技术领域Technical Field
本发明涉及成像流式细胞检测与计数技术领域，具体而言，尤其涉及一种基于双向背景差分法的成像流式细胞粘连计数与活性检测方法、装置。The present invention relates to the technical field of imaging flow cell detection and counting, and in particular to an imaging flow cell adhesion counting and activity detection method and device based on a two-way background difference method.
背景技术Background technique
单细胞计数和成像是医学、环境科学、生物学等相关领域的关键技术，在疾病预测、病情诊断、细胞培养、环境监测等方面具有重要应用价值，细胞活性检测也是进行细胞生物学实验的一项重要指标，是评价细胞培养、细胞毒性及生理研究的基础。在医学领域，细胞计数具有非常重要的临床意义，如淋巴细胞，成人淋巴细胞约占白细胞的1/4，是人体重要的免疫活性细胞，淋巴细胞计数增多见于病毒性感染、结核病、百日咳、传染性单核细胞增多症、传染性淋巴细胞增多症、淋巴细胞白血病、淋巴肉瘤等。淋巴细胞计数减低可见于细胞免疫缺陷病、某些传染病的急性期、放射病、应用肾上腺皮质激素、抗淋巴细胞球蛋白治疗、淋巴细胞减少症、免疫缺陷病、丙种球蛋白缺乏症等等；再如常见的血红细胞，它是人体中最多的一类细胞，主要功能是通过细胞内含有的血红蛋白进行氧气于二氧化碳的交换，红细胞计数增多，多见于真性红细胞增多症、脱水、先天性心脏病等，红细胞计数降低多见于再生障碍性贫血、缺血性贫血、白血病等。因此，对一些人体关键细胞的检测在医学领域具有重大临床意义。Single cell counting and imaging are key technologies in medicine, environmental science, biology and other related fields. They have important application value in disease prediction, disease diagnosis, cell culture, environmental monitoring, etc. Cell activity detection is also an important indicator for cell biology experiments and the basis for evaluating cell culture, cytotoxicity and physiological research. In the medical field, cell counting has very important clinical significance. For example, lymphocytes account for about 1/4 of white blood cells in adults and are important immune active cells in the human body. Increased lymphocyte counts are seen in viral infections, tuberculosis, whooping cough, infectious mononucleosis, infectious lymphocytosis, lymphocytic leukemia, lymphosarcoma, etc. Decreased lymphocyte counts can be seen in cellular immunodeficiency diseases, the acute phase of certain infectious diseases, radiation sickness, the use of adrenocortical hormones, antilymphocyte globulin therapy, lymphocytopenia, immunodeficiency diseases, agammaglobulin deficiency, etc. Another example is common red blood cells, which are the most numerous type of cells in the human body. Their main function is to exchange oxygen and carbon dioxide through the hemoglobin contained in the cells. Increased red blood cell counts are often seen in polycythemia vera, dehydration, congenital heart disease, etc., and decreased red blood cell counts are often seen in aplastic anemia, ischemic anemia, leukemia, etc. Therefore, the detection of some key human cells has great clinical significance in the medical field.
在细胞培养方面，对细胞的计数和活性检测非常关键，如酵母细胞的培养，几千年来，酵母一直被用来制造啤酒、面包和葡萄酒。在现代，它已成为生产燃料、化学品和药物(如胰岛素、人血清白蛋白)以及肝炎病毒和人乳头瘤病毒疫苗的商业主力军。酵母也被设计成工业规模的化学品(如琥珀酸、乳酸、白藜芦醇)和先进的生物燃料(如异丁醇)，对酵母细胞计数和快速检测并对酵母菌进行拍照和算法分析，为酵母细胞生长情况提供数据基础，可以有效避免发酵失败造成的经济损失，因此对酵母细胞进行计数和活性检测具有重大意义。In terms of cell culture, counting and activity detection of cells are very critical, such as the culture of yeast cells. For thousands of years, yeast has been used to make beer, bread and wine. In modern times, it has become a commercial workhorse in the production of fuels, chemicals and drugs (such as insulin, human serum albumin), as well as hepatitis virus and human papillomavirus vaccines. Yeast has also been engineered into industrial-scale chemicals (such as succinic acid, lactic acid, resveratrol) and advanced biofuels (such as isobutanol). Counting and rapid detection of yeast cells, taking pictures of yeast and performing algorithmic analysis provide a data basis for yeast cell growth, which can effectively avoid economic losses caused by fermentation failures. Therefore, counting and activity detection of yeast cells is of great significance.
