Cell fluorescence microscopy image scanning and target cell marker screening device and its application

Abstract

The present invention belongs to the technical field of cell fluorescence detection methods and equipment, and specifically relates to cell fluorescence microscopy image scanning and target cell marker screening devices and applications. The system comprises an optical imaging system, an image acquisition system, and an image processing system. The system emits excitation light of different wavelengths through an excitation module to illuminate the sample to be tested, uses an imaging camera to capture fluorescence images under multiple excitation conditions, and combines the natural light images captured by the preview camera to achieve the acquisition of the fluorescence response of the target cell. The image processing system intercepts, indexes, and overlays the fluorescence signal to form a multi-light source overlay image. The system calls a screening module to complete cell classification and identification by comparing it with known fluorescence features. The system can efficiently and accurately extract the multi-channel fluorescence features of target cells, and achieve rapid screening of target cells, pending target cells, and non-target cells. The system is suitable for fields such as tumor detection and immune labeling.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell fluorescence detection methods and equipment, and in particular relates to a cell fluorescence microscopic image scanning and target cell marker screening device and application. Background Art

[0002] Cell fluorescence microscopy is a cellular analysis method based on the combination of fluorescent labeling and high-resolution optical imaging. It is widely used in life science research, drug screening, disease diagnosis, and the exploration of therapeutic mechanisms. By labeling specific cellular structures or proteins with specific fluorescent probes or antibodies, biological characteristics such as cell morphology, distribution, and functional status can be visualized.

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

[0004] On the other hand, the identification and screening of target cells typically relies on manual image reading or image post-processing software analysis. This process is not only time-consuming and susceptible to subjective judgment, but also has low accuracy and robustness, especially in the presence of multiple fluorescent markers or weak signal backgrounds. Furthermore, there is currently a lack of a device system that can integrate cell image acquisition, fluorescent marker identification, and automated screening output. Consequently, practical applications require the coordinated operation of multiple devices, increasing costs and operational complexity.

[0005] Therefore, designing a comprehensive device system that integrates image scanning, fluorescence identification, target cell screening, and automated processing to improve the efficiency and accuracy of cell image acquisition and analysis has become a key technical challenge that urgently needs to be addressed in this field. This invention was proposed in this context, aiming to achieve highly integrated and intelligent cell fluorescence imaging and screening analysis. Summary of the Invention

[0006] In view of the above problems, the present invention aims to provide: a cell fluorescence microscopy image scanning and target cell marker screening device, the device comprising an optical imaging system, an image acquisition system and an image processing system;

[0007] The optical imaging system comprises:

[0008] An excitation module, configured to emit predetermined excitation light to illuminate the sample to be tested;

[0009] A motion platform, the motion platform being used to place a slide carrying a sample to be tested, wherein the sample to be tested includes target cells and non-target cells;

[0010] Objective lens, trinocular, eyepiece; the trinocular has three light output channels, including an eyepiece channel, a preview camera channel, and an imaging camera channel;

[0011] The image acquisition system includes: a preview camera and an imaging camera;

[0012] The image processing system comprises:

[0013] 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;

[0014] 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;

[0015] The 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.

[0016] In a preferred technical solution, the excitation module includes:

[0017] An excitation unit, the excitation unit comprising a plurality of excitation light sources, the plurality of excitation light sources being configured to respectively emit excitation light of different wavelengths to perform fluorescence excitation on the sample to be tested;

[0018] An electric filter converter outputs different excitation lights by switching filters.

[0019] 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;

[0020] 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.

[0021] In a preferred technical solution, the marking module includes:

[0022] An interception unit is used to intercept a fluorescence microscopic image generated under a single excitation light from a cell fluorescence microscopic image inputted with multiple excitation lights;

[0023] An indexing unit, configured to generate a plurality of single fluorescent labeling layers based on the intercepted fluorescence microscopic image produced under a single excitation light;

[0024] 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 superimposed image.

