CN111399208B - Implementation method, microscope and storage medium for focused photographing of biofluorescence samples - Google Patents
Implementation method, microscope and storage medium for focused photographing of biofluorescence samplesDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明涉及光学聚焦技术领域，尤指一种生物荧光样本的聚焦拍摄实现方法、显微镜和存储介质。The invention relates to the technical field of optical focusing, in particular to a method, microscope and storage medium for realizing focusing and shooting of biological fluorescence samples.
背景技术Background technique
荧光染色是一种应用于医学研究已经临床检验的染色剂，它可以对目标进行靶向染色标记，从而有利于后续目标的识别和检测。Fluorescent staining is a dye that has been clinically tested in medical research. It can target and label targets, which is beneficial to the identification and detection of subsequent targets.
使用显微镜对用荧光染色试剂制备的生物样本进行拍摄时，所需的曝光时间远远多于明场下对图像采集时所需的曝光时间，其高达几百毫秒，甚至几十秒，同时荧光条件下的背景多为黑色，纹理较少，使用清晰度评价函数进行图像的清晰度评价时难度，也远远大于在明场条件下进行评判，易发生聚焦失败的情况，所以直接对荧光涂片进行自动或者手动聚焦都将耗费大量时间，从而大大降低了采图效率。When using a microscope to photograph biological samples prepared with fluorescent staining reagents, the exposure time required is far more than the exposure time required for image acquisition in brightfield, which is as high as hundreds of milliseconds or even tens of seconds. Under the condition, the background is mostly black and has few textures. It is much more difficult to evaluate the sharpness of the image using the sharpness evaluation function than it is to evaluate it under bright field conditions, and it is prone to focus failure. It will take a lot of time to focus automatically or manually, which will greatly reduce the efficiency of image acquisition.
同时，在激发光长时间的照射下，生物样本上的荧光物质易发生衰减和猝灭，因此，若在聚焦过程中花费过多时间，使得目标因荧光物质的淬灭而被噪声淹没，将妨碍后续人员的观察或计算机的图像处理，因此如何快速在当前视野下对生物荧光样本进行聚焦扫描十分必要。At the same time, the fluorescent substance on the biological sample is prone to decay and quenching under the long-term irradiation of the excitation light. Therefore, if too much time is spent in the focusing process, the target will be overwhelmed by noise due to the quenching of the fluorescent substance, and the It hinders the observation of the follow-up personnel or the image processing of the computer, so it is necessary to quickly focus and scan the biofluorescence sample in the current field of view.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种生物荧光样本的聚焦拍摄实现方法、显微镜和存储介质，实现对生物荧光样本进行聚焦扫描，提升对生物荧光样本的图像成像质量和效果。The purpose of the present invention is to provide a method, microscope and storage medium for focusing and photographing biofluorescence samples, so as to realize focused scanning of biofluorescence samples and improve the image imaging quality and effect of biofluorescence samples.
本发明提供的技术方案如下：The technical scheme provided by the present invention is as follows:
本发明提供一种生物荧光样本的聚焦拍摄实现方法，包括步骤：The present invention provides a method for realizing focusing and photographing of biological fluorescence samples, comprising the steps of:
在明场条件下点亮普通光源，并控制摄像头对载物台所放置生物荧光样本的荧光涂片区域上任一涂片预选点进行自动聚焦直至聚焦成功；所述涂片预选点数量为至少三个；Light up a common light source under brightfield conditions, and control the camera to automatically focus on any smear preselected point on the fluorescent smear area of the biological fluorescence sample placed on the stage until the focus is successful; the number of smear preselected points is at least three ;
获取聚焦成功时的聚焦参数，根据所述聚焦参数进行平面拟合得到基准平面；Acquiring focusing parameters when focusing is successful, and performing plane fitting according to the focusing parameters to obtain a reference plane;
切换所述普通光源为荧光光源，根据预设扫描点的坐标和所述基准平面计算得到对应的目标移动高度；Switching the common light source to a fluorescent light source, and calculating the corresponding target moving height according to the coordinates of the preset scanning point and the reference plane;
根据所述目标移动高度按照预设扫描策略，控制所述摄像头进行自动扫描采集目标图像。According to the moving height of the target, according to a preset scanning strategy, the camera is controlled to perform automatic scanning to collect target images.
进一步的，所述在明场条件下点亮普通光源，并控制摄像头对载物台所放置生物荧光样本的荧光涂片区域上任一涂片预选点进行自动聚焦直至聚焦成功包括步骤：Further, the step of lighting up a common light source under brightfield conditions and controlling the camera to automatically focus on any pre-selected point of the smear on the fluorescent smear area of the biological fluorescence sample placed on the stage until the focusing is successful includes the steps:
在明场条件下点亮普通光源，根据预设聚焦策略控制所述摄像头移动对所述涂片预选点进行自动聚焦，直至所述摄像头输入的图像清晰度数值最大时确定聚焦成功。A common light source is lit under bright field conditions, and the camera is controlled to move according to a preset focusing strategy to automatically focus on the pre-selected points of the smear, until the camera inputs a maximum image clarity value to determine that the focusing is successful.
进一步的，所述在明场条件下点亮普通光源之后，所述根据预设聚焦策略控制所述摄像头移动对所述涂片预选点进行自动聚焦之前包括步骤：Further, after the common light source is turned on under bright field conditions, the steps of controlling the camera to move according to the preset focusing strategy to automatically focus on the smear preselected point include:
控制显微镜头移动和/或载物台移动，使得上述任一涂片预选点位于所述摄像头镜头的正下方。Control the movement of the microscope head and/or the movement of the stage, so that any of the above-mentioned smear preselected points are located directly below the camera lens.
进一步的，所述根据预设聚焦策略控制所述摄像头移动对所述涂片预选点进行自动聚焦，直至所述摄像头输入的图像的清晰度数值最大时确定聚焦成功包括步骤：Further, controlling the camera to move to automatically focus on the smear pre-selected points according to a preset focusing strategy, and determining that the focusing is successful until the image input by the camera has a maximum sharpness value includes the steps of:
根据预设聚焦策略控制所述摄像头移动靠向所述涂片预选点，并控制所述摄像头的镜头在测量量程中进行聚焦；Control the camera to move toward the preselected point of the smear according to the preset focusing strategy, and control the lens of the camera to focus in the measurement range;
在整个聚焦过程中根据清晰度评价函数计算输入图像的清晰度，在清晰度停止增大时确定聚焦成功。During the entire focusing process, the sharpness of the input image is calculated according to the sharpness evaluation function, and it is determined that the focusing is successful when the sharpness stops increasing.
进一步的，所述获取聚焦成功时的聚焦参数，根据所述聚焦参数进行平面拟合得到基准平面包括步骤：Further, obtaining the focusing parameters when the focusing is successful, and performing plane fitting according to the focusing parameters to obtain the reference plane includes the steps of:
获取聚焦成功时所述载物台相对于预设原点在高度方向上的目标高度值，以及所有涂片预选点相对于所述预设原点的预选点坐标；Obtain the target height value of the stage relative to the preset origin in the height direction when the focusing is successful, and the preselected point coordinates of all smear preselected points relative to the preset origin;
根据所述目标高度值和预选点坐标拟合得到近似焦平面，确定经过所述涂片预选点最多的近似焦平面为所述基准平面。The approximate focal plane is obtained by fitting the target height value and the coordinates of the preselected points, and the approximate focal plane passing through the most preselected points of the smear is determined as the reference plane.
进一步的，所述获取聚焦成功时的聚焦参数，根据所述聚焦参数进行平面拟合得到基准平面包括步骤：Further, obtaining the focusing parameters when the focusing is successful, and performing plane fitting according to the focusing parameters to obtain the reference plane includes the steps of:
获取聚焦成功时所述载物台相对于预设原点在高度方向上的目标高度值，以及所有涂片预选点相对于所述预设原点的预选点坐标；Obtain the target height value of the stage relative to the preset origin in the height direction when the focusing is successful, and the preselected point coordinates of all smear preselected points relative to the preset origin;
从所述预选点坐标中筛选根据预设异常检验算法，查找并剔除所述涂片预选点中的异常涂片预选点，根据目标高度值以及在剔除后结果中任选三个涂片预选点拟合得到基准平面。Screening from the coordinates of the preselected points according to the preset abnormality inspection algorithm, finding and eliminating the abnormal smear preselected points in the smear preselected points, and selecting three smear preselected points according to the target height value and the result after the elimination Fitted to get the datum plane.
