CN115876811A - A method for quantitative analysis of the degree of aggregation of colloidal gold labels in electron microscopy - Google Patents
A method for quantitative analysis of the degree of aggregation of colloidal gold labels in electron microscopyDownload PDFInfo
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Abstract
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Translated fromChinese技术领域technical field
本发明涉及电镜胶体金标记技术领域，具体是一种电子显微镜胶体金标记聚集程度定量分析的方法。The invention relates to the technical field of electron microscope colloidal gold labeling, in particular to a method for quantitative analysis of the aggregation degree of electron microscope colloidal gold labels.
背景技术Background technique
物质在生物体内的分布大都具有时空差异性。电子显微镜技术是微观尺度下获取样品位置信息的重要方法。人们用纳米尺度的颗粒状胶体金当做标记物来标识目标物在电镜下的位置，其分布方式存在以下模式：随机、聚集、离散等。而显微条件下获取含量信息的方法主要基于对显微图像的测量，即对特定区域做胶体金颗粒的计数并计算出现频率。在抗原抗体反应的范畴下，目标物-胶体金标签之间的数量关系基本呈线性。基于这种线性关系，在胶体金标记法中不仅可以用胶体金的位置定性描述抗原的位置，还可以用单位面积内金颗粒的数量(即密度)来定量描述抗原的含量。因此利用电镜胶体金标记技术可以同时获取生物样品中特定组分的位置信息和含量信息。但是，现有电镜胶体金标记技术对位置的描述只能是定性的，没有定量描述以及比较各处理组之间聚集程度的方法。电子显微镜拍摄的图像中蕴含着丰富的信息，聚集程度的信息是包含在上述图片当中的。但受限于电镜胶体金标记的图像处理方法的种类不足，以往无法基于电镜胶体金标记的图像对处理组之间的聚集程度进行定量比较。The distribution of substances in organisms mostly has temporal and spatial differences. Electron microscopy is an important method to obtain information on the position of samples at the microscopic scale. People use nanoscale granular colloidal gold as a marker to mark the position of the target under the electron microscope, and its distribution mode has the following modes: random, aggregated, discrete, etc. The method of obtaining content information under microscopic conditions is mainly based on the measurement of microscopic images, that is, counting colloidal gold particles in a specific area and calculating the frequency of occurrence. Under the category of antigen-antibody reaction, the quantitative relationship between the target substance and the colloidal gold label is basically linear. Based on this linear relationship, in the colloidal gold labeling method, not only the position of the colloidal gold can be used to qualitatively describe the position of the antigen, but also the number of gold particles per unit area (ie density) can be used to quantitatively describe the content of the antigen. Therefore, the electron microscope colloidal gold labeling technology can simultaneously obtain the position information and content information of specific components in biological samples. However, the existing electron microscope colloidal gold labeling technology can only describe the position qualitatively, and there is no method for quantitative description and comparison of the degree of aggregation between treatment groups. The images taken by the electron microscope contain a wealth of information, and the information on the degree of aggregation is included in the above pictures. However, limited by the lack of image processing methods for colloidal gold labeling in electron microscopy, it was not possible to quantitatively compare the degree of aggregation between treatment groups based on images of colloidal gold labeling in electron microscopy.
常用于定量描述和比较目标物的聚集程度的方法是平均最近邻分析法(averagenearest neighboring analysis,ANNA)，平均最近邻分析(ANNA法)是一个回归分析，即将实测值和期望值做比较，最后呈现的结果是数值，数值等于1表示目标物随机分布，数值小于1表示聚集，绝对值越小，聚集程度越大；数值大于1表示离散，绝对值越大，离散程度越大。但是，ANNA法不能直接套用到电镜胶体金标记领域，相比数量地理学场景中用到的ANNA法，在电镜胶体金标记中应用ANNA法有其独特性。无论在植物生态学中还是在数量地理学中，用于定量分析的目标物(比如树木、火山口、居民点等)基本都是真实的，是对该研究领域的科学意义有正向贡献的，而电镜胶体金标记由于采用了金颗粒作为标记物代表目标物蛋白的位置，金颗粒作为二次信号是有假阳性的，这给ANNA值的计算带来了困扰，尤其是完全没有阳性，只有假阳性的数据组，其ANNA值是没有生物学意义的，甚至有负面贡献。因此需要找到能够使得平均最近邻分析(ANNA法)适用于电镜胶体金标记场景进行定量分析的方法。The method commonly used to quantitatively describe and compare the aggregation degree of target objects is the average nearest neighbor analysis (ANNA). The result is a value. A value equal to 1 means that the target is randomly distributed. A value less than 1 means aggregation. The smaller the absolute value, the greater the degree of aggregation. The value greater than 1 means discreteness. The larger the absolute value, the greater the degree of dispersion. However, the ANNA method cannot be directly applied to the field of colloidal gold labeling for electron microscopy. Compared with the ANNA method used in quantitative geography scenarios, the application of ANNA method in colloidal gold labeling for electron microscopy has its uniqueness. No matter in plant ecology or in quantitative geography, the targets used for quantitative analysis (such as trees, craters, settlements, etc.) are basically real and have a positive contribution to the scientific significance of this research field , and colloidal gold labeling in electron microscopy uses gold particles as markers to represent the position of the target protein, and gold particles as a secondary signal have false positives, which brings trouble to the calculation of ANNA values, especially no positives at all. For data sets with only false positives, the ANNA value has no biological significance or even has a negative contribution. Therefore, it is necessary to find a method that can make the average nearest neighbor analysis (ANNA method) suitable for quantitative analysis in the electron microscope colloidal gold labeling scene.