[0025] In a preferred technical solution, intercepting a fluorescence microscopic image generated under a single excitation light specifically includes:

[0026] T1. Analyze the distribution of various fluorescent signals in fluorescence microscopy images under multiple excitation lights, and count the pixel proportions of each fluorescent signal in different single excitation light images;

[0027] T2. When the ratio of the number of effective pixels of a certain fluorescent signal in a certain single excitation light image to the total number of pixels in the image reaches the maximum, the fluorescent signal is determined to be the main fluorescent signal of the single excitation light;

[0028] In a preferred technical solution, generating multiple single fluorescent marker layers specifically includes:

[0029] T3, the indexing unit receives all main fluorescence signals of any single excitation light and generates a main fluorescence image for each main fluorescence signal;

[0030] T4. Superimpose the main fluorescence images of the single excitation light to generate a single fluorescence labeling layer of the single excitation light.

[0031] In a preferred technical solution, the screening module uses the microscopic image under natural light at the same location as the basis, calls all single fluorescent labeling layers, compares them with known cell fluorescence reactions, and performs cell classification; the cell classification includes known target cells, undetermined target cells, and non-target cells.

[0032] The present invention also provides a screening method based on the cell fluorescence microscopic image scanning and target cell marker screening device, comprising the following steps:

[0033] S1. Sample preparation and platform positioning: placing a slide carrying a sample to be tested, which includes target cells and non-target cells, on a motion platform;

[0034] S2. Multi-light source excitation and image acquisition: The excitation module controls the excitation unit to emit excitation light of different wavelengths, sequentially irradiating the sample to be tested, and the imaging camera in the image acquisition system receives the fluorescence image generated by the sample under each excitation light, and simultaneously receives the natural light image captured by the preview camera, thereby generating a cell fluorescence microscopic image under multiple excitation lights;

[0035] S3, fluorescence signal labeling and layer generation: extracting the fluorescence signal of the target cell from the cell fluorescence microscopy image of the multiple excitation lights and labeling it through a labeling module in the image processing system;

[0036] S4. Cell classification and identification based on layer comparison: Through the screening module, based on the microscopic image under natural light at the same location, all single fluorescent labeling layers are called and compared with the known cell fluorescence response to perform cell classification; the cell classification includes known target cells, undetermined target cells and non-target cells.

[0037] In a preferred technical solution, step S3 includes:

[0038] S31, extracting, by an interception unit, a single fluorescence image under each single excitation light condition from the cell fluorescence microscopic image under multiple excitation lights;

[0039] S32, indexing the fluorescence signal at each spatial position in the single fluorescence image by an indexing unit according to the wavelength of the fluorescence signal;

[0040] S33, superimposing all the labeled fluorescence images under the single excitation light conditions onto the natural light image at the same coordinate position to form a multi-light source superimposed image with labeling information.

[0041] In a preferred technical solution, in step S4, the screening module calls all single fluorescent labeling layers based on the microscopic image under natural light at the same position, compares them with the known cell fluorescence response, performs cell classification, and obtains a cell classification list; the cell classification includes known target cells, undetermined target cells, and non-target cells.

[0042] Beneficial effects:

[0043] The present invention provides a cell fluorescence microscopic image scanning and target cell marker screening device, which has the following beneficial effects:

[0044] 1. Combining multi-band fluorescence excitation with natural light acquisition improves cell identification accuracy: By setting multiple excitation light sources of different wavelengths and combining them with the natural light channel to collect the background image of the sample, image information acquisition of the same location under different excitation conditions is achieved, which helps to comprehensively compare and analyze the fluorescence response characteristics of the target cells, significantly improving the accuracy of cell labeling and identification.

[0045] 2. Adopting the main fluorescence signal determination and layer overlay mechanism, multi-source information fusion expression is achieved: the main fluorescence signal is extracted through an analysis algorithm based on clustering or pixel distribution, and the main fluorescence image under various excitation conditions is superimposed on the natural light image to form a multi-light source superimposed image. This enhances the intuitiveness and visual expression of the image, facilitating subsequent screening and recognition processing by the operator or algorithm.