进一步的，所述切换所述普通光源为荧光光源，根据预设扫描点的坐标和所述基准平面计算得到对应的目标移动高度包括步骤：Further, the switching of the common light source to a fluorescent light source, and calculating and obtaining the corresponding moving height of the target according to the coordinates of the preset scanning point and the reference plane includes the steps of:
控制关闭所述普通光源且打开所述荧光光源并插入滤光片；Controlling to turn off the common light source and turn on the fluorescent light source and insert a filter;
控制显微镜头移动和/或载物台移动，使得所述载物台所放置生物荧光样本的荧光涂片区域上的任一预设扫描点位于所述摄像头的正下方；Controlling the movement of the microscope head and/or the movement of the stage, so that any preset scanning point on the fluorescent smear area of the biological fluorescence sample placed on the stage is located directly below the camera;
根据所述预设扫描点的坐标，以及所述基准平面对应的平面表达式计算得到所述预设扫描点对应的目标移动高度。The target moving height corresponding to the preset scanning point is obtained by calculating according to the coordinates of the preset scanning point and the plane expression corresponding to the reference plane.
进一步的，所述根据所述目标移动高度按照预设扫描策略，控制所述摄像头进行自动扫描采集目标图像包括步骤：Further, according to the moving height of the target, according to the preset scanning strategy, controlling the camera to automatically scan and collect the target image includes the steps:
在距离当前预设扫描点所对应目标移动高度的预设步长范围内，控制所述摄像头和/或载物台进行往复移动扫描采集包括所述当前预设扫描点对应的图像；Control the camera and/or the stage to perform reciprocating scanning scanning to collect images including the image corresponding to the current preset scanning point within a preset step size range from the target moving height corresponding to the current preset scanning point;
根据清晰度评价函数计算所采集图像的清晰度，确定清晰度数值最大的图像为所述当前预设扫描点对应的目标图像；Calculate the sharpness of the collected image according to the sharpness evaluation function, and determine that the image with the largest sharpness value is the target image corresponding to the current preset scanning point;
切换至下一预设扫描点进行聚焦扫描，直至采集到所有预设扫描点对应的目标图像为止。Switch to the next preset scanning point for focused scanning until all target images corresponding to the preset scanning points are acquired.
本发明还提供一种显微镜，包括处理器、存储器以及存储在所述存储器中并可在所述处理器上运行的计算机程序，所述处理器，用于执行所述存储器上所存放的计算机程序，实现如所述的生物荧光样本的聚焦拍摄实现方法所执行的操作。The present invention also provides a microscope, comprising a processor, a memory, and a computer program stored in the memory and executable on the processor, the processor being used to execute the computer program stored in the memory , to realize the operations performed by the method for realizing the focus shooting of the biofluorescence sample.
本发明还提供一种存储介质，所述存储介质中存储有至少一条指令，所述指令由处理器加载并执行以实现如所述的生物荧光样本的聚焦拍摄实现方法所执行的操作。The present invention also provides a storage medium, wherein at least one instruction is stored in the storage medium, and the instruction is loaded and executed by a processor to implement the operations performed by the method for implementing the focused photographing of a biological fluorescence sample.
通过本发明提供的一种生物荧光样本的聚焦拍摄实现方法、显微镜和存储介质，能够对生物荧光样本进行聚焦扫描，提升对生物荧光样本的图像成像质量和效果。The method, microscope and storage medium for focusing and photographing a biofluorescence sample provided by the present invention can focus and scan the biofluorescence sample, and improve the image imaging quality and effect of the biofluorescence sample.
附图说明Description of drawings
下面将以明确易懂的方式，结合附图说明优选实施方式，对一种生物荧光样本的聚焦拍摄实现方法、显微镜和存储介质的上述特性、技术特征、优点及其实现方式予以进一步说明。In the following, the preferred embodiments will be described in a clear and easy-to-understand manner with reference to the accompanying drawings, and further description will be given of the above-mentioned characteristics, technical features, advantages and implementation methods of a method for realizing focused photography of a biofluorescence sample, a microscope and a storage medium.
图1是本发明一种生物荧光样本的聚焦拍摄实现方法的一个实施例的流程图；FIG. 1 is a flow chart of an embodiment of a method for realizing focused photography of a biofluorescence sample of the present invention;
图2是本发明一种生物荧光样本的聚焦拍摄实现方法的另一个实施例的流程图；FIG. 2 is a flow chart of another embodiment of a method for realizing focused photography of a biofluorescence sample according to the present invention;
图3是本发明一种生物荧光样本的聚焦拍摄实现方法的另一个实施例的流程图；FIG. 3 is a flow chart of another embodiment of a method for realizing focused photography of a biofluorescence sample according to the present invention;
图4是本发明一种生物荧光样本的聚焦拍摄实现方法的另一个实施例的流程图；FIG. 4 is a flow chart of another embodiment of a method for implementing focused photography of a biofluorescence sample according to the present invention;
图5是本发明一种生物荧光样本的聚焦拍摄实现方法的另一个实施例的流程图；FIG. 5 is a flowchart of another embodiment of a method for realizing focused photography of a biofluorescence sample of the present invention;
图6是本发明一种显微镜的一个实施例的结构示意图；6 is a schematic structural diagram of an embodiment of a microscope of the present invention;
图7是本发明一种显微镜自动聚焦结果的示意图；Fig. 7 is the schematic diagram of a kind of microscope automatic focusing result of the present invention;
图8是本发明一种样本的近似焦平面的示意图；8 is a schematic diagram of an approximate focal plane of a sample of the present invention;
图9是本发明载物台移动将选定的扫描点移动到摄像头正下方的示意图。FIG. 9 is a schematic diagram of the movement of the stage of the present invention to move the selected scanning point directly below the camera.
具体实施方式Detailed ways
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，下面将对照附图说明本发明的具体实施方式。显而易见地，下面描述中的附图仅仅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的前提下，还可以根据这些附图获得其他的附图，并获得其他的实施方式。In order to more clearly describe the embodiments of the present invention or the technical solutions in the prior art, the specific embodiments of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative efforts, and obtain other implementations.
为使图面简洁，各图中只示意性地表示出了与本发明相关的部分，它们并不代表其作为产品的实际结构。另外，以使图面简洁便于理解，在有些图中具有相同结构或功能的部件，仅示意性地绘示了其中的一个，或仅标出了其中的一个。在本文中，“一个”不仅表示“仅此一个”，也可以表示“多于一个”的情形。In order to keep the drawings concise, the drawings only schematically show the parts related to the present invention, and they do not represent its actual structure as a product. In addition, in order to make the drawings concise and easy to understand, in some drawings, only one of the components having the same structure or function is schematically shown, or only one of them is marked. As used herein, "one" not only means "only one", but also "more than one".
本发明的一个实施例，如图1所示，一种生物荧光样本的聚焦拍摄实现方法，包括：An embodiment of the present invention, as shown in FIG. 1 , is a method for realizing focusing and photographing a biofluorescence sample, including:
S100在明场条件下点亮普通光源，并控制摄像头对载物台所放置生物荧光样本的荧光涂片区域上任一涂片预选点进行自动聚焦直至聚焦成功；涂片预选点数量为至少三个；The S100 lights up a common light source under brightfield conditions, and controls the camera to automatically focus on any smear preselected point on the fluorescent smear area of the biofluorescence sample placed on the stage until the focus is successful; the number of smear preselected points is at least three;
具体的，本发明显微镜(全自动镜检平台)包括用于放置生物荧光样本等待测样本的载物台，位于所述载物台下方并用于对待测物进行观测的显微镜头，与显微镜头连接并从显微镜头获取其观测的图像的摄像头，固定在载物台上且用于跟踪载物台的位置坐标的位置传感器。固定在显微镜头上且用于跟踪显微镜头的位置坐标的编码器，以及与显微镜头和载物台连接且用于驱动显微镜头和载物台移动，以使得摄像头或显微镜头观测视野改变的驱动装置。Specifically, the microscope (automatic microscope inspection platform) of the present invention includes a stage for placing the biological fluorescence samples to be tested, a microscope head located under the stage and used for observing the object to be tested, connected with the microscope head A camera that acquires its observed image from the microscope head, and a position sensor that is fixed on the stage and used to track the position coordinates of the stage. An encoder fixed on the microscope head and used to track the position coordinates of the microscope head, and a drive connected to the microscope head and the stage and used to drive the microscope head and the stage to move, so that the camera or microscope head observes changes in the field of view device.
驱动装置包括横向移动电机、纵向移动电机、垂直移动电机，横向移动电机、纵向移动电机分别与显微镜头连接，控制显微镜头进行水平和/或垂直移动。垂直移动电机分别与载物台连接，控制载物台进行垂直移动。The driving device includes a lateral movement motor, a longitudinal movement motor, and a vertical movement motor. The lateral movement motor and the longitudinal movement motor are respectively connected with the microscope head to control the microscope head to move horizontally and/or vertically. The vertical movement motors are respectively connected with the stage to control the stage to move vertically.