目前的细胞计数检测方法有：人工显微计数法，库尔特细胞计数法，基于图像的荧光计数法；人工显微计数法作为传统计数方法，一直是一件繁琐而费时的工作，操作人员观察时间久导致眼疲劳，进而可能引入计数误差。而库尔特细胞计数法虽然可行，但该方法无法获取细胞的形态学信息，如形状、尺寸等；基于图像的荧光计数法，虽然可以获取到细胞图像，但是通量低，细胞容易聚集；公开的商用图像流式细胞仪如Millipore公司的ImageStream和FlowSight，其光路和空间结构及其复杂，制造成本也比较高昂，限制了它的广泛使用。The current cell counting detection methods include: manual microscopic counting, Coulter cell counting, and image-based fluorescence counting. As a traditional counting method, manual microscopic counting has always been a tedious and time-consuming task. The operator's long observation time leads to eye fatigue, which may introduce counting errors. Although the Coulter cell counting method is feasible, it cannot obtain morphological information of cells, such as shape and size. Although the image-based fluorescence counting method can obtain cell images, it has low throughput and cells are prone to aggregation. The publicly available commercial image flow cytometers such as Millipore's ImageStream and FlowSight have extremely complex optical paths and spatial structures, and the manufacturing cost is also relatively high, which limits its widespread use.
综合以上分析，现有的一些计数检测方法具有耗时耗力、功能不全、通量低、价格昂贵等缺点。Based on the above analysis, some existing counting detection methods have the disadvantages of being time-consuming and labor-intensive, incomplete in function, low in throughput, and expensive.
发明内容Summary of the invention
根据上述提出现有检测方法具有耗时耗力、功能不全、通量低、价格昂贵等技术问题，而提供一种基于双向背景差分法的成像流式细胞粘连计数与活性检测方法、装置。本发明可以实现对微藻细胞、293T淋巴细胞、小鼠细胞等多种细胞的计数和活性判断，应用范围广泛，鉴定指标稳定。According to the above technical problems that the existing detection methods are time-consuming, labor-intensive, incomplete, low-throughput, and expensive, a method and device for imaging flow cell adhesion counting and activity detection based on a two-way background difference method is provided. The present invention can realize the counting and activity judgment of various cells such as microalgae cells, 293T lymphocytes, and mouse cells, with a wide range of applications and stable identification indicators.
本发明采用的技术手段如下：The technical means adopted by the present invention are as follows:
一种基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，包括如下步骤：An imaging flow cytometry adhesion counting and activity detection method based on a two-way background difference method comprises the following steps:
S1、获取待测样品的明视场图像和细胞荧光图像及空测时的背景图像，并对细胞荧光图像进行二值化处理；S1, obtaining a bright field image and a cell fluorescence image of the sample to be tested and a background image during an empty test, and performing a binarization process on the cell fluorescence image;
S2、利用双向背景差分法提取目标轮廓差分图像和目标中心亮斑差分图像，并对目标轮廓差分图像和目标中心亮斑差分图像进行融合及二值化处理，确定运动目标的位置；S2, using a bidirectional background difference method to extract a target contour difference image and a target center bright spot difference image, and fusing and binarizing the target contour difference image and the target center bright spot difference image to determine the position of the moving target;
S3、对二值化处理后的图像进行形态学闭运算，再进行形态学开运算；S3, performing a morphological closing operation on the image after the binary processing, and then performing a morphological opening operation;
S4、对二值图像矩阵进行遍历，实现待测细胞的数量计算以及待测细胞种类的识别和分类。S4. Traverse the binary image matrix to calculate the number of cells to be tested and identify and classify the types of cells to be tested.