[0046] 3. Introducing a trinocular structure and dual-camera acquisition path to achieve parallel processing of observation and imaging: The trinocular structure includes an eyepiece observation channel, a preview camera channel, and an imaging camera channel, allowing the operator to complete high-definition image acquisition while observing in real time, effectively improving the performance of the equipment in terms of operational convenience and data acquisition efficiency.

[0047] 4. Modular image processing process improves screening efficiency and adaptability: The present invention divides the image processing process into multiple modules of interception, indexing, superposition and classification, which has good system scalability and facilitates the integration of different algorithm strategies to adapt to various types of target cell screening needs.

[0048] 5. The cell classification dimension is more refined and the results are more medically valuable: By fitting and analyzing the fluorescence response of each target cell under multiple excitation channels, accurate classification of known target cells, undetermined target cells and non-target cells is achieved, which helps to build a high-throughput and highly specific cell screening system suitable for scenarios such as circulating tumor cell (CTC) identification, immunophenotyping and drug sensitivity screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the system structure of the present invention;

[0050] Figure 2 Schematic diagram of the system appearance of the present invention;

[0051] Figure 3 Schematic diagram of the method of the present invention. DETAILED DESCRIPTION

[0052] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0053] Example 1

[0054] according to Figure 1 and Figure 2 As shown, this embodiment provides a cell fluorescence microscopy image scanning and target cell marker screening device, which includes an optical imaging system, an image acquisition system and an image processing system.

[0055] Among them, the optical imaging system includes the following components:

[0056] An excitation module, used to emit predetermined excitation light to illuminate the sample to be tested;

[0057] A motion platform for placing a slide carrying samples to be tested, wherein the samples to be tested include target cells and non-target cells;

[0058] The objective lens, trinocular and eyepiece, wherein the trinocular has three light output channels, which are respectively connected to the eyepiece channel, preview camera channel and imaging camera channel, for realizing parallel work of observation and image acquisition.

[0059] The excitation module includes an excitation unit and an electric filter converter. The excitation unit is equipped with multiple excitation light sources, each emitting excitation light of different wavelengths to achieve multi-channel fluorescence excitation. The electric filter converter is equipped with multiple filters that can adjust the filter path through electric switching, achieving precise control of the excitation light wavelength and improving the specificity and sensitivity of fluorescence excitation.

[0060] The image acquisition system includes a preview camera and an imaging camera. Preferably, the preview camera integrates a natural light receiver, while the imaging camera is equipped with both a natural light receiver and a fluorescence receiver. The natural light receiver is used to capture brightfield microscopic images of the sample under natural light at the same coordinate on the slide; the fluorescence receiver is used to capture fluorescence images produced at the same location under different excitation light conditions, thereby achieving full coverage of fluorescence information for the same target area under multiple excitation conditions.

[0061] The image processing system includes a marking module, a multi-light source superposition module and a screening module.

[0062] The marking module is used to extract and mark the target cell fluorescence signal in the cell fluorescence image collected under multiple excitation light conditions.

[0063] The marking module includes a truncation unit and an indexing unit:

[0064] The interception unit is responsible for extracting the fluorescence microscopy image formed under single excitation light conditions from the input multiple excitation light images. The specific process includes the following steps:

[0065] T1. Analyze the fluorescence signals frame by frame under multiple excitation light conditions and count the number of effective pixels of each fluorescence signal in different images.

[0066] T2. Determine in which excitation light image the pixel ratio of a certain fluorescence signal is the largest, and use the corresponding image as the main fluorescence image of the signal.

[0067] The indexing unit is used to process the captured single excitation light image to generate the main fluorescence layer. The processing flow is as follows:

[0068] T3, the indexing unit receives all main fluorescence signals under each single excitation light and generates a corresponding main fluorescence image for each main fluorescence signal;

[0069] T4. Perform spatial alignment and grayscale normalization on multiple main fluorescence images belonging to the same excitation light channel, and finally superimpose them to form a single fluorescent labeling layer corresponding to the excitation light channel.