明场是让光束透过样本后直接进入物镜，视场是明亮的，暗场是却是强而窄的光束照射样本，而不让光束直接进入到物镜，但是样本中的颗粒能够散射光线，这些散射光线，有一部分进入到物镜，所以在黑暗的背景上，也能够看到样本中的颗粒在闪光点。一般来说，观察形貌都比较喜欢用明场像，因为成像衬度好(尤其是加了合适的光阑)，形变小。Bright field is to let the light beam pass through the sample and directly enter the objective lens, the field of view is bright, dark field is to illuminate the sample with a strong and narrow beam, and the beam does not directly enter the objective lens, but the particles in the sample can scatter the light, Some of this scattered light enters the objective lens, so even on a dark background, particles in the sample can be seen as shining spots. Generally speaking, bright-field images are preferred to observe the morphology, because the imaging contrast is good (especially when a suitable diaphragm is added) and the deformation is small.
荧光涂片区域是生物荧光样本的涂片染色区域，生物荧光样本是用荧光染色试剂制备的生物样本。涂片预选点的数量为至少三个，示例性的，观察员根据需求选择5个涂片预选点，这5个涂片预选点设为生物荧光样本的扫描区域最大内接矩形的四个顶点以及中心点，此处只是示例，其他选择涂片预选点的方式也在本发明保护范围内。The fluorescent smear area is the smear-stained area of a biofluorescent sample, which is a biological sample prepared with a fluorescent staining reagent. The number of smear pre-selected points is at least three. Exemplarily, the observer selects 5 smear pre-selected points according to requirements. The center point is only an example here, and other ways of selecting the preselected point of the smear are also within the protection scope of the present invention.
将显微镜切换为明场条件下，然后点亮设置在显微镜上的普通光源，控制摄像头对生物荧光样本的荧光涂片区域上的任一涂片预选点，利用物体光反射的原理，将反射的光被摄像头上的CCD传感器接受，通过计算机处理，带动电动聚焦装置自动进行对焦变焦直至聚焦成功。Switch the microscope to brightfield condition, then turn on the common light source set on the microscope, control the camera to pre-select any smear point on the fluorescent smear area of the biological fluorescence sample, and use the principle of object light reflection to convert the reflected light. The light is received by the CCD sensor on the camera, and processed by the computer, the electric focusing device is driven to automatically focus and zoom until the focusing is successful.
S200获取聚焦成功时的聚焦参数，根据聚焦参数进行平面拟合得到基准平面；S200 obtains the focusing parameters when the focusing is successful, and performs plane fitting according to the focusing parameters to obtain the reference plane;
S300切换普通光源为荧光光源，根据预设扫描点的坐标和基准平面计算得到对应的目标移动高度；S300 switches the ordinary light source to the fluorescent light source, and calculates the corresponding target moving height according to the coordinates of the preset scanning point and the reference plane;
S400根据目标移动高度按照预设扫描策略，控制摄像头进行自动扫描采集目标图像。The S400 controls the camera to automatically scan and collect the target image according to the preset scanning strategy according to the moving height of the target.
具体的，在明场下对生物荧光样本的荧光涂片区域的至少一个进行自动聚焦，在聚焦成功时获取聚焦参数，根据聚焦参数进行平面拟合得到基准平面，随后将普通光源切换为荧光光源，将拟合出的基准平面作为荧光扫描的基准，根据基准平面和预设扫描点的坐标计算得到对应的目标移动高度，根据目标移动高度按照预设扫描策略进行小范围的扫描和采集目标图像，即在目标移动高度上下进行小范围的扫描和采集目标图像。Specifically, at least one of the fluorescent smear areas of the biofluorescence sample is automatically focused in bright field, focusing parameters are obtained when focusing is successful, and a reference plane is obtained by plane fitting according to the focusing parameters, and then the ordinary light source is switched to the fluorescent light source , using the fitted datum plane as the benchmark for fluorescence scanning, calculate the corresponding target moving height according to the coordinates of the datum plane and the preset scanning point, perform a small-scale scanning and collect target images according to the target moving height according to the preset scanning strategy , that is, a small range of scanning is performed up and down the moving height of the target and the target image is collected.
本发明可适用于结核分歧杆菌的荧光痰涂片扫描过程，当然也可用于其他生物荧光样本采集目标图像。本实施例先在显微镜明场条件下对多个涂片预选点进行快速的自动聚焦，并拟合出一个基准平面，该基准平面即可视为当前生物荧光样本的近似平面，随后将普通光源切换为荧光光源，在所求近似平面的基础上，进行小范围的扫描，可以在荧光条件下快速、精确的找到当前生物荧光样本的基准平面，相比直接在荧光条件下逐个视野进行自动聚焦，能大大缩短聚焦时间，同时降低荧光的猝灭率，保证了生物荧光样本对应目标图像采集的有效性与准确性，提高了镜检效率。The present invention can be applied to the fluorescent sputum smear scanning process of Mycobacterium tuberculosis, and of course can also be used to collect target images of other biological fluorescent samples. In this example, a number of preselected smear points are quickly and automatically focused under the microscope brightfield condition, and a reference plane is fitted, which can be regarded as the approximate plane of the current biofluorescence sample, and then the ordinary light source Switch to the fluorescent light source, and perform a small-scale scan on the basis of the desired approximate plane, which can quickly and accurately find the reference plane of the current biofluorescence sample under fluorescent conditions, compared to direct autofocusing on a field-by-field basis under fluorescent conditions , which can greatly shorten the focusing time and at the same time reduce the quenching rate of fluorescence, which ensures the validity and accuracy of the acquisition of the target image corresponding to the biological fluorescence sample, and improves the efficiency of microscopy.
本发明的一个实施例，如图2所示，一种生物荧光样本的聚焦拍摄实现方法，包括：An embodiment of the present invention, as shown in FIG. 2 , is a method for realizing focusing and photographing a biofluorescence sample, including:
S110在明场条件下点亮普通光源；S110 lights up ordinary light sources under brightfield conditions;
S130根据预设聚焦策略控制摄像头移动对涂片预选点进行自动聚焦，直至摄像头输入的图像清晰度数值最大时确定聚焦成功；S130 controls the movement of the camera according to the preset focusing strategy to automatically focus on the pre-selected points of the smear, until the image clarity value input by the camera is the largest, and it is determined that the focusing is successful;
具体的，预设聚焦策略包括爬坡聚焦策略和图像处理聚焦策略。Specifically, the preset focusing strategy includes a climbing focusing strategy and an image processing focusing strategy.
爬坡聚焦策略具体分为粗聚焦阶段和细聚焦阶段，初试时显微镜的显微镜头处的摄像头距离载物台较远，粗聚焦时载物台在Z轴方向(高度方向)上移动较大步长，使得摄像头以快速靠近显微镜的焦平面(以下为了便于叙述，均简称为焦平面)，当靠近焦平面时再调整为较小的步长进行细聚焦。The climbing focusing strategy is divided into a coarse focusing stage and a fine focusing stage. In the initial test, the camera at the microscope head of the microscope is far away from the stage, and the stage moves a larger step in the Z-axis direction (height direction) during coarse focusing. long, so that the camera quickly approaches the focal plane of the microscope (hereinafter referred to as the focal plane for ease of description), and then adjusts to a smaller step size for fine focusing when approaching the focal plane.
图像处理聚焦策略具体分为粗聚焦阶段和细聚焦阶段，启动摄像头采集视频，采用灰度差分评价函数和快速爬山回溯搜索算法进行粗略聚焦，然后采用基于laplace算子的评价函数和小步长搜索策略进行精细聚焦。具体的图像处理聚焦策略过程参见专利CN200910234988.7-基于图像处理的显微镜快速自动聚焦方法，在此不再详细说明。The image processing focusing strategy is divided into a coarse focusing stage and a fine focusing stage. Start the camera to collect video, use the grayscale difference evaluation function and the fast hill-climbing backtracking search algorithm for rough focusing, and then use the laplace operator-based evaluation function and small step search. Strategies are finely focused. For the specific image processing focusing strategy process, please refer to the patent CN200910234988.7-Fast automatic focusing method of microscope based on image processing, which will not be described in detail here.
根据预设聚焦策略控制摄像头移动对涂片预选点进行自动聚焦，然后根据图像处理方法计算获取摄像头当前视野下图像的清晰度，直至摄像头当前视野下输入图像的清晰度数值最大时确定聚焦成功。Control the camera to move according to the preset focusing strategy to automatically focus on the preselected points of the smear, and then calculate and obtain the clarity of the image under the current field of view of the camera according to the image processing method, until the input image under the current field of view of the camera has the maximum clarity value to determine that the focus is successful.