发明内容Contents of the invention
本发明要解决的技术问题是提供一种电子显微镜胶体金标记聚集程度定量分析的方法，用以使得平均最近邻分析法法能够适用于电镜胶体金标记场景进行定量分析。The technical problem to be solved by the present invention is to provide a method for quantitative analysis of the aggregation degree of colloidal gold labels in electron microscopy, so that the average nearest neighbor analysis method can be applied to quantitative analysis of colloidal gold labeling scenes in electron microscopy.
为了解决上述技术问题，本发明提供一种电子显微镜胶体金标记聚集程度定量分析的方法，包括的具体过程为：获取透射电镜胶体金标记图片，然后在图形工作站中对电镜胶体金标记图片进行预处理，对于预处理后的电镜胶体金标记图片采用参数依赖的半自动分割金颗粒并手动选取感兴趣区域，然后计算感兴趣区域内的真实金颗粒的面密度，面密度在60-140范围内的电镜胶体金标记被认定为适合后续平均最近邻分析法的阳性标记数据，以此采用平均最近邻分析法计算金颗粒的聚集程度，最终认定ANNA数值在0.6-1.4之间的电镜胶体金标记为合格的阳性标记数据。In order to solve the above-mentioned technical problems, the present invention provides a method for quantitative analysis of the aggregation degree of colloidal gold markers in electron microscopy, which includes the following specific processes: obtaining the colloidal gold marker pictures in transmission electron microscope, and then pre-processing the colloidal gold marker pictures in electron microscope Processing, for the preprocessed electron microscope colloidal gold marked images, use parameter-dependent semi-automatic segmentation of gold particles and manually select the region of interest, and then calculate the surface density of the real gold particles in the region of interest, and the surface density is in the range of 60-140 Electron microscope colloidal gold labeling was identified as positive labeling data suitable for the follow-up average nearest neighbor analysis method, and the average nearest neighbor analysis method was used to calculate the aggregation degree of gold particles, and the electron microscope colloidal gold labeling with an ANNA value between 0.6-1.4 was finally identified as Qualified positive marker data.
作为本发明的一种电子显微镜胶体金标记聚集程度定量分析的方法的改进：As an improvement of the method for the quantitative analysis of the colloidal gold label aggregation degree of an electron microscope of the present invention:
所述预处理包括对所述电镜胶体金标记图片的亮度和对比度进行归一化处理和图片相位反转处理。The preprocessing includes normalizing the brightness and contrast of the electron microscope colloidal gold marked picture and performing picture phase inversion processing.
作为本发明的一种电子显微镜胶体金标记聚集程度定量分析的方法的进一步改进：As a further improvement of the method for the quantitative analysis of the colloidal gold label aggregation degree of an electron microscope of the present invention:
所述半自动分割金颗粒的具体过程为：The concrete process of described semi-automatically dividing gold particle is:
通过三组滤波器对待分割的真实金颗粒和假信号按尺寸因子参数进行筛选获得电镜图片中所有真实胶体金颗粒模型gold total，三组滤波器分别为综合尺寸和信号峰值、金颗粒与背景边界的清晰度以及信号峰值。Through three sets of filters, the real gold particles and false signals to be segmented are screened according to the size factor parameters to obtain the gold total of all real colloidal gold particle models in the electron microscope picture. The three sets of filters are the comprehensive size and signal peak value, gold particles and background boundary. clarity and signal peaks.
作为本发明的一种电子显微镜胶体金标记聚集程度定量分析的方法的进一步改进：As a further improvement of the method for the quantitative analysis of the colloidal gold label aggregation degree of an electron microscope of the present invention:
所述感兴趣区域包括内层感兴趣区域ROI inner和由内层感兴趣区域ROI inner向外环形扩展的环形区域，向外扩展的环形区域为质量控制区ROI peripheral；内层感兴趣区域ROI inner与质量控制区ROI peripheral之和为外层感兴趣区域ROI outer。The region of interest includes an inner layer region of interest ROI inner and an annular area extending outward from the inner layer region of interest ROI inner, and the outwardly expanding annular region is a quality control area ROI peripheral; the inner layer region of interest ROI inner The sum of ROI peripheral and the quality control area is the outer ROI outer.