[0070] The multi-light source overlay module superimposes these individual fluorescent marker layers onto the natural light image, creating a multi-light source overlay image that combines multi-channel fluorescence information with brightfield structural information. This image preserves the sample's structural morphology while enhancing fluorescence localization, facilitating subsequent screening and identification.

[0071] The screening module uses natural light images as a basis, calls all single fluorescent marker layers, compares and matches them with a preset library of known cell fluorescence response characteristics, and performs the task of identifying and classifying target cells. Preferably, the module determines whether each cell is:

[0072] Known target cells;

[0073] Undetermined target cells (potential biological characteristics exist but have not yet been matched to the database);

[0074] Non-target cells (not fitting any known response pattern).

[0075] 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 realize efficient and accurate cell classification and identification through a modular processing flow, and is suitable for various biomedical application scenarios such as tumor cell screening, immune labeling analysis, and live cell tracking.

[0076] Example 2

[0077] like Figure 3 As shown, this embodiment provides a screening method based on the above-mentioned cell fluorescence microscopy image scanning and target cell marker screening device. This method combines multi-band excitation imaging technology with image processing algorithms to achieve automatic recognition and classification of target cells in a sample. Specifically, it includes the following steps:

[0078] S1. Sample preparation and platform positioning:

[0079] Stably place the slide containing the sample to be measured on a motion platform, ensuring that the slide is within the focal plane of the imaging system. The sample to be measured may contain target and non-target cells of unknown species. This step is intended to provide a stable foundation for subsequent fluorescence excitation and image acquisition.

[0080] S2. Multi-light source excitation and image acquisition:

[0081] The excitation module in the optical imaging system controls multiple excitation light sources, sequentially emitting excitation light of different wavelengths toward the sample under test. After each excitation light exposure, the imaging camera captures the corresponding fluorescence image, recording the fluorescence response characteristics of the cells at specific wavelengths. Simultaneously, the preview camera simultaneously captures brightfield microscopic images of the sample under natural light, generating corresponding background structure information. This process generates a multi-channel, multispectral sequence of cell fluorescence microscopic images, which serves as the foundation for subsequent image processing.

[0082] S3. Fluorescent signal labeling and layer generation:

[0083] The image processing system starts the labeling module to analyze the acquired multiple excitation light images, extract the fluorescence signals of various target cells, and generate identifiable layer information. This step includes the following preferred sub-steps:

[0084] S31, fluorescence image capture: The capture unit analyzes multiple excitation light images in sequence, separates the single fluorescence image produced under each excitation light, and ensures that images under different wavelength conditions are processed independently;

[0085] S32, fluorescence signal labeling: The labeling unit labels the target signal according to the wavelength and position coordinates of the fluorescence signal in different fluorescence images, that is, labels each pixel position with a corresponding fluorescence channel label;

[0086] S33. Layer generation and overlay: The image layers that have been indexed under each single excitation light are superimposed on the structural image under natural light to form a multi-light source overlay image with both comprehensive structure and fluorescence signals, significantly improving the image contrast and interpretability.

[0087] S4. Cell classification and recognition based on layer comparison:

[0088] The image processing system further utilizes a screening module to overlay and analyze each single fluorescent marker layer using the natural light image as a coordinate reference, and then compares it with a pre-set library of cell fluorescence signatures. Automatic cell classification is achieved by matching and analyzing the fluorescence response combinations of target cells under multiple channels.

[0089] The screening module generates a cell classification list based on the above comparison results. The list can be divided into three categories:

[0090] Known target cells: cells with typical fluorescent characteristics that have been successfully matched;

[0091] Undetermined target cells: Partial feature matching, possible expression of novel markers;

[0092] Non-target cells: Background cells with no obvious fluorescent response or inconsistent characteristics.