S200获取聚焦成功时的聚焦参数，根据聚焦参数进行平面拟合得到基准平面；S200 obtains the focusing parameters when the focusing is successful, and performs plane fitting according to the focusing parameters to obtain the reference plane;
S300切换普通光源为荧光光源，根据预设扫描点的坐标和基准平面计算得到对应的目标移动高度；S300 switches the ordinary light source to the fluorescent light source, and calculates the corresponding target moving height according to the coordinates of the preset scanning point and the reference plane;
S400根据目标移动高度按照预设扫描策略，控制摄像头进行自动扫描采集目标图像。The S400 controls the camera to automatically scan and collect the target image according to the preset scanning strategy according to the moving height of the target.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。本实施例先在显微镜明场条件下，根据预设聚焦策略控制摄像头移动对多个涂片预选点进行快速的自动聚焦，并拟合出一个基准平面，该基准平面即可视为当前生物荧光样本的近似平面，随后将普通光源切换为荧光光源，在所求近似平面的基础上，进行小范围的扫描，可以在荧光条件下快速、精确的找到当前生物荧光样本的基准平面，相比直接在荧光条件下逐个视野进行自动聚焦，能大大缩短聚焦时间，同时降低荧光的猝灭率，保证了生物荧光样本对应目标图像采集的有效性与准确性，提高了镜检效率。此外，由于采用预设聚焦策略实现聚焦，能够实现快速自动聚焦及解决聚焦精度不高的问题，具有很强的实时性、很高的可靠性和实用性。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here. In this example, under the condition of microscope bright field, the camera is controlled to move according to the preset focusing strategy to perform fast automatic focusing on the pre-selected points of the smear, and a reference plane is fitted, and the reference plane can be regarded as the current biological fluorescence. The approximate plane of the sample, then switch the ordinary light source to the fluorescent light source, and perform a small-scale scan on the basis of the obtained approximate plane, which can quickly and accurately find the reference plane of the current biofluorescence sample under fluorescent conditions. The automatic focusing of each field of view under fluorescent conditions can greatly shorten the focusing time and reduce the quenching rate of fluorescence, which ensures the effectiveness and accuracy of the biofluorescence sample corresponding to the target image acquisition, and improves the efficiency of microscopy. In addition, due to the use of a preset focusing strategy to achieve focusing, fast automatic focusing can be achieved and the problem of low focusing accuracy can be solved, which has strong real-time performance, high reliability and practicability.
本发明的一个实施例，如图3所示，一种生物荧光样本的聚焦拍摄实现方法，包括：An embodiment of the present invention, as shown in FIG. 3 , is a method for realizing focusing and photographing a biological fluorescence sample, including:
S110在明场条件下点亮普通光源；S110 lights up ordinary light sources under brightfield conditions;
S120控制显微镜头移动和/或载物台移动，使得任一涂片预选点位于摄像头镜头的正下方；S120 controls the movement of the microscope head and/or the stage, so that any smear preselected point is located directly under the camera lens;
S131根据预设聚焦策略控制摄像头移动靠向涂片预选点，并控制摄像头的镜头在测量量程中进行聚焦；S131 controls the camera to move toward the smear preselected point according to the preset focusing strategy, and controls the lens of the camera to focus in the measurement range;
S132在整个聚焦过程中根据清晰度评价函数计算输入图像的清晰度，在清晰度停止增大时确定聚焦成功；S132 calculates the sharpness of the input image according to the sharpness evaluation function in the entire focusing process, and determines that the focusing is successful when the sharpness stops increasing;
具体的，在聚焦的过程中，利用清晰度评价函数计算得到的数值，对聚过程中摄像头当前视野下图像的清晰度进行评估，在粗聚焦阶段与细聚焦阶段清晰度数值不断变大，当清晰度数值开始减小时认为聚焦成功。其中，清晰度评价函数具体为采用Canny算子或Sobel算子计算图像梯度，将梯度值累加结果作为清晰度数值，或者根据Tenengrad评价函数计算得到清晰度数值。Specifically, during the focusing process, the sharpness of the image under the current field of view of the camera during the focusing process is evaluated by using the value calculated by the sharpness evaluation function. Focusing is considered successful when the sharpness value begins to decrease. The sharpness evaluation function is specifically calculated by using the Canny operator or the Sobel operator to calculate the image gradient, and using the cumulative result of the gradient values as the sharpness value, or calculating the sharpness value according to the Tenengrad evaluation function.
对摄像头进行聚焦过程中每个视野下输入图像，根据清晰度评价函数计算对应的清晰度数值，然后当清晰度数值开始减小时确定获取到最大清晰度值则认为聚焦成功。具体的移动聚焦过程如下：In the process of focusing the camera, input the image in each field of view, and calculate the corresponding sharpness value according to the sharpness evaluation function. Then, when the sharpness value begins to decrease, it is determined that the maximum sharpness value is obtained, and the focusing is considered successful. The specific moving focus process is as follows:
对于相同视野下对焦最准确时清晰度数值最大，通过控制垂直移动电机使显微镜头上下移动来寻找此位置，首先垂直移动电机根据预设步长朝向一个方向运动，如果清晰度数值一直递增，说明还没到最大值，保持这个预设步长和方向继续运动，直到出现清晰度数值变小，说明已经超过最大值，此时减小步长并往反方向移动，随着清晰度数值越来越逼近最大值，垂直移动电机的运动方向会做多次改变，电机移动步长也会逐渐减小至预设最低步长，直到垂直移动电机的步长减小到预设最低步长，此时可认定清晰度数值已达到最大从而确定聚焦成功。自动聚焦结果如图7所示。For the most accurate focusing in the same field of view, the sharpness value is the largest. By controlling the vertical movement motor to move the microscope head up and down to find this position, first the vertical movement motor moves in one direction according to the preset step size. If the sharpness value keeps increasing, it means If the maximum value has not been reached, keep the preset step size and direction and continue to move until the sharpness value becomes smaller, indicating that the maximum value has been exceeded. At this time, reduce the step size and move in the opposite direction. The closer it is to the maximum value, the movement direction of the vertical movement motor will be changed many times, and the motor movement step size will gradually decrease to the preset minimum step size until the vertical movement motor step size is reduced to the preset minimum step size. It can be determined that the sharpness value has reached the maximum and the focusing is successful. The autofocus results are shown in Figure 7.
S210获取聚焦成功时载物台相对于预设原点在高度方向上的目标高度值，以及所有涂片预选点相对于预设原点的预选点坐标；S210 obtains the target height value of the stage relative to the preset origin in the height direction when the focusing is successful, and the preselected point coordinates of all smear preselected points relative to the preset origin;
具体的，预设原点为根据需求预先设置的参考坐标系的原点。可以通过位置传感器获取聚焦成功时载物台相对于预设原点在高度方向上的目标高度值，通过参考坐标系以及相机坐标系进行转换运算得到各个涂片预选点相对于预设原点的空间坐标得到预选点坐标。由于目标高度值接近等于涂片预选点的Z轴坐标值，此处也可以只获取涂片预选点的三维空间坐标(x，y，z)，将涂片预选点的Z轴坐标值作为载物台相对于预设原点在高度方向上的目标高度值。Specifically, the preset origin is the origin of the reference coordinate system preset according to requirements. The target height value of the stage relative to the preset origin in the height direction can be obtained through the position sensor, and the spatial coordinates of each smear pre-selected point relative to the preset origin can be obtained by converting the reference coordinate system and the camera coordinate system. Get the coordinates of the preselected point. Since the target height value is nearly equal to the Z-axis coordinate value of the smear pre-selection point, it is also possible to obtain only the three-dimensional space coordinates (x, y, z) of the smear pre-selection point, and use the Z-axis coordinate value of the smear pre-selection point as the carrier The target height value of the stage relative to the preset origin in the height direction.
S220根据目标高度值和预选点坐标拟合得到近似焦平面，确定经过涂片预选点最多的近似焦平面为基准平面；S220 obtains the approximate focal plane by fitting the target height value and the coordinates of the preselected point, and determines the approximate focal plane with the most preselected points through the smear as the reference plane;
具体的，预先选定待扫描区域的多个涂片预选点，根据任意一种聚焦策略以及清晰度评价函数对涂片预选点进行自动聚焦，并记录聚焦成功时载物台相对于预设原点的高度以及涂片预选点的X、Y轴坐标即平面坐标(xn，yn)。根据获得的目标高度值以及相应的平面坐标(xn，yn)拟合出得到若干个近似焦平面，这些近似焦平面对应的空间平面表达式为z＝Axn+Byn+C，然后将所有代入至近似焦平面，确定筛选出经过涂片预选点最多的近似焦平面作为基准平面。Specifically, pre-select multiple smear pre-selected points in the area to be scanned, automatically focus on the smear pre-selected points according to any focusing strategy and sharpness evaluation function, and record the stage relative to the preset origin when the focusing is successful The height of the smear and the X and Y axis coordinates of the preselected point of the smear are plane coordinates (xn, yn). According to the obtained target height value and the corresponding plane coordinates (xn, yn), several approximate focal planes are obtained by fitting, and the spatial plane expression corresponding to these approximate focal planes is z=Axn+Byn+C, and then all are substituted into Approximate focal plane, determine the approximate focal plane with the most pre-selected smear points as the reference plane.