进一步地，所述步骤S2的具体实现过程如下：Furthermore, the specific implementation process of step S2 is as follows:
S21、对所述明视场图像与所述背景图像进行差分，得到含有目标中心亮斑的正向差分图像Img1；S21, performing a difference between the bright field image and the background image to obtain a forward difference image Img1 containing a bright spot at the center of the target;
S22、对所述背景图像与所述明视场图像做差分，得到含有目标轮廓的反向差分图像Img2；S22, performing a difference between the background image and the bright field image to obtain a reverse difference image Img2 containing a target contour;
S23、根据公式ImgF(i,j)＝Img1(i,j)×μ1+Img2(i,j)×μ2+B对正向差分图像Img1和反向差分图像Img2进行图像融合，其中，ImgF表示计算后的融合图像，μ1、μ2为加权系数，(i,j)为图像像素点的坐标，B为亮度调节量；S23, performing image fusion on the forward difference image Img1 and the reverse difference image Img2 according to the formula ImgF(i,j)=Img1(i,j)×μ1+Img2(i,j)×μ2+B, wherein ImgF represents the calculated fused image, μ1 and μ2 are weighting coefficients, (i,j) is the coordinate of the image pixel point, and B is the brightness adjustment amount;
S24、对融合图像ImgF进行二值化处理，确定二值化融合图像。S24, binarizing the fused image ImgF to determine a binary fused image.
进一步地，所述步骤S3的具体实现过程如下：Furthermore, the specific implementation process of step S3 is as follows:
S31、根据公式对所述二值化融合图像进行闭运算处理，填充所述二值化融合图像中出现的细小孔洞缝隙；S31, according to the formula Performing a closing operation on the binary fused image to fill small holes and gaps in the binary fused image;
S32、根据公式对所述二值化融合图像进行开运算处理，去掉所述二值化融合图像中细小的噪声颗粒。S32, according to the formula An opening operation is performed on the binary fused image to remove fine noise particles in the binary fused image.
进一步地，所述步骤S4的具体实现过程如下：Furthermore, the specific implementation process of step S4 is as follows:
S41、对二值图像矩阵进行遍历，得到图像中所有细胞的面积和AreaS；S41, traverse the binary image matrix to obtain the area and AreaS of all cells in the image;
S42、确定单个细胞的面积为二值图像中连通域面积的中值m，将细胞的面积和AreaS除以单个细胞的面积m，计算得到细胞数量；S42, determining the area of a single cell as the median m of the area of the connected domain in the binary image, dividing the area of the cell and AreaS by the area m of the single cell, and calculating the number of cells;
S43、对二值图像矩阵进行遍历，得到细胞的周长L,通过公式计算获取细胞的圆度信息；S43, traverse the binary image matrix to obtain the cell perimeter L, through the formula Calculate and obtain the roundness information of cells;
S44、通过公式计算得到细胞的粒径，其中S为细胞轮廓区域的面积，L为细胞轮廓的周长，实现对待测细胞种类的识别和分类。S44, through the formula The cell size is calculated, where S is the area of the cell outline and L is the perimeter of the cell outline, to achieve identification and classification of the cell types to be tested.
本发明还提供了一种基于上述基于双向背景差分法的成像流式细胞粘连计数与活性检测方法的基于双模态成像流式细胞检测装置，包括：依次连接的光源模块、微流控芯片模块，液压驱动系统、光检测组件、图像采集组件和图像处理组件；The present invention also provides a dual-modality imaging flow cell detection device based on the above-mentioned imaging flow cell adhesion counting and activity detection method based on the bidirectional background difference method, comprising: a light source module, a microfluidic chip module, a hydraulic drive system, a light detection component, an image acquisition component and an image processing component connected in sequence;
光源模块发出蓝光，光源照射细胞样品激发被碘化丙啶染色的细胞发出荧光，照射未被碘化丙啶染色的样品发出反射光；两种波长的光经过光检测组件传至图像采集组件得到细胞荧光图像和明视场图像，图像处理组件对图像进行处理实现细胞粘连计数与活性检测。The light source module emits blue light, which illuminates the cell sample to excite the cells stained with propidium iodide to emit fluorescence, and illuminates the sample not stained with propidium iodide to emit reflected light; the two wavelengths of light are transmitted to the image acquisition component through the light detection component to obtain the cell fluorescence image and bright field image, and the image processing component processes the image to realize cell adhesion counting and activity detection.