作为本发明的一种电子显微镜胶体金标记聚集程度定量分析的方法的进一步改进：As a further improvement of the method for the quantitative analysis of the colloidal gold label aggregation degree of an electron microscope of the present invention:
所述真实金颗粒的面密度的计算过程为：The calculation process of the areal density of the real gold particles is:
获取所述电镜胶体金标记图片的内层感兴趣区域的体素inner Number ofvoxel、外层感兴趣区域的体素outer Number of voxel、体素尺寸voxel size、内层感兴趣区域的金颗粒数number of gold inner、外层感兴趣区域的金颗粒数number of goldouter，voxel size包括voxel X和voxel Y；然后依次进行如下计算：Obtain the voxel inner Number of voxel of the inner region of interest, the voxel outer Number of voxel of the outer layer of interest, the voxel size of the voxel size, and the number of gold particles in the inner region of interest of the electron microscope colloidal gold marked picture of gold inner, the number of gold particles in the outer region of interest, and the voxel size includes voxel X and voxel Y; then perform the following calculations in turn:
(1)内层感兴趣区域ROI inner的面积：(1) The area of ROI inner in the inner region of interest:
area of inner＝inner Number of voxel*voxel X*voxel Y (1)area of inner＝inner Number of voxel*voxel X*voxel Y (1)
(2)外层感兴趣区域ROI outer的面积：(2) The area of the outer region of interest ROI outer:
area of outer＝outer Number of voxel*voxel X*voxel Y (2)area of outer=outer Number of voxel*voxel X*voxel Y (2)
(3)质量控制区ROI peripheral的面积：(3) The area of ROI peripheral in the quality control area:
area of peripheral＝area of outer-area of inner (3)area of peripheral＝area of outer-area of inner (3)
(4)基于距离阈值从所述所有真实胶体金颗粒模型gold total中分拣出内层感兴趣区域(ROI inner)中分布的金颗粒模型，包括spots close to ROI inner模型和spotsfar to ROI inner模型；(4) Sorting out the gold particle models distributed in the inner region of interest (ROI inner) from the all real colloidal gold particle models gold total based on the distance threshold, including the spots close to ROI inner model and the spotsfar to ROI inner model ;
基于距离阈值从所述所有真实胶体金颗粒模型中分拣出外层感兴趣区域ROIouter中分布的金颗粒模型，包括spots close to ROI outer和spots far to ROI outer模型；Sorting out the gold particle models distributed in the outer region of interest ROIouter from all the real colloidal gold particle models based on the distance threshold, including spots close to ROI outer and spots far to ROI outer models;
(5)质量控制区ROI peripheral中真实金颗粒模型的数量：(5) The number of real gold particle models in the quality control area ROI peripheral:
number of gold peripheral＝number of gold outer-number of gold inner(4)number of gold peripheral＝number of gold outer-number of gold inner (4)
(6)内层感兴趣区域ROI inner中真实金颗粒的面密度：(6) Surface density of real gold particles in ROI inner of the inner region of interest:
areal density inner＝number of gold inner/area of inner (5)areal density inner=number of gold inner/area of inner (5)
(7)质量控制区ROI peripheral中真实金颗粒的面密度：(7) Surface density of real gold particles in ROI peripheral in the quality control area:
areal density peripheral＝number of gold peripheral/area ofperipheral (6)areal density peripheral＝number of gold peripheral/area of peripheral (6)
(8)背底扣除参数的计算：(8) Calculation of background deduction parameters:
ratio peripheral/inner＝areal density peripheral/areal density inner(7)ratio peripheral/inner＝areal density peripheral/areal density inner (7)
(9)扣除完背底后的真实金颗粒模型的面密度：(9) The surface density of the real gold particle model after deducting the background:
areal density＝ areal density inner-areal density peripheral (8)areal density＝ areal density inner-areal density peripheral (8)
(10)真实金颗粒的面密度areal density大于30个/平方微米的样本被认定为阳性，选取areal density数值为60-140个/平方微米的样本用于所述金颗粒的聚集程度的计算。(10) Samples with an areal density of real gold particles greater than 30 per square micron are considered positive, and samples with an areal density of 60-140 per square micron are selected for the calculation of the aggregation degree of the gold particles.
作为本发明的一种电子显微镜胶体金标记聚集程度定量分析的方法的进一步改进：As a further improvement of the method for the quantitative analysis of the colloidal gold label aggregation degree of an electron microscope of the present invention:
所述计算金颗粒的聚集程度的过程为：The process of calculating the degree of aggregation of gold particles is:
(1)、显示所述内层感兴趣区域ROI inner内spots close to ROI inner模型并作为平均最近邻分析法计算的金颗粒对象gold ANNA，以最外围金颗粒模型的点连线形成一个封闭的折线区域作为平均最近邻分析法的计算区域ROI ANNA；(1) Display the spots close to ROI inner model in the ROI inner of the inner region of interest and calculate it as the gold particle object gold ANNA calculated by the average nearest neighbor analysis method, and form a closed circle with the point connection line of the outermost gold particle model The broken line area is used as the calculation area ROI ANNA of the average nearest neighbor analysis method;
(2)获取平均最近邻分析法的计算区域ROI ANNA的number of voxel参数；(2) Obtain the number of voxel parameters of the calculation region ROI ANNA of the average nearest neighbor analysis method;
(3)平均最近邻分析法的计算区域ROI ANNA的面积计算：(3) Calculate the area of ROI ANNA in the calculation area of the average nearest neighbor analysis method:
area of ANNA＝number of voxel*voxel size (9)area of ANNA＝number of voxel*voxel size (9)
(4)平均最近邻分析法中回归分析期望值De的计算：(4) Calculation of regression analysis expectation value De in average nearest neighbor analysis method:
其中Nr gold表示平均最近邻分析法的计算区域ROI ANNA中金颗粒的个数；Where Nr gold represents the number of gold particles in the calculation area ROI ANNA of the average nearest neighbor analysis method;
(5)对平均最近邻分析法中金颗粒的实际间距值
(6)、计算金颗粒的聚集程度参数：(6), calculate the degree of aggregation parameters of gold particles:
(7)、ANNA值在0.6-1.4之间的样本被认定为有效样本。(7) The samples with ANNA value between 0.6-1.4 are considered valid samples.