S300切换普通光源为荧光光源，根据预设扫描点的坐标和基准平面计算得到对应的目标移动高度；S300 switches the ordinary light source to the fluorescent light source, and calculates the corresponding target moving height according to the coordinates of the preset scanning point and the reference plane;
S400根据目标移动高度按照预设扫描策略，控制摄像头进行自动扫描采集目标图像。The S400 controls the camera to automatically scan and collect the target image according to the preset scanning strategy according to the moving height of the target.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。自适应的改变步长，在粗聚焦时选用长步长能缩短对焦时间，细聚焦时选用小步长进行搜索能够提高对焦精度，加快了对焦速度，在聚焦区域内采取小步长，并在粗聚焦和细聚焦过程中结合清晰度评价函数控制聚焦过程，提高了自动聚焦的速度以及精度，节约了自动对焦时间，大大的提高对焦效率。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here. Adaptively changing the step size, selecting a long step size during coarse focusing can shorten the focusing time, and selecting a small step size for searching during fine focusing can improve the focusing accuracy and speed up the focusing speed. In the process of coarse focusing and fine focusing, the focusing process is controlled by combining the sharpness evaluation function, which improves the speed and accuracy of the automatic focusing, saves the automatic focusing time, and greatly improves the focusing efficiency.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。本实施例先在显微镜明场条件下对多个涂片预选点进行快速的自动聚焦，并目标高度值和预选点坐标拟合得到近似焦平面，确定经过涂片预选点最多的近似焦平面为基准平面，提升基准平面拟合准确率，随后将普通光源切换为荧光光源，在所求基准平面的基础上，进行小范围的扫描，可以在荧光条件下快速、精确的找到当前生物荧光样本的基准平面，相比直接在荧光条件下逐个视野进行自动聚焦，能大大缩短聚焦时间，同时降低荧光的猝灭率，保证了生物荧光样本对应目标图像采集的有效性与准确性，提高了镜检效率。此外，由于拟合得到的基准平面准确率提升，从而间接使得目标图像采集的有效性与准确性大大提升。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here. In this example, a number of smear preselected points are quickly auto-focused under the microscope brightfield condition, and the approximate focal plane is obtained by fitting the target height value and the preselected point coordinates, and the approximate focal plane with the most smear preselected points is determined as The reference plane improves the fitting accuracy of the reference plane, and then switches the ordinary light source to the fluorescent light source. Based on the obtained reference plane, a small-scale scan can be performed to quickly and accurately find the current biofluorescence sample under fluorescent conditions. The reference plane can greatly shorten the focusing time and reduce the quenching rate of fluorescence compared with the automatic focusing directly on each field of view under fluorescent conditions, which ensures the effectiveness and accuracy of the biofluorescence sample corresponding to the target image acquisition, and improves the microscope inspection. efficiency. In addition, due to the improved accuracy of the datum plane obtained by fitting, the effectiveness and accuracy of target image acquisition are indirectly greatly improved.
本发明的一个实施例，如图4所示，一种生物荧光样本的聚焦拍摄实现方法，包括：An embodiment of the present invention, as shown in FIG. 4 , is a method for realizing focusing and photographing a biofluorescence sample, including:
S110在明场条件下点亮普通光源；S110 lights up ordinary light sources under brightfield conditions;
S120控制显微镜头移动和/或载物台移动，使得任一涂片预选点位于摄像头镜头的正下方；S120 controls the movement of the microscope head and/or the stage, so that any smear preselected point is located directly under the camera lens;
S131根据预设聚焦策略控制摄像头移动靠向涂片预选点，并控制摄像头的镜头在测量量程中进行聚焦；S131 controls the camera to move toward the smear preselected point according to the preset focusing strategy, and controls the lens of the camera to focus in the measurement range;
S132在整个聚焦过程中根据清晰度评价函数计算输入图像的清晰度，在清晰度停止增大时确定聚焦成功；S132 calculates the sharpness of the input image according to the sharpness evaluation function in the entire focusing process, and determines that the focusing is successful when the sharpness stops increasing;
S210获取聚焦成功时载物台相对于预设原点在高度方向上的目标高度值，以及所有涂片预选点相对于预设原点的预选点坐标；S210 obtains the target height value of the stage relative to the preset origin in the height direction when the focusing is successful, and the preselected point coordinates of all smear preselected points relative to the preset origin;
S230从预选点坐标中筛选根据预设异常检验算法，查找并剔除涂片预选点中的异常涂片预选点，根据目标高度值以及在剔除后结果中任选三个涂片预选点拟合得到基准平面；S230 selects from the coordinates of the preselected points and selects the abnormal smear preselected points in the preselected smear points according to the preset abnormal inspection algorithm, and obtains by fitting according to the target height value and any three preselected smear points in the eliminated result. datum plane;
具体的，设生物荧光样本的空间平面方程为z＝Ax+By+C。采用预设异常检验算法(包括但是不限于Grubbs算法或者T Test算法)查找剔除5个涂片预选点中z值异常的点，在剩下的点中任选3个点，分别为p1，p2，p3，可得到如下方程组：Specifically, the space plane equation of the bioluminescence sample is set as z=Ax+By+C. The preset abnormality test algorithm (including but not limited to Grubbs algorithm or T Test algorithm) is used to find and eliminate the points with abnormal z value in the 5 preselected points of the smear, and choose 3 points from the remaining points, which are p1 and p2 respectively. , p3, the following equations can be obtained:
其中x，y，z分别为聚焦成功时涂片预选点的平面坐标，以及聚焦成功时载物台相对于预设原点的高度。A、B、C是待求的参数。此时若对生物荧光样本进行拍摄可得到较为清楚的荧光图像即目标图像，如图8所示，说明距荧光条件下的生物荧光样本较近。Among them, x, y, and z are the plane coordinates of the preselected point of the smear when the focusing is successful, and the height of the stage relative to the preset origin when the focusing is successful. A, B, and C are the parameters to be sought. At this time, if the biofluorescence sample is photographed, a clearer fluorescent image, that is, the target image, can be obtained, as shown in FIG. 8 , indicating that it is relatively close to the biofluorescence sample under fluorescent conditions.
S300切换普通光源为荧光光源，根据预设扫描点的坐标和基准平面计算得到对应的目标移动高度；S300 switches the ordinary light source to the fluorescent light source, and calculates the corresponding target moving height according to the coordinates of the preset scanning point and the reference plane;
S400根据目标移动高度按照预设扫描策略，控制摄像头进行自动扫描采集目标图像。The S400 controls the camera to automatically scan and collect the target image according to the preset scanning strategy according to the moving height of the target.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。本实施例先在显微镜明场条件下对多个涂片预选点进行快速的自动聚焦，并根据剔除异常涂片预选点后的涂片预选点和目标高度值拟合出一个基准平面，相对于上一实施例，剔除异常涂片预选点能够排除干扰点，更进一步提升基准平面拟合准确率，随后将普通光源切换为荧光光源，在所求基准平面的基础上，进行小范围的扫描，可以在荧光条件下快速、精确的找到当前生物荧光样本的基准平面，相比直接在荧光条件下逐个视野进行自动聚焦，能大大缩短聚焦时间，同时降低荧光的猝灭率，保证了生物荧光样本对应目标图像采集的有效性与准确性，提高了镜检效率。此外，由于拟合得到的基准平面准确率提升，从而间接使得目标图像采集的有效性与准确性大大提升。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here. In this example, a number of smear pre-selected points are quickly and automatically focused under the microscope brightfield condition, and a reference plane is fitted according to the smear pre-selected points after removing abnormal smear pre-selected points and the target height value. In the previous embodiment, eliminating the abnormal smear preselected points can eliminate the interference points, and further improve the accuracy of the reference plane fitting, and then switch the ordinary light source to the fluorescent light source, and perform a small-scale scan on the basis of the obtained reference plane. It can quickly and accurately find the reference plane of the current biofluorescence sample under fluorescent conditions. Compared with automatic focusing one by one under fluorescent conditions, the focusing time can be greatly shortened, and the quenching rate of fluorescence can be reduced at the same time, ensuring the biofluorescence sample. Corresponding to the effectiveness and accuracy of target image acquisition, the efficiency of microscopy is improved. In addition, due to the improved accuracy of the datum plane obtained by fitting, the effectiveness and accuracy of target image acquisition are indirectly greatly improved.