[0093] The above method can achieve rapid screening and classification identification of multiple types of cells in high-throughput samples without the help of external manual interpretation. It is suitable for various application scenarios such as tumor cell screening, immune marker analysis, live cell identification, and drug sensitivity testing.

[0094] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A cell fluorescence microscopy 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 comprises: An excitation module, configured to emit predetermined excitation light to illuminate the sample to be tested; A motion platform, the motion platform being used to place a slide carrying a sample to be tested, wherein the sample to be tested includes target cells and non-target cells; Objective lens, trinocular, eyepiece; the trinocular 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 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; The 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.

2. The cell fluorescence microscopic image scanning and target cell marker screening device according to claim 1, characterized in that: The excitation module includes: An excitation unit, the excitation unit comprising a plurality of excitation light sources, the plurality of excitation light sources being configured to respectively emit excitation light of different wavelengths to perform fluorescence excitation on the sample to be tested; An electric filter converter outputs different excitation lights by switching filters.

3. The cell fluorescence microscopic image scanning and target cell marker screening device according to claim 2, characterized in that: 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.

4. The cell fluorescence microscopic image scanning and target cell marker screening device according to any one of claims 1 to 3, characterized in that: The marking module includes: An interception unit is used to intercept a fluorescence microscopic image generated under a single excitation light from a cell fluorescence microscopic image inputted with multiple excitation lights; An indexing unit, configured to generate a plurality of single fluorescent labeling layers based on the intercepted fluorescence microscopic image produced under a 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 superimposed image.

5. The cell fluorescence microscopic image scanning and target cell marker screening device according to claim 4, characterized in that: Capturing a fluorescence microscopic image produced under a single excitation light specifically includes: T1. Analyze the distribution of various fluorescent signals in 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 ratio of the number of effective pixels of a certain fluorescent signal in a certain single excitation light image to the total number of pixels of the image reaches a maximum, the fluorescent signal is determined as the main fluorescent signal of the single excitation light.

6. The cell fluorescence microscopic image scanning and target cell marker screening device according to claim 5, characterized in that: Generating multiple single fluorescent marker layers specifically includes: T3, the indexing unit receives all 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 labeling layer of the single excitation light.

7. The cell fluorescence microscopic image scanning and target cell marker screening device according to claim 6, characterized in that: The screening module uses the microscopic image under natural light at the same position as the sample to call all single fluorescent labeling layers, compare them with known cell fluorescence reactions, and perform cell classification; the cell classification includes known target cells, undetermined target cells, and non-target cells.

8. A screening method based on the cell fluorescence microscopic image scanning and target cell marker screening device according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. placing a slide carrying a sample to be tested, wherein the sample to be tested includes target cells and non-target cells, on a motion platform; S2, controlling the excitation unit to emit excitation light of different wavelengths through the excitation module to sequentially illuminate the sample to be tested, and receiving the fluorescence image generated by the sample under each excitation light through the imaging camera in the image acquisition system, and simultaneously receiving the natural light image captured by the preview camera, to generate a cell fluorescence microscopic image under multiple excitation lights; S3, extracting the fluorescence signals of target cells from the cell fluorescence microscopy image of the multiple excitation lights and marking them through a marking module in the image processing system; S4. Using a screening module, based on microscopic images of the same location under natural light, all single fluorescent labeling layers are called up, compared with known cell fluorescence responses, and cell classification is performed; the cell classification includes known target cells, undetermined target cells, and non-target cells.

9. The screening method according to claim 8, characterized in that The step S3 comprises: S31, extracting, by an interception unit, a single fluorescence image under each single excitation light condition from the cell fluorescence microscopic image under multiple excitation lights; S32, indexing the fluorescence signal at each spatial position in the single fluorescence image by an indexing unit according to the wavelength of the fluorescence signal; S33, superimposing all the labeled fluorescence images under the single excitation light conditions onto the natural light image at the same coordinate position to form a multi-light source superimposed image with labeling information.

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

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