本发明的有益效果主要体现在：The beneficial effects of the present invention are mainly reflected in:
1、本发明相比数量地理学场景中用到的ANNA法，通过金颗粒的面密度和聚集程度的计算筛选出合适的金颗粒的阳性标记数据，排除了假阳性对ANNA定量分析的干扰；1. Compared with the ANNA method used in the quantitative geography scene, the present invention screens out the positive label data of suitable gold particles through the calculation of the surface density and aggregation degree of the gold particles, and eliminates the interference of false positives on the quantitative analysis of ANNA;
2、植物生态学和数量地理学中目标物的密度较难做出方便的调整，因此ANNA计算会受到目标物密度过高或过低的干扰，而本发明建立了金颗粒浓度(以参数面密度表示)的适用范围为60-140个/平方微米，此参数既能排除假阳性对ANNA计算的干扰，又能避免标记阳性率过高，金颗粒过分密集导致真实的聚集情况被掩盖，反映为表征聚集程度的ANNA值向离散方向偏移；2, the density of target object in plant ecology and quantitative geography is more difficult to make convenient adjustment, so ANNA calculates and can be subject to the interference of target object density too high or too low, and the present invention establishes gold particle concentration (with parameter surface The applicable range of density is 60-140 per square micron. This parameter can not only eliminate the interference of false positives on ANNA calculation, but also avoid the high positive rate of markers. The excessive density of gold particles will cause the real aggregation to be covered up, reflecting the The ANNA value representing the degree of aggregation is shifted to the discrete direction;
3、电镜胶体金标记的目标物是人为加入的标记物，其密度可调节，本发明通过适宜体现ANNA效果的电镜胶体金标记的面密度值的区间范围，将目标物调整到合适的密度，以利于ANNA计算达成最优条件。3. The target object marked by electron microscope colloidal gold is an artificially added marker, and its density can be adjusted. The present invention adjusts the target object to a suitable density through the interval range of the area density value of the electron microscope colloidal gold mark that is suitable for reflecting the ANNA effect. In order to facilitate ANNA calculation to achieve the optimal conditions.
附图说明Description of drawings
下面结合附图对本发明的具体实施方式作进一步详细说明。The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.
图1为本发明一种电子显微镜胶体金标记聚集程度定量分析的方法的流程示意图；Fig. 1 is a schematic flow sheet of the method for the quantitative analysis of the colloidal gold marker aggregation degree of an electron microscope of the present invention;
图2为平均最近邻分析(ANNA)法的计算区域ROI ANNA的示意图；Fig. 2 is the schematic diagram of the calculation area ROI ANNA of average nearest neighbor analysis (ANNA) method;
图3为一个特定的金颗粒模型与金颗粒对象gold ANNA中其他每一个金颗粒模型的间距的示意图；Fig. 3 is a schematic diagram of the spacing between a specific gold particle model and every other gold particle model in the gold particle object gold ANNA;
图4为利用已知聚集/分散模式的蛋白收集数据应用于本发明的方法进行验证的实验策略示意；Fig. 4 is a schematic diagram of the experimental strategy of using the protein collection data of known aggregation/dispersion mode to verify the method of the present invention;
图5为控制膜弯曲形成囊泡的蛋白甾醇甲基转移酶的电镜胶体金标记结果的示意图；Figure 5 is a schematic diagram of electron microscope colloidal gold labeling results of protein sterol methyltransferase that controls membrane bending to form vesicles;
图6为番茄斑萎病毒TSWV内含体蛋白Nss的电镜胶体金标记结果的示意图；Fig. 6 is the schematic diagram of electron microscope colloidal gold labeling result of tomato spotted wilt virus TSWV inclusion body protein Nss;
图7为运用ANNA法对已知分布模式的蛋白做电镜胶体金标记聚集程度定量分析的结果的示意图；Figure 7 is a schematic diagram of the results of the quantitative analysis of the colloidal gold-labeled aggregation degree of electron microscopy for proteins with known distribution patterns using the ANNA method;
图8为ANNA法应用于电镜胶体金标记聚集程度定量分析中不可信的假阳性组(Gn,Gc)的ANNA值分布的示意图Figure 8 is a schematic diagram of the ANNA value distribution of the unreliable false positive group (Gn, Gc) in the quantitative analysis of the colloidal gold labeled aggregation degree using the ANNA method
具体实施方式Detailed ways
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:
实施例1、一种电子显微镜胶体金标记聚集程度定量分析的方法，如图1所示，具体过程如下：Embodiment 1. A method for quantitative analysis of the degree of aggregation of colloidal gold labels by electron microscopy, as shown in Figure 1, the specific process is as follows:
1、图片预处理1. Image preprocessing
通过透射电子显微镜获取电镜胶体金标记图片，然后在图形工作站对电镜胶体金标记图片进行预处理，预处理包括亮度和对比度的归一化，然后对图片进行相位反转，使金颗粒成为信号峰值。Obtain electron microscope colloidal gold-labeled images through a transmission electron microscope, and then preprocess the electron microscope colloidal gold-labeled images on a graphics workstation. Preprocessing includes normalization of brightness and contrast, and then phase inverts the images to make gold particles become signal peaks .
归一化的方法是通过Imaris软件中Image process菜单中的normalize命令；相位反转的方法是通过Imaris软件中Image process菜单中的invert命令。常用图像处理软件均可对图像进行归一化和相位反转的操作。本案使用的Imaris为牛津仪器集团(OxfordInstrument Group)的交互式显微镜图像分析软件。The normalization method is through the normalize command in the Image process menu in the Imaris software; the phase inversion method is through the invert command in the Image process menu in the Imaris software. Commonly used image processing software can perform normalization and phase inversion operations on images. Imaris used in this case is an interactive microscope image analysis software from Oxford Instrument Group.