本发明的一个实施例，如图5所示，一种生物荧光样本的聚焦拍摄实现方法，包括：An embodiment of the present invention, as shown in FIG. 5 , is a method for realizing focusing and photographing a biofluorescence sample, including:
S100在明场条件下点亮普通光源，并控制摄像头对载物台所放置生物荧光样本的荧光涂片区域上任一涂片预选点进行自动聚焦直至聚焦成功；涂片预选点数量为至少三个；The S100 lights up a common light source under brightfield conditions, and controls the camera to automatically focus on any smear preselected point on the fluorescent smear area of the biofluorescence sample placed on the stage until the focus is successful; the number of smear preselected points is at least three;
S200获取聚焦成功时的聚焦参数，根据聚焦参数进行平面拟合得到基准平面；S200 obtains the focusing parameters when the focusing is successful, and performs plane fitting according to the focusing parameters to obtain the reference plane;
S310控制关闭普通光源且打开荧光光源并插入滤光片；S310 controls to turn off the common light source and turn on the fluorescent light source and insert the filter;
S320控制显微镜头移动和/或载物台移动，使得载物台所放置生物荧光样本的荧光涂片区域上的任一预设扫描点位于摄像头的正下方；S320 controls the movement of the microscope head and/or the movement of the stage, so that any preset scanning point on the fluorescent smear area of the biological fluorescence sample placed on the stage is located directly below the camera;
S330根据预设扫描点的坐标，以及基准平面对应的平面表达式计算得到预设扫描点对应的目标移动高度；S330 calculates the target moving height corresponding to the preset scan point according to the coordinates of the preset scan point and the plane expression corresponding to the reference plane;
S410在距离当前预设扫描点所对应目标移动高度的预设步长范围内，控制摄像头和/或载物台在高度方向上进行往复移动扫描采集包括当前预设扫描点对应的图像；S410, within a preset step size range from the target moving height corresponding to the current preset scanning point, control the camera and/or the stage to perform reciprocating movement scanning in the height direction to collect images including the image corresponding to the current preset scanning point;
S420根据清晰度评价函数计算所采集图像的清晰度，确定清晰度数值最大的图像为当前预设扫描点对应的目标图像；S420 calculates the sharpness of the collected image according to the sharpness evaluation function, and determines that the image with the largest sharpness value is the target image corresponding to the current preset scanning point;
S430切换至下一预设扫描点进行聚焦扫描，直至采集到所有预设扫描点对应的目标图像为止。S430 switches to the next preset scanning point to perform focus scanning until the target images corresponding to all the preset scanning points are collected.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。如图9所示，移动将选定的预设扫描点移动到摄像头正下方。当载物台移动将选定的预设扫描点移动到摄像头正下方时，根据预设扫描点的平面坐标，以及根据上述实施例获取得到基准平面对应的空间平面表达式z＝Ax+By+C，可求得该预设扫描点对应的目标移动高度。其中预设扫描点为在生物荧光样本上需要进行采集图像的扫描区域均匀采样获得。其中预设扫描点为在样本的待扫描区域均匀采样获得，具体为，根据需要采集的水平方向和竖直方向的视野数H和V，将样本扫描区域的最大内接矩形R划分为H*V个小区域，在每个小区域中随机生成一个坐标点作为扫描点，共生成H*V个扫描点。示例性的，如图9所示为选定的一个预设扫描点。此后，控制显微镜头移动和/或载物台移动，在目标移动高度对应高度的上下一定步长范围内沿着高度方向进行从上到下或从下到上的扫描，并根据清晰度评价函数计算采集图像的清晰度。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here. As shown in Figure 9, the move moves the selected preset scan point directly below the camera. When the stage moves to move the selected preset scanning point directly below the camera, the space plane expression z=Ax+By+ corresponding to the reference plane is obtained according to the plane coordinates of the preset scanning point and the above-mentioned embodiment. C, the target moving height corresponding to the preset scanning point can be obtained. The preset scanning point is obtained by uniform sampling of the scanning area where the image needs to be collected on the biological fluorescence sample. The preset scanning points are obtained by uniform sampling in the to-be-scanned area of the sample. Specifically, the maximum inscribed rectangle R of the sample scanning area is divided into H* according to the number of fields of view H and V in the horizontal and vertical directions to be collected. There are V small areas, and a coordinate point is randomly generated in each small area as a scanning point, and a total of H*V scanning points are generated. Exemplarily, as shown in FIG. 9 , a preset scanning point is selected. After that, control the movement of the microscope head and/or the stage to perform scanning from top to bottom or from bottom to top along the height direction within a certain step range above and below the height of the target moving height corresponding to the height, and according to the definition evaluation function Calculate the sharpness of the acquired image.
其中，该预设步长范围较小，且范围内包含该预设扫描点对应的真正空间平面，所以可迅速的找到清晰度最大值对应的图像，取该图像作为最后采集的目标图像。如果有需求则自动移动到下一个预设扫描点重复该步骤进行聚焦扫描，直至采集到所有预设扫描点对应的目标图像为止。The range of the preset step size is small, and the range includes the real space plane corresponding to the preset scanning point, so the image corresponding to the maximum sharpness value can be quickly found, and the image is taken as the final collected target image. If there is a need, it will automatically move to the next preset scanning point and repeat this step to perform focused scanning until the target images corresponding to all the preset scanning points are collected.
在显微镜下对荧光样本进行拍摄时，所需的曝光时间较长，且聚焦困难，导致在对样本的每个视野进行自动聚焦时耗时较长，同时曝光时间过长，易使荧光物质发生衰减和淬灭，使得目标被噪声淹没。因此，先将载物台放置于全自动镜检平台上，并将荧光涂片区域对准显微镜物镜(或显微镜头)的正下方。在在显微镜明场条件下，载物台或者显微镜头依次移动使得任意一个涂片预选点置于摄像头正下方，对荧光染色试剂制备的生物样本即生物荧光样本进行多点自动聚焦，并根据多点聚焦后的信息确定生物荧光样本的基准平面，随后将普通光源切换到荧光光源，将上一步得到的基准平面作为荧光扫描的基准平面，在根据基准平面再次进行小范围的自动扫描并采集图像，可以在荧光条件下快速、精确的找到当前生物荧光样本的基准平面，相比直接在荧光条件下逐个视野进行自动聚焦，能大大缩短聚焦时间，同时降低荧光的猝灭率，保证了生物荧光样本对应目标图像采集的有效性与准确性，提高了镜检效率。When photographing a fluorescent sample under a microscope, the required exposure time is long and focusing is difficult, which leads to a long time-consuming autofocusing on each field of view of the sample. At the same time, the exposure time is too long, which is easy to cause the occurrence of fluorescent substances. Attenuation and quenching, so that the target is overwhelmed by noise. Therefore, first place the stage on the automatic microscope platform, and align the fluorescent smear area directly under the microscope objective lens (or microscope head). Under the bright field condition of the microscope, the stage or the microscope head is moved in sequence so that any pre-selected point of the smear is placed directly under the camera, and the biological sample prepared by the fluorescent staining reagent, that is, the biological fluorescence sample, is automatically focused on multiple points, and according to the multi-point automatic focusing The information after point focusing determines the reference plane of the biofluorescence sample, then switches the ordinary light source to the fluorescent light source, uses the reference plane obtained in the previous step as the reference plane for fluorescence scanning, and performs a small-scale automatic scan again according to the reference plane and collects images. , which can quickly and accurately find the reference plane of the current biofluorescence sample under fluorescent conditions. Compared with direct autofocusing on a field-by-field basis under fluorescent conditions, it can greatly shorten the focusing time and reduce the quenching rate of fluorescence, ensuring the bioluminescence The effectiveness and accuracy of the sample corresponding to the target image acquisition improves the efficiency of microscopy.