进一步地，所述微流控芯片模块包括样品孔、鞘液孔、废液孔、检测区，所述微流控芯片模块上还凹刻连通样品孔、鞘液孔、废液孔、检测区的流道。Furthermore, the microfluidic chip module comprises a sample hole, a sheath fluid hole, a waste fluid hole, and a detection area, and a flow channel connecting the sample hole, the sheath fluid hole, the waste fluid hole, and the detection area is also concavely engraved on the microfluidic chip module.
进一步地，所述液压驱动系统包括正压压力源、负压压力源和电磁阀组，用于控制鞘液和待测样品形成样品，流经流道且连续流过所述检测区。Furthermore, the hydraulic drive system includes a positive pressure source, a negative pressure source and a solenoid valve group, which are used to control the sheath fluid and the sample to be tested to form a sample, flow through the flow channel and continuously flow through the detection area.
进一步地，所述光检测组件包括双光路镜头、二向色镜和滤光片；所述图像采集组件为CMOS传感器；所述图像处理组件为Jetson Nano处理器。Furthermore, the light detection component includes a dual-light path lens, a dichroic mirror and a filter; the image acquisition component is a CMOS sensor; and the image processing component is a Jetson Nano processor.
本发明还提供了一种存储介质，其特征在于，所述存储介质包括存储的程序，其中，所述程序运行时，执行上述基于双向背景差分法的成像流式细胞粘连计数与活性检测方法。The present invention also provides a storage medium, characterized in that the storage medium includes a stored program, wherein when the program is run, the above-mentioned imaging flow cell adhesion counting and activity detection method based on the two-way background difference method is executed.
本发明还提供了电子装置，包括存储器、处理器及存储在所述存储器上并可在所述处理器上运行的计算机程序，所述处理器通过所述计算机程序运行执行上述基于双向背景差分法的成像流式细胞粘连计数与活性检测方法。The present invention also provides an electronic device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the above-mentioned imaging flow cell adhesion counting and activity detection method based on the bidirectional background difference method through the operation of the computer program.
较现有技术相比，本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:
1、本发明提供的基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，能够实现对微藻细胞、293T淋巴细胞、小鼠细胞等多种细胞的计数和活性判断，应用范围广泛，鉴定指标稳定。1. The imaging flow cytometry adhesion counting and activity detection method based on the two-way background difference method provided by the present invention can realize the counting and activity judgment of various cells such as microalgae cells, 293T lymphocytes, mouse cells, etc., has a wide range of applications and stable identification indicators.
2、本发明提供的基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，采用双向背景差分法并对差分得到的图像进行图像融合，有效解决了噪声对细胞计数的影响；2. The imaging flow cell adhesion counting and activity detection method based on the bidirectional background difference method provided by the present invention adopts the bidirectional background difference method and performs image fusion on the differential images, which effectively solves the influence of noise on cell counting;
3、本发明提供的基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，将连通域的面积和作为图像中所有细胞的面积和AreaS，将二值图像中连通域面积的中值作为单个细胞的面积m，将细胞面积和AreaS除以单个细胞面积m计算得到细胞数，有效解决了细胞粘连对细胞计数的影响。3. The imaging flow cytometry adhesion counting and activity detection method based on the bidirectional background difference method provided by the present invention takes the area sum of the connected domain as the area sum AreaS of all cells in the image, takes the median of the area of the connected domain in the binary image as the area m of a single cell, and calculates the cell number by dividing the cell area sum AreaS by the single cell area m, thereby effectively solving the influence of cell adhesion on cell counting.