2、对于预处理后的电镜胶体金标记图片计算金颗粒的面密度2. Calculate the surface density of gold particles for the preprocessed electron microscope colloidal gold marked image
金颗粒的面密度包括内层感兴趣区域(ROI inner)的面密度和内层感兴趣区域外周100纳米环形区域内的面密度，其中内层感兴趣区域外周100纳米环形区域作为质量控制区(ROI peripheral)，用于背底扣除，提高电镜胶体金标记的信噪比，具体过程为：The areal density of gold particles comprises the areal density of the inner region of interest (ROI inner) and the areal density in the 100 nanometer ring region around the inner region of interest, wherein the 100 nanometer ring region around the inner region of interest is used as the quality control area ( ROI peripheral), used for background subtraction to improve the signal-to-noise ratio of colloidal gold labeling in electron microscopy, the specific process is:
2.1、金颗粒参数依赖的半自动分割渲染2.1. Semi-automatic segmentation rendering based on parameters of gold particles
通过Imaris软件的spots算法分割金颗粒，尺寸因子参数为10纳米，使用3个滤波器对待分割的真实金颗粒和假信号进行筛选：其中quality函数通过综合尺寸和信号峰值两个参数进行筛选；intensity STDEV函数通过检测金颗粒与背景边界的清晰度来筛选；intensity median函数通过检测信号峰值来进行筛选。最终得到一张电镜图片中所有真实胶体金颗粒模型gold total。The gold particles are segmented by the spots algorithm of the Imaris software, and the size factor parameter is 10 nanometers. Three filters are used to screen the real gold particles and false signals to be segmented: the quality function is used to screen the two parameters of the integrated size and signal peak value; intensity The STDEV function is screened by detecting the definition of the boundary between gold particles and the background; the intensity median function is screened by detecting the peak value of the signal. Finally, the gold total of all real colloidal gold particle models in an electron microscope picture is obtained.
2.2、通过Imaris软件的surface算法手动分割各个感兴趣区域(ROI)，包括内层感兴趣区域(ROI inner)和由内层感兴趣区域向外环形扩展100纳米的环形区域，向外环形扩展100纳米的环形区域作为背底扣除的质量控制区(ROI peripheral)(质量控制区不包含内层感兴趣区域)。具体方法如下：2.2. Manually segment each region of interest (ROI) through the surface algorithm of Imaris software, including the inner region of interest (ROI inner) and the ring area extending 100 nanometers from the inner region of interest to the outside, and extending 100 nanometers to the outside. The nano ring area is used as the quality control area (ROI peripheral) for background subtraction (the quality control area does not include the inner region of interest). The specific method is as follows:
在Imaris软件中打开surface模块的edit标签页下的contour功能，使鼠标处于select模式下点击draw，沿感兴趣区域边界绘制折线直至封闭，得到内层感兴趣区域(ROIinner)的surface模型。然后再新建一个surface算法，沿内层感兴趣区域(ROI inner)边界向外推约100纳米，绘制封闭折线，得到外层感兴趣区域(ROI outer)的surface模型，其中，向外扩展100纳米的环形区域为质量控制区(ROI peripheral)，外层感兴趣区域(ROIouter)为内层感兴趣区域(ROI inner)与质量控制区(ROI peripheral)之和。Open the contour function under the edit tab of the surface module in the Imaris software, put the mouse in the select mode and click draw, draw a polyline along the boundary of the region of interest until it is closed, and obtain the surface model of the inner region of interest (ROIinner). Then create a new surface algorithm, extrapolate about 100 nanometers along the boundary of the inner region of interest (ROI inner), draw a closed polyline, and obtain the surface model of the outer region of interest (ROI outer), where the outward extension is 100 nanometers The circular area of is the quality control area (ROI peripheral), and the outer ROI (ROIouter) is the sum of the inner ROI (ROI inner) and the quality control area (ROI peripheral).
2.3、通过Imaris软件获取图片的内层感兴趣区域的体素inner Number ofvoxel、外层感兴趣区域的体素outer Number of voxel、体素尺寸voxel size、内层感兴趣区域的金颗粒数number of gold inner、外层感兴趣区域的金颗粒数number of goldouter等参数。2.3. Obtain the inner Number of voxel of the inner region of interest, the outer Number of voxel of the outer region of interest, voxel size, and the number of gold particles in the inner region of interest through the Imaris software parameters such as gold inner, the number of gold particles in the outer region of interest, and the number of goldouter.
其中，voxel size包括voxel X、voxel Y，为Imaris软件打开图片属性的geometry后读取图片参数自动获得。Among them, the voxel size includes voxel X and voxel Y, which are automatically obtained by reading the image parameters after opening the geometry of the image attribute for the Imaris software.
inner Number of voxel,outer Number of voxel和number of gold inner、number of gold outer分别为实际测量的金颗粒聚集区域的相应数值。Inner Number of voxel, outer Number of voxel and number of gold inner, number of gold outer are the corresponding values of the actually measured gold particle aggregation area.
