells, to-be-determined target cells, and non-target cells.

8. The screening method of the cell fluorescence microscopic image scanning and target cell labeling and screening device according to any one of claims 1-7, characterized in that, It includes the following steps: S1. Place the specimen slide carrying the sample to be tested on the moving platform, where the sample to be tested includes target cells and non-target cells; S2. Control the excitation unit through the excitation module to emit excitation lights of different wavelengths, irradiate the sample to be tested in sequence, and receive the fluorescence images generated by the sample under each excitation light through the imaging camera in the image acquisition system, and at the same time receive the natural light image collected by the preview camera to generate a cell fluorescence microscopic image with multiple excitation lights; S3. Through the marking module in the image processing system, extract the fluorescence signals of the target cells from the cell fluorescence microscopic image with multiple excitation lights and mark them; S4. Based on the microscopic images under natural light at the same position, the screening module calls all single fluorescence-labeled layers, compares them with known cell fluorescence reactions, and classifies cells; the cell classification includes known target cells, to-be-determined target cells, and non-target cells.

9. The screening method according to claim 8, wherein The step S3 includes: S31. Extract the single fluorescence images under each single excitation light condition from the cell fluorescence microscopic image with multiple excitation lights through the intercepting unit; S32. The indexing unit indexes the fluorescence signals at each spatial position in the single fluorescence image according to the wavelength of the fluorescence signal; S33. Superimpose the indexed fluorescence images under all single excitation light conditions on the natural light image at the same coordinate position to form a multi-light-source superimposed image with indexing information.

10. The screening method according to claim 9, characterized in that, In the step S4, based on the microscopic images under natural light at the same position, the screening module calls all single fluorescence-labeled layers, compares them with known cell fluorescence reactions, classifies cells, and obtains a cell classification list; the cell classification includes known target cells, to-be-determined target cells, and non-target cells.

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