本发明的一个实施例，一种生物荧光样本的聚焦拍摄实现方法，包括：One embodiment of the present invention, a method for implementing focused photography of a biofluorescence sample, includes:
S110在明场条件下点亮普通光源；S110 lights up ordinary light sources under brightfield conditions;
S120控制显微镜头移动和/或载物台移动，使得任一涂片预选点位于摄像头镜头的正下方；S120 controls the movement of the microscope head and/or the stage, so that any smear preselected point is located directly under the camera lens;
S131根据预设聚焦策略控制摄像头移动靠向涂片预选点，并控制摄像头的镜头在测量量程中进行聚焦；S131 controls the camera to move toward the smear preselected point according to the preset focusing strategy, and controls the lens of the camera to focus in the measurement range;
S132在整个聚焦过程中根据清晰度评价函数计算输入图像的清晰度，在清晰度停止增大时确定聚焦成功；S132 calculates the sharpness of the input image according to the sharpness evaluation function in the entire focusing process, and determines that the focusing is successful when the sharpness stops increasing;
S210获取聚焦成功时载物台相对于预设原点在高度方向上的目标高度值，以及所有涂片预选点相对于预设原点的预选点坐标；S210 obtains the target height value of the stage relative to the preset origin in the height direction when the focusing is successful, and the preselected point coordinates of all smear preselected points relative to the preset origin;
S220根据目标高度值和预选点坐标拟合得到近似焦平面，确定经过涂片预选点最多的近似焦平面为基准平面；S220 obtains the approximate focal plane by fitting the target height value and the coordinates of the preselected point, and determines the approximate focal plane with the most preselected points through the smear as the reference plane;
S310控制关闭普通光源且打开荧光光源并插入滤光片；S310 controls to turn off the common light source and turn on the fluorescent light source and insert the filter;
S320控制显微镜头移动和/或载物台移动，使得载物台所放置生物荧光样本的荧光涂片区域上的任一预设扫描点位于摄像头的正下方；S320 controls the movement of the microscope head and/or the movement of the stage, so that any preset scanning point on the fluorescent smear area of the biological fluorescence sample placed on the stage is located directly below the camera;
S330根据预设扫描点的坐标，以及基准平面对应的平面表达式计算得到预设扫描点对应的目标移动高度；S330 calculates the target moving height corresponding to the preset scan point according to the coordinates of the preset scan point and the plane expression corresponding to the reference plane;
S410在距离当前预设扫描点所对应目标移动高度的预设步长范围内，控制摄像头和/或载物台在高度方向上进行往复移动扫描采集包括当前预设扫描点对应的图像；S410, within a preset step size range from the target moving height corresponding to the current preset scanning point, control the camera and/or the stage to perform reciprocating movement scanning in the height direction to collect images including the image corresponding to the current preset scanning point;
S420根据清晰度评价函数计算所采集图像的清晰度，确定清晰度数值最大的图像为当前预设扫描点对应的目标图像；S420 calculates the sharpness of the collected image according to the sharpness evaluation function, and determines that the image with the largest sharpness value is the target image corresponding to the current preset scanning point;
S430切换至下一预设扫描点进行聚焦扫描，直至采集到所有预设扫描点对应的目标图像为止。S430 switches to the next preset scanning point to perform focus scanning until the target images corresponding to all the preset scanning points are collected.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here.
本发明的一个实施例，一种生物荧光样本的聚焦拍摄实现方法，包括：One embodiment of the present invention, a method for implementing focused photography of a biofluorescence sample, includes:
S110在明场条件下点亮普通光源；S110 lights up ordinary light sources under brightfield conditions;
S120控制显微镜头移动和/或载物台移动，使得任一涂片预选点位于摄像头镜头的正下方；S120 controls the movement of the microscope head and/or the stage, so that any smear preselected point is located directly under the camera lens;
S131根据预设聚焦策略控制摄像头移动靠向涂片预选点，并控制摄像头的镜头在测量量程中进行聚焦；S131 controls the camera to move toward the smear preselected point according to the preset focusing strategy, and controls the lens of the camera to focus in the measurement range;
S132在整个聚焦过程中根据清晰度评价函数计算输入图像的清晰度，在清晰度停止增大时确定聚焦成功；S132 calculates the sharpness of the input image according to the sharpness evaluation function in the entire focusing process, and determines that the focusing is successful when the sharpness stops increasing;
S210获取聚焦成功时载物台相对于预设原点在高度方向上的目标高度值，以及所有涂片预选点相对于预设原点的预选点坐标；S210 obtains the target height value of the stage relative to the preset origin in the height direction when the focusing is successful, and the preselected point coordinates of all smear preselected points relative to the preset origin;
S230从预选点坐标中筛选根据预设异常检验算法，查找并剔除涂片预选点中的异常涂片预选点，根据目标高度值以及在剔除后结果中任选三个涂片预选点拟合得到基准平面；S230 selects from the coordinates of the preselected points and selects the abnormal smear preselected points in the preselected smear points according to the preset abnormal inspection algorithm, and obtains by fitting according to the target height value and any three preselected smear points in the eliminated result. datum plane;
S310控制关闭普通光源且打开荧光光源并插入滤光片；S310 controls to turn off the common light source and turn on the fluorescent light source and insert the filter;
S320控制显微镜头移动和/或载物台移动，使得载物台所放置生物荧光样本的荧光涂片区域上的任一预设扫描点位于摄像头的正下方；S320 controls the movement of the microscope head and/or the movement of the stage, so that any preset scanning point on the fluorescent smear area of the biological fluorescence sample placed on the stage is located directly below the camera;
S330根据预设扫描点的坐标，以及基准平面对应的平面表达式计算得到预设扫描点对应的目标移动高度；S330 calculates the target moving height corresponding to the preset scan point according to the coordinates of the preset scan point and the plane expression corresponding to the reference plane;
S410在距离当前预设扫描点所对应目标移动高度的预设步长范围内，控制摄像头和/或载物台在高度方向上进行往复移动扫描采集包括当前预设扫描点对应的图像；S410, within a preset step size range from the target moving height corresponding to the current preset scanning point, control the camera and/or the stage to perform reciprocating movement scanning in the height direction to collect images including the image corresponding to the current preset scanning point;
S420根据清晰度评价函数计算所采集图像的清晰度，确定清晰度数值最大的图像为当前预设扫描点对应的目标图像；S420 calculates the sharpness of the collected image according to the sharpness evaluation function, and determines that the image with the largest sharpness value is the target image corresponding to the current preset scanning point;
S430切换至下一预设扫描点进行聚焦扫描，直至采集到所有预设扫描点对应的目标图像为止。S430 switches to the next preset scanning point to perform focus scanning until the target images corresponding to all the preset scanning points are collected.
具体的，与上述实施例相同的部分参见上述实施例，在此不再一一赘述。Specifically, for the same parts as the above-mentioned embodiments, refer to the above-mentioned embodiments, which will not be repeated here.
所属领域的技术人员可以清楚地了解到，为了描述的方便和简洁，仅以上述各程序模块的划分进行举例说明，实际应用中，可以根据需要而将上述功能分配由不同的程序模块完成，即将所述装置的内部结构划分成不同的程序单元或模块，以完成以上描述的全部或者部分功能。实施例中的各程序模块可以集成在一个处理单元中，也可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处理单元中，上述集成的单元既可以采用硬件的形式实现，也可以采用软件程序单元的形式实现。另外，各程序模块的具体名称也只是为了便于相互区分，并不用于限制本申请的保护范围。Those skilled in the art can clearly understand that, for the convenience and conciseness of the description, only the division of the above-mentioned program modules is used as an example. The internal structure of the device is divided into different program units or modules to complete all or part of the functions described above. Each program module in the embodiment may be integrated in one processing unit, or each unit may exist physically alone, or two or more units may be integrated in one processing unit, and the above-mentioned integrated units may be implemented in the form of hardware. , can also be implemented in the form of software program units. In addition, the specific names of each program module are only for the convenience of distinguishing from each other, and are not used to limit the protection scope of the present application.
本发明的一个实施例，如图6所示，一种显微镜100，包括处理器110、存储器120，其中，存储器120，用于存放计算机程序；处理器110，用于执行存储器120上所存放的计算机程序，实现上述方法实施例中的生物荧光样本的聚焦拍摄实现方法。An embodiment of the present invention, as shown in FIG. 6 , a
所述显微镜100可以为桌上型计算机、笔记本、掌上电脑、平板型计算机、手机、人机交互屏等设备。所述显微镜100可包括，但不仅限于处理器110、存储器120。本领域技术人员可以理解，图6仅仅是显微镜100的示例，并不构成对显微镜100的限定，可以包括比图示更多或更少的部件，或者组合某些部件，或者不同的部件，例如：显微镜100还可以包括输入/输出接口、显示设备、网络接入设备、通信总线、通信接口等。通信接口和通信总线，还可以包括输入/输出接口，其中，处理器110、存储器120、输入/输出接口和通信接口通过通信总线完成相互间的通信。该存储器120存储有计算机程序，该处理器110用于执行存储器120上所存放的计算机程序，实现上述方法实施例中的生物荧光样本的聚焦拍摄实现方法。The
所述处理器110可以是中央处理单元(Central Processing Unit，CPU)，还可以是其他通用处理器、数字信号处理器(Digital Signal Processor，DSP)、专用集成电路(Application Specific Integrated Circuit，ASIC)、现场可编程门阵列(Field-Programmable Gate Array，FPGA)或者其他可编程逻辑器件、分立门或者晶体管逻辑器件、分立硬件组件等。通用处理器可以是微处理器或者该处理器也可以是任何常规的处理器等。The processor 110 may be a central processing unit (Central Processing Unit, CPU), or other general-purpose processors, a digital signal processor (Digital Signal Processor, DSP), an application specific integrated circuit (Application Specific Integrated Circuit, ASIC), Field-Programmable Gate Array (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. A general purpose processor may be a microprocessor or the processor may be any conventional processor or the like.