2.4、计算内层感兴趣区域(ROI inner)的面积：2.4. Calculate the area of the inner region of interest (ROI inner):
area of inner＝inner Number of voxel*voxel X*voxel Y (1)area of inner＝inner Number of voxel*voxel X*voxel Y (1)
2.5、计算外层感兴趣区域(ROI outer)的面积：2.5. Calculate the area of the outer region of interest (ROI outer):
area of outer＝outer Number of voxel*voxel X*voxel Y (2)area of outer=outer Number of voxel*voxel X*voxel Y (2)
2.6、计算质量控制区(ROI peripheral)的面积：2.6. Calculate the area of the quality control area (ROI peripheral):
area of peripheral＝area of outer-area of inner (3)area of peripheral＝area of outer-area of inner (3)
2.7、利用基于距离阈值对模型进行分拣的算法(Imaris软件的XTension模块中的find spots close to surface插件)对所有真实胶体金颗粒模型gold total进行分拣，对于内层感兴趣区域(ROI inner)和外层感兴趣区域(ROI outer)均需进行分拣操作，分别得到分布于内层感兴趣区域(ROI inner)内部和外部的金颗粒模型组，以及分布于外层感兴趣区域(ROI outer)内部和外部的金颗粒模型组。2.7. Use the algorithm for sorting models based on the distance threshold (the find spots close to surface plug-in in the XTension module of Imaris software) to sort all real colloidal gold particle models gold total, and for the inner region of interest (ROI inner ) and the outer region of interest (ROI outer) need to be sorted to obtain the gold particle model groups distributed inside and outside the inner region of interest (ROI inner), and the gold particle model group distributed in the outer region of interest (ROI inner) outer) The inner and outer gold particle model groups.
操作对象为总spots和相应的surface，输入距离因子(放大倍数4万倍，voxelsize 9纳米时为0.1，不同倍数下略有变动)。分拣操作自动生成四个新spots模型，即spotsclose to ROI inner、spots far to ROI inner、spots close to ROI outer和spots farto ROI outer四个模型。The operation object is the total spots and the corresponding surface, and the distance factor is input (the magnification is 40,000 times, and the voxelsize is 0.1 when the voxelsize is 9 nanometers, and it changes slightly under different magnifications). The sorting operation automatically generates four new spots models, namely spots close to ROI inner, spots far to ROI inner, spots close to ROI outer and spots farto ROI outer.
2.8、计算用于背底扣除的质量控制区(ROI peripheral)中真实金颗粒模型的数量：2.8. Calculate the number of real gold particle models in the quality control area (ROI peripheral) for background subtraction:
number of gold peripheral＝number of gold outer-number of gold inner(4)number of gold peripheral＝number of gold outer-number of gold inner (4)
2.9、计算内层感兴趣区域(ROI inner)中真实金颗粒的面密度：2.9. Calculate the surface density of real gold particles in the inner region of interest (ROI inner):
areal density inner＝number of gold inner/area of inner (5)areal density inner=number of gold inner/area of inner (5)
2.10、计算用于背底扣除的质量控制区(ROI peripheral)中真实金颗粒的面密度：2.10. Calculate the surface density of real gold particles in the quality control area (ROI peripheral) for background subtraction:
areal density peripheral＝number of gold peripheral/area ofperipheral (6)areal density peripheral＝number of gold peripheral/area of peripheral (6)
2.11、背底扣除参数的计算：2.11. Calculation of background deduction parameters:
ratio peripheral/inner＝areal density peripheral/areal density inner(7)ratio peripheral/inner＝areal density peripheral/areal density inner (7)
2.12、最终得到扣除完背底后的真实金颗粒的面密度：2.12. Finally, the surface density of the real gold particles after deducting the background is obtained:
areal density＝ areal density inner-areal density peripheral (8)areal density＝ areal density inner-areal density peripheral (8)
2.13、扣除完背底后真实金颗粒的面密度areal density大于30个/平方微米的样本被认定为阳性，选取areal density数值中为60-140个/平方微米的样本用于后续聚集程度定量分析。2.13. Samples with an areal density of real gold particles greater than 30/square micron after deducting the background are considered positive, and samples with an areal density of 60-140/square micron are selected for subsequent quantitative analysis of the degree of aggregation .
3.对于预处理后的电镜胶体金标记图片进行平均最近邻分析，计算金颗粒的聚集程度，具体过程为3. Perform average nearest neighbor analysis on the preprocessed electron microscope colloidal gold-labeled images to calculate the degree of aggregation of gold particles. The specific process is
3.1、利用Imaris软件确定平均最近邻分析(ANNA法)的计算区域：显示内层感兴趣区域(ROI inner)内spots close to ROI inner模型，将其作为ANNA计算的金颗粒对象(gold ANNA)，以最外围金颗粒模型的点连线(使得围成的面积尽可能大)形成一个封闭的折线区域，如图2中虚线围成的区域所示，得到一个surface模型作为平均最近邻分析(ANNA)法的计算区域(ROI ANNA)；3.1. Use Imaris software to determine the calculation area of the average nearest neighbor analysis (ANNA method): display the spots close to ROI inner model in the inner region of interest (ROI inner), and use it as the gold particle object (gold ANNA) calculated by ANNA, Form a closed polyline area with the dotted line of the outermost gold particle model (so that the enclosed area is as large as possible), as shown in the area enclosed by the dotted line in Figure 2, obtain a surface model as the average nearest neighbor analysis (ANNA ) calculation area (ROI ANNA);
3.2、通过Imaris软件获取平均最近邻分析(ANNA法)的计算区域(ROI ANNA)的体素number of voxel参数；3.2. Obtain the voxel number of voxel parameter of the calculation area (ROI ANNA) of the average nearest neighbor analysis (ANNA method) by Imaris software;
3.3、平均最近邻分析(ANNA)法的计算区域(ROI ANNA)的面积计算：3.3. Calculate the area of the calculation region (ROI ANNA) of the average nearest neighbor analysis (ANNA) method:
area of ANNA＝number of voxel*voxel size (9)area of ANNA＝number of voxel*voxel size (9)
3.4、平均最近邻分析(ANNA)法分析中回归分析期望值De的计算：3.4. Calculation of expected value De of regression analysis in average nearest neighbor analysis (ANNA) analysis:
其中Nr gold表示平均最近邻分析(ANNA)法的计算区域(ROI ANNA)中金颗粒的个数，由Imaris对每张电镜胶体金标记图片实测而获得，area of ANNA表示平均最近邻分析(ANNA)法的计算区域(ROI ANNA)的面积，由Imaris对每张电镜胶体金标记图片实测而获得。Wherein Nr gold represents the number of gold particles in the calculation area (ROI ANNA) of the average nearest neighbor analysis (ANNA) method, which is obtained by Imaris on the actual measurement of each electron microscope colloidal gold marked picture, and area of ANNA represents the average nearest neighbor analysis (ANNA ) method to calculate the area (ROI ANNA), which is obtained by Imaris from the actual measurement of each electron microscope colloidal gold marked picture.