4、本发明提供的基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，通过对二值图像遍历得到细胞面积和周长信息，计算得出细胞的圆度和粒径，实现对待测细胞种类的识别和分类；4. The imaging flow cell adhesion counting and activity detection method based on the bidirectional background difference method provided by the present invention obtains the cell area and perimeter information by traversing the binary image, calculates the roundness and particle size of the cell, and realizes the identification and classification of the cell types to be tested;
5、本发明提供的基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，其双模态成像流式细胞检测装置可同时获取检测样品的细胞荧光图像和明视场图像，具有检测的效率高和准确性高的优点。5. The imaging flow cytometric adhesion counting and activity detection method based on the bidirectional background difference method provided by the present invention has a dual-modality imaging flow cytometric detection device that can simultaneously obtain the cell fluorescence image and bright field image of the detection sample, and has the advantages of high detection efficiency and high accuracy.
基于上述理由本发明可在成像流式细胞检测与计数等领域广泛推广。Based on the above reasons, the present invention can be widely promoted in the fields of imaging flow cytometry detection and counting.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图做以简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
图1为本发明方法流程图。FIG1 is a flow chart of the method of the present invention.
图2为本发明装置结构示意图。FIG. 2 is a schematic diagram of the structure of the device of the present invention.
图3为本发明装置中微流控芯片模块结构示意图。FIG. 3 is a schematic diagram of the structure of the microfluidic chip module in the device of the present invention.
图中：1、光源模块；2、微流控芯片模快；3、液压驱动系统；4、光检测组件模块；5、图像采集组件；6、图像处理组件；7、样品孔；8、鞘液孔；9、废液孔；10、检测区。In the figure: 1. light source module; 2. microfluidic chip module; 3. hydraulic drive system; 4. light detection component module; 5. image acquisition component; 6. image processing component; 7. sample hole; 8. sheath liquid hole; 9. waste liquid hole; 10. detection area.
具体实施方式Detailed ways
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
需要说明的是，本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.
如图1所示，本发明提供了一种基于双向背景差分法的成像流式细胞粘连计数与活性检测方法，包括如下步骤：As shown in FIG1 , the present invention provides an imaging flow cytometry adhesion counting and activity detection method based on a two-way background difference method, comprising the following steps:
S1、获取待测样品的明视场图像和细胞荧光图像及空测时的背景图像，并对细胞荧光图像进行二值化处理；S1, obtaining a bright field image and a cell fluorescence image of the sample to be tested and a background image during an empty test, and performing a binarization process on the cell fluorescence image;
S2、利用双向背景差分法提取目标轮廓差分图像和目标中心亮斑差分图像，并对目标轮廓差分图像和目标中心亮斑差分图像进行融合及二值化处理，确定运动目标的位置；S2, using a bidirectional background difference method to extract a target contour difference image and a target center bright spot difference image, and fusing and binarizing the target contour difference image and the target center bright spot difference image to determine the position of the moving target;
具体实施时，作为本发明优选的实施方式，所述步骤S2的具体实现过程如下：In specific implementation, as a preferred embodiment of the present invention, the specific implementation process of step S2 is as follows:
S21、对所述明视场图像与所述背景图像进行差分，得到含有目标中心亮斑的正向差分图像Img1；S21, performing a difference between the bright field image and the background image to obtain a forward difference image Img1 containing a bright spot at the center of the target;
S22、对所述背景图像与所述明视场图像做差分，得到含有目标轮廓的反向差分图像Img2；S22, performing a difference between the background image and the bright field image to obtain a reverse difference image Img2 containing a target contour;
S23、根据公式ImgF(i,j)＝Img1(i,j)×μ1+Img2(i,j)×μ2+B对正向差分图像Img1和反向差分图像Img2进行图像融合，其中，ImgF表示计算后的融合图像，μ1、μ2为加权系数，(i,j)为图像像素点的坐标，B为亮度调节量；S23, performing image fusion on the forward difference image Img1 and the reverse difference image Img2 according to the formula ImgF(i,j)=Img1(i,j)×μ1+Img2(i,j)×μ2+B, wherein ImgF represents the calculated fused image, μ1 and μ2 are weighting coefficients, (i,j) is the coordinate of the image pixel point, and B is the brightness adjustment amount;
S24、对融合图像ImgF进行二值化处理，确定二值化融合图像。S24, binarizing the fused image ImgF to determine a binary fused image.