所述存储器120可以是所述显微镜100的内部存储单元，例如：显微镜的硬盘或内存。所述存储器也可以是所述显微镜的外部存储设备，例如：所述显微镜上配备的插接式硬盘，智能存储卡(Smart Media Card，SMC)，安全数字(Secure Digital，SD)卡，闪存卡(Flash Card)等。进一步地，所述存储器120还可以既包括所述显微镜100的内部存储单元也包括外部存储设备。所述存储器120用于存储所述计算机程序以及所述显微镜100所需要的其他程序和数据。所述存储器还可以用于暂时地存储已经输出或者将要输出的数据。The memory 120 may be an internal storage unit of the
通信总线是连接所描述的元素的电路并且在这些元素之间实现传输。例如，处理器110通过通信总线从其它元素接收到命令，解密接收到的命令，根据解密的命令执行计算或数据处理。存储器120可以包括程序模块，例如内核(kernel)，中间件(middleware)，应用程序编程接口(Application Programming Interface，API)和应用。该程序模块可以是有软件、固件或硬件、或其中的至少两种组成。输入/输出接口转发用户通过输入/输出接口(例如感应器、键盘、触摸屏)输入的命令或数据。通信接口将该显微镜100与其它网络设备、用户设备、网络进行连接。例如，通信接口可以通过有线或无线连接到网络以连接到外部其它的网络设备或用户设备。无线通信可以包括以下至少一种：无线保真(WiFi)，蓝牙(BT)，近距离无线通信技术(NFC)，全球卫星定位系统(GPS)和蜂窝通信等等。有线通信可以包括以下至少一种：通用串行总线(USB)，高清晰度多媒体接口(HDMI)，异步传输标准接口(RS-232)等等。网络可以是电信网络和通信网络。通信网络可以为计算机网络、因特网、物联网、电话网络。显微镜100可以通过通信接口连接网络，显微镜100和其它网络设备通信所用的协议可以被应用、应用程序编程接口(API)、中间件、内核和通信接口至少一个支持。A communication bus is a circuit that connects the described elements and enables transmission between these elements. For example, the processor 110 receives commands from other elements through a communication bus, decrypts the received commands, and performs computation or data processing according to the decrypted commands. The memory 120 may include program modules such as kernel, middleware, Application Programming Interface (API), and applications. The program module may be composed of software, firmware or hardware, or at least two of them. The input/output interface forwards commands or data entered by the user through the input/output interface (eg, sensor, keyboard, touch screen). The communication interface connects the
本发明的一个实施例，一种存储介质，存储介质中存储有至少一条指令，指令由处理器加载并执行以实现上述生物荧光样本的聚焦拍摄实现方法对应实施例所执行的操作。例如，计算机可读存储介质可以是只读内存(ROM)、随机存取存储器(RAM)、只读光盘(CD-ROM)、磁带、软盘和光数据存储设备等。An embodiment of the present invention is a storage medium, the storage medium stores at least one instruction, and the instruction is loaded and executed by a processor to implement the operations performed by the corresponding embodiments of the above-mentioned method for implementing focused photography of a biological fluorescence sample. For example, the computer-readable storage medium may be read only memory (ROM), random access memory (RAM), compact disk read only (CD-ROM), magnetic tapes, floppy disks, optical data storage devices, and the like.
它们可以用计算装置可执行的程序代码来实现，从而，可以将它们存储在存储装置中由计算装置来执行，或者将它们分别制作成各个集成电路模块，或者将它们中的多个模块或步骤制作成单个集成电路模块来实现。这样，本发明不限制于任何特定的硬件和软件结合。They can be implemented with program code executable by a computing device, so that they can be stored in a storage device and executed by the computing device, or they can be separately fabricated into individual integrated circuit modules, or a plurality of modules or steps in them Made into a single integrated circuit module to achieve. As such, the present invention is not limited to any particular combination of hardware and software.
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详细描述或记载的部分，可以参见其他实施例的相关描述。In the above-mentioned embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference may be made to the relevant descriptions of other embodiments.
本领域普通技术人员可以意识到，结合本文中所公开的实施例描述的各示例的单元及算法步骤，能够以电子硬件、或者计算机软件和电子硬件的结合来实现。这些功能究竟以硬件还是软件来执行，取决于技术方案的特定应用和设计约束条件。专业技术人员可以对每个特定的应用来使用不同方法来实现所描述的功能，但是这种实现不应认为超出本申请的范围。Those of ordinary skill in the art can realize that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Skilled artisans may implement the described functionality using different methods for each particular application, but such implementations should not be considered beyond the scope of this application.
在本申请所提供的实施例中，应该理解到，所揭露的装置/显微镜和方法，可以通过其他的方式实现。例如，以上所描述的装置/显微镜实施例仅仅是示意性的，例如，所述模块或单元的划分，仅仅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讯连接可以是通过一些接口，装置或单元的间接耦合或通讯连接，可以是电性、机械或其他的形式。In the embodiments provided in this application, it should be understood that the disclosed apparatus/microscope and method may be implemented in other ways. For example, the device/microscope embodiments described above are only illustrative. For example, the division of the modules or units is only a logical function division. In actual implementation, there may be other division methods, for example, multiple units. Or components may be combined or may be integrated into another system, or some features may be omitted, or not implemented. On the other hand, the shown or discussed mutual coupling or direct coupling or communication connection may be through some interfaces, indirect coupling or communication connection of devices or units, which may be in electrical, mechanical 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网络单元上。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 components displayed as units may or may not be physical units, that is, may be located in one place, or may be distributed to multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution in this embodiment.
另外，在本申请各个实施例中的各功能单元可能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 each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically alone, or two or more units may be integrated into one unit. The above-mentioned integrated units may be implemented in the form of hardware, or may be implemented in the form of software functional units.
所述集成的模块/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存储介质中。基于这样的理解，本发明实现上述实施例方法中的全部或部分流程，也可以通过计算机程序发送指令给相关的硬件完成，所述的计算机程序可存储于一计算机可读存储介质中，该计算机程序在被处理器执行时，可实现上述各个方法实施例的步骤。其中，所述计算机程序包括：计算机程序代码，所述计算机程序代码可以为源代码形式、对象代码形式、可执行文件或某些中间形式等。所述计算机可读存储介质可以包括：能够携带所述计算机程序代码的任何实体或装置、记录介质、U盘、移动硬盘、磁碟、光盘、计算机存储器、只读存储器(ROM，Read-Only Memory)、随机存取存储器(RAM，Random Access Memory)、电载波信号、电信信号以及软件分发介质等。需要说明的是，所述计算机可读存储介质包含的内容可以根据司法管辖区内立法和专利实践的要求进行适当的增减，例如：在某些司法管辖区，根据立法和专利实践，计算机可读介质不包括电载波信号和电信信号。The integrated modules/units, if implemented in the form of software functional units and sold or used as independent products, may be stored in a computer-readable storage medium. Based on this understanding, the present invention realizes all or part of the processes in the methods of the above embodiments, and can also be completed by sending instructions to relevant hardware through a computer program, and the computer program can be stored in a computer-readable storage medium. When the program is executed by the processor, the steps of the foregoing method embodiments can be implemented. Wherein, the computer program includes: computer program code, and the computer program code may be in the form of source code, object code, executable file or some intermediate form, and the like. The computer-readable storage medium may include: any entity or device capable of carrying the computer program code, a recording medium, a U disk, a removable hard disk, a magnetic disk, an optical disk, a computer memory, a read-only memory (ROM, Read-Only Memory) ), random access memory (RAM, Random Access Memory), electric carrier signal, telecommunication signal and software distribution medium, etc. It should be noted that the content contained in the computer-readable storage medium may be appropriately increased or decreased according to the requirements of legislation and patent practice in the jurisdiction, for example: in some jurisdictions, according to legislation and patent practice, the computer can The read medium does not include electrical carrier signals and telecommunication signals.
应当说明的是，上述实施例均可根据需要自由组合。以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。It should be noted that the above embodiments can be freely combined as required. The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
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