3.5、利用软件Imaris对ANNA分析中回归分析实测值
3.6、ANNA值的最终计算，即计算金颗粒的聚集程度：3.6. The final calculation of the ANNA value, that is, the calculation of the degree of aggregation of gold particles:
ANNA＝Do/De (11)ANNA＝Do/De (11)
3.7、将步骤2获得的areal density数值为60-140个/平方微米的样本依次按步骤3.1-3.6计算获得样本的金颗粒的聚集程度ANNA。3.7. The samples whose areal density value obtained in step 2 is 60-140 pieces/square micron are calculated according to steps 3.1-3.6 to obtain the aggregation degree ANNA of the gold particles of the sample.
3.8、聚集程度ANNA值在0.6-1.4之间的样本被认定为有效样本，最终共选取50个有效样本用于后续科学意义的分析。3.8. The samples whose aggregation degree ANNA value is between 0.6-1.4 are identified as effective samples, and finally a total of 50 effective samples are selected for subsequent analysis of scientific significance.
4、电镜胶体金标记的ANNA方法使用4. Use of the ANNA method labeled with colloidal gold for electron microscopy
基于步骤2获得的金颗粒的面密度和步骤3获得的金颗粒的聚集程度，采用面密度是否在60-140个/平方微米范围内来判断该电镜胶体金标记的数据适合用步骤3的平均最近邻分析，然后计算并获得ANNA数值，采用ANNA数值是否在0.6-1.4之间的电镜胶体金标记以此来判断电镜胶体金标记实验是否为合格的阳性标记，以及金颗粒所标识的目标蛋白是否以聚集的形式分布于囊泡中，或是以离散的形式分布于细胞器基质中，亦或是二种分布模式兼具。Based on the areal density of the gold particles obtained in step 2 and the degree of aggregation of the gold particles obtained in step 3, whether the areal density is in the range of 60-140/square micron is used to judge whether the electron microscope colloidal gold label data is suitable for the average of step 3 Nearest neighbor analysis, and then calculate and obtain the ANNA value, and use the electron microscope colloidal gold labeling with the ANNA value between 0.6-1.4 to judge whether the electron microscope colloidal gold labeling experiment is a qualified positive label, and the target protein identified by the gold particles Whether it is distributed in vesicles in an aggregated form, distributed in the organelle matrix in a discrete form, or both.
相比其他场景使用ANNA方法(例如数量地理学中用到的ANNA法)，在电镜胶体金标记中应用ANNA法有其独特性，即需要用针对电镜胶体金标记的方法排除假阳性标记数据的干扰。而数量地理学中假阳性数据对ANNA值可靠性的干扰一般被忽略。本方法用面密度参数的高低，以及ANNA数值所在的区间，判断电镜胶体金标记是否为阳性标记。面密度(单位：个/平方微米)值高于30认可为阳性，低于30为假阳性，高于60为典型阳性。预实验结果表明，典型阳性数据组的ANNA值在0.6-1.4之间，而假阳性数据组的ANNA值高于1.7。应用于电镜胶体金标记的ANNA法只在面密度高于60，ANNA值在0.6-1.4之间的数据组中进行；面密度低于60，ANNA值高于1.7的数据组不被信任，弃之不用。Compared with the ANNA method used in other scenarios (such as the ANNA method used in quantitative geography), the application of the ANNA method in colloidal gold labeling for electron microscopy has its uniqueness, that is, it is necessary to use the method for colloidal gold labeling for electron microscopy to exclude false positive labeling data. interference. However, in quantitative geography, the interference of false positive data on the reliability of ANNA values is generally ignored. This method judges whether the electron microscope colloidal gold label is a positive label by using the level of the surface density parameter and the interval of the ANNA value. Area density (unit: piece/square micron) value higher than 30 is recognized as positive, lower than 30 is false positive, and higher than 60 is typical positive. The pre-experimental results show that the ANNA value of the typical positive data set is between 0.6-1.4, while the ANNA value of the false positive data set is higher than 1.7. The ANNA method applied to electron microscope colloidal gold labeling is only performed in data sets with an areal density higher than 60 and an ANNA value between 0.6 and 1.4; data sets with an areal density lower than 60 and an ANNA value higher than 1.7 are not trusted and discarded. no need.