S3、对二值化处理后的图像进行形态学闭运算，再进行形态学开运算；S3, performing a morphological closing operation on the binarized image, and then performing a morphological opening operation;
具体实施时，作为本发明优选的实施方式，所述步骤S3的具体实现过程如下：In specific implementation, as a preferred embodiment of the present invention, the specific implementation process of step S3 is as follows:
S31、根据公式对所述二值化融合图像进行闭运算处理，填充所述二值化融合图像中出现的细小孔洞缝隙；S31, according to the formula Performing a closing operation on the binary fused image to fill small holes and gaps in the binary fused image;
S32、根据公式对所述二值化融合图像进行开运算处理，去掉所述二值化融合图像中细小的噪声颗粒。S32, according to the formula An opening operation is performed on the binary fused image to remove fine noise particles in the binary fused image.
S4、对二值图像矩阵进行遍历，实现待测细胞的数量计算以及待测细胞种类的识别和分类。S4. Traverse the binary image matrix to calculate the number of cells to be tested and identify and classify the types of cells to be tested.
具体实施时，作为本发明优选的实施方式，所述步骤S4的具体实现过程如下：In specific implementation, as a preferred embodiment of the present invention, the specific implementation process of step S4 is as follows:
S41、对二值图像矩阵进行遍历，得到图像中所有细胞的面积和AreaS；S41, traverse the binary image matrix to obtain the area and AreaS of all cells in the image;
S42、确定单个细胞的面积为二值图像中连通域面积的中值m，将细胞的面积和AreaS除以单个细胞的面积m，计算得到细胞数量；S42, determining the area of a single cell as the median m of the area of the connected domain in the binary image, dividing the area of the cell and AreaS by the area m of the single cell, and calculating the number of cells;
S43、对二值图像矩阵进行遍历，得到细胞的周长L,通过公式计算获取细胞的圆度信息；S43, traverse the binary image matrix to obtain the cell perimeter L, through the formula Calculate and obtain the roundness information of cells;
S44、通过公式计算得到细胞的粒径，其中S为细胞轮廓区域的面积，L为细胞轮廓的周长，实现对待测细胞种类的识别和分类。S44, through the formula The cell size is calculated, where S is the area of the cell outline and L is the perimeter of the cell outline, to achieve identification and classification of the cell types to be tested.
如图2所示，本发明提供了一种基于上述基于双向背景差分法的成像流式细胞粘连计数与活性检测方法的基于双模态成像流式细胞检测装置，包括：依次连接的光源模块1、微流控芯片模块2，液压驱动系统3、光检测组件4、图像采集组件5和图像处理组件6。光源模块1发出蓝光，光源照射细胞样品激发被碘化丙啶(PI)染色的细胞发出荧光，照射未被碘化丙啶(PI)染色的样品发出反射光；两种波长的光经过光检测组件4传至图像采集组件5得到细胞荧光图像和明视场图像，图像处理组件6对图像进行处理实现细胞粘连计数与活性检测。As shown in FIG2 , the present invention provides a dual-modality imaging flow cell detection device based on the above-mentioned imaging flow cell adhesion counting and activity detection method based on the bidirectional background difference method, comprising: a light source module 1, a microfluidic chip module 2, a hydraulic drive system 3, a light detection component 4, an image acquisition component 5 and an image processing component 6 connected in sequence. The light source module 1 emits blue light, and the light source irradiates the cell sample to excite the cells stained with propidium iodide (PI) to emit fluorescence, and irradiates the sample not stained with propidium iodide (PI) to emit reflected light; the two wavelengths of light are transmitted to the image acquisition component 5 through the light detection component 4 to obtain a cell fluorescence image and a bright field image, and the image processing component 6 processes the image to realize cell adhesion counting and activity detection.
具体实施时，作为本发明优选的实施方式，如图3所示，所述微流控芯片模块2包括样品孔7、鞘液孔8、废液孔9、检测区10，所述微流控芯片模块2上还凹刻连通样品孔7、鞘液孔8、废液孔9、检测区10的流道。In specific implementation, as a preferred embodiment of the present invention, as shown in Figure 3, the microfluidic chip module 2 includes a sample hole 7, a sheath liquid hole 8, a waste liquid hole 9, and a detection area 10, and the microfluidic chip module 2 is also engraved with a flow channel connecting the sample hole 7, the sheath liquid hole 8, the waste liquid hole 9, and the detection area 10.
具体实施时，作为本发明优选的实施方式，所述液压驱动系统3包括正压压力源、负压压力源和电磁阀组，用于控制鞘液和待测样品形成样品，流经流道且连续流过所述检测区10。In specific implementation, as a preferred embodiment of the present invention, the hydraulic drive system 3 includes a positive pressure source, a negative pressure source and a solenoid valve group, which are used to control the sheath fluid and the sample to be tested to form a sample, flow through the flow channel and continuously flow through the detection area 10.
具体实施时，作为本发明优选的实施方式，所述光检测组件包括双光路镜头、二向色镜和滤光片；所述图像采集组件为CMOS传感器；所述图像处理组件为Jetson Nano处理器。In specific implementation, as a preferred embodiment of the present invention, the light detection component includes a dual-light path lens, a dichroic mirror and a filter; the image acquisition component is a CMOS sensor; and the image processing component is a Jetson Nano processor.
在本申请所提供的实施例中，应该理解到，所揭露的技术内容，可通过其它的方式实现。其中，以上所描述的装置实施例仅仅是示意性的，例如所述单元的划分，可以为一种逻辑功能划分，实际实现时可以有另外的划分方式，例如多个单元或组件可以结合或者可以集成到另一个系统，或一些特征可以忽略，或不执行。另一点，所显示或讨论的相互之间的耦合或直接耦合或通信连接可以是通过一些接口，单元或模块的间接耦合或通信连接，可以是电性或其它的形式。In the embodiments provided in the present application, it should be understood that the disclosed technical content can be implemented in other ways. Among them, the device embodiments described above are only schematic. For example, the division of the units can be a logical function division. There may be other division methods in actual implementation. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of units or modules, which can be electrical or other forms.
所述作为分离部件说明的单元可以是或者也可以不是物理上分开的，作为单元显示的部件可以是或者也可以不是物理单元，即可以位于一个地方，或者也可以分布到多个单元上。可以根据实际的需要选择其中的部分或者全部单元来实现本实施例方案的目的。The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple units. Some or all of the units may be selected according to actual needs to achieve the purpose of the present embodiment.
另外，在本发明实施例中的各功能单元可以集成在一个处理单元中，也可以是各个单元单独物理存在，也可以两个或两个以上单元集成在一个单元中。上述集成的单元既可以采用硬件的形式实现，也可以采用软件功能单元的形式实现。In addition, each functional unit in the embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above integrated unit may be implemented in the form of hardware or in the form of software functional units.
所述集成的单元如果以软件功能单元的形式实现并作为独立的产品销售或使用时，可以存储在一个计算机可读取存储介质中。基于这样的理解，本发明的技术方案本质上或者说对现有技术做出贡献的部分或者该技术方案的全部或部分可以以软件产品的形式体现出来，该计算机软件产品存储在一个存储介质中，包括若干指令用以使得一台计算机设备(可为个人计算机、服务器或者网络设备等)执行本发明实施例所述方法的全部或部分步骤。而前述的存储介质包括：U盘、只读存储器(ROM，Read-Only Memory)、随机存取存储器(RAM，Random Access Memory)、移动硬盘、磁碟或者光盘等各种可以存储程序代码的介质。If the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or all or part of the technical solution can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including a number of instructions for a computer device (which can be a personal computer, a server or a network device, etc.) to perform all or part of the steps of the method described in the embodiment of the present invention. The aforementioned storage medium includes: U disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), mobile hard disk, magnetic disk or optical disk and other media that can store program codes.
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
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