在电镜胶体金标记中应用ANNA法定量分析目标物聚集程度，可以人为调节标记物金颗粒的标记效率(体现为面密度)至适宜体现ANNA效果的区间范围。面密度(单位：个/平方微米)60-140为适宜体现ANNA效果的区间范围。面密度低于30为假阳性，数据不可信；面密度高于140，目标物太密集，ANNA有从聚集向随机偏移的趋势，造成假象。因此在电镜胶体金标记的操作中，控制抗体的浓度和标记的条件，使得标记的面密度在60-140范围内，方能较好体现ANNA分析的效果。Applying the ANNA method to quantitatively analyze the aggregation degree of the target substance in electron microscope colloidal gold labeling can artificially adjust the labeling efficiency (reflected as surface density) of the marker gold particles to an interval suitable for reflecting the effect of ANNA. Area density (unit: piece/square micron) 60-140 is the interval range suitable for reflecting the effect of ANNA. If the area density is lower than 30, it is a false positive, and the data is not credible; if the area density is higher than 140, the target is too dense, and ANNA tends to shift from aggregation to random, causing false impressions. Therefore, in the operation of electron microscope colloidal gold labeling, the concentration of the antibody and the conditions of labeling should be controlled so that the surface density of the label is in the range of 60-140, which can better reflect the effect of ANNA analysis.
综上所述，ANNA法应用于电镜胶体金标记过程中，在确保真实阳性的情况下，金颗粒浓度(以参数面密度表示)的适用范围为60-140个/平方微米。此参数既能排除假阳性对ANNA计算的干扰，又能避免标记阳性率过高，金颗粒过分密集导致真实的聚集情况被掩盖，反映为表征聚集程度的ANNA值向离散方向偏移。To sum up, the ANNA method is applied in the colloidal gold labeling process of electron microscopy. Under the condition of ensuring true positive, the applicable range of gold particle concentration (expressed by parameter surface density) is 60-140 particles/square micron. This parameter can not only eliminate the interference of false positives on ANNA calculation, but also avoid the high positive rate of markers. The excessive density of gold particles will cause the real aggregation to be covered up, which is reflected in the deviation of the ANNA value representing the degree of aggregation to the discrete direction.
实验：experiment:
本实验为利用已知聚集/分散模式的蛋白收集数据进行ANNA法应用于本发明的电子显微镜胶体金标记聚集程度定量分析的方法的验证。选用囊泡相关(具有聚集的性质)或不相关(不具有聚集的性质)的目标物进行金标记，利用本发明的电子显微镜胶体金标记聚集程度定量分析的方法进行计算获得其聚集程度的定量数据，实验策略如图4所示。囊泡相关的目标物包括：内质网定位信号四氨基酸短肽(名称为HDEL)、控制膜弯曲形成囊泡的蛋白甾醇甲基转移酶(名称为SMT1)(电镜胶体金标记结果见图5，金颗粒呈聚集分布状态)、生物质膜相关的细胞骨架微丝蛋白(名称为actin)。非囊泡相关的目标物包括：番茄斑萎病毒TSWV内含体蛋白(名称为Nss)(电镜胶体金标记结果见图6，金颗粒呈离散分布状态)。与囊泡和非囊泡都有关系的目标物双链RNA(名称为J2)，它既存在囊泡定位(TSWV是RNA病毒，其核酸复制需在囊泡中完成，因此在囊泡中存在病毒产生双链RNA中间体)又存在非囊泡定位(寄主的双链RNA)。另外还设置两个完全没有特异性的阴性对照：TSWV编码蛋白，名称为Gc和Gn(其定位与囊泡无关，也与纤维状内含体无关，在纤维状物质的目标区域上没有阳性标记，只有非特异的假阳性标记)用于验证非特异标记金颗粒数量稀少时该ANNA方法是否与阳性标记没有区分度。双链RNA(J2)做了两个标记浓度，用于达到差异明显的面密度。一个标记叫J2-dilute30，电镜胶体金标记的面密度为220±120；一个标记叫J2-dilute50，电镜胶体金标记的面密度为100±40。共计7组数据，每组重复拍摄50张图片，分别用本发明的电子显微镜胶体金标记聚集程度定量分析的方法进行金颗粒聚集程度的分析，对应地获得HDEL、SMT1、Nss、J2-dilute30、J2-dilute50、Gc和Gn共7组金颗粒的聚集程度数据，绘制成小提琴图，如图7所示，其中SMT1的电镜胶体金标记为典型的金颗粒聚集分布的数据；而Nss的电镜胶体金标记为典型的金颗粒离散分布的数据，如图7和图8所示。分布于囊泡中的HDEL蛋白和SMT1蛋白，其ANNA值显著小于1，表明其有强烈的聚集趋势。SMT1蛋白的聚集程度更甚于HDEL蛋白。且代表SMT1蛋白位置的金颗粒ANNA值呈多簇现象，表明其聚集程度的多样性，可以分为明显的三类。分布于纤维上的Nss，其ANNA值显著大于1，表明其有强烈的离散趋势。而双链RNA(J2-dilu50)从理论上讲有两种分布模式，病毒来源的双链RNA可能分布于囊泡中，寄主来源的双链RNA则不分布于囊泡中。ANNA结果显示其值横跨1，呈现明显的两簇，既有聚集又有离散，暗示其由分布模式不同的两类组成(图7)。在目标物纤维状物质上电镜胶体金标记呈假阳性的蛋白Gn和Gc，其ANNA均值中位数高于1.8，且在小提琴图中峰形怪异(图8Gn和Gc组)，该结果与真实阳性结果的区分度很大，能够轻易被筛选出来弃之不用。这是电镜胶体金标记中本发明应用ANNA方法相对于其他领域应用ANNA方法的独特处理。其中分布于囊泡中的HDEL和SMT1，其ANNA值显著小于1，表明其有强烈的聚集趋势；分布于纤维上的Nss，其ANNA值显著大于1，表明其有强烈的离散趋势；不同来源的双链RNA(J2-dilu50)有两种分布模式，其ANNA值横跨1，在聚集和离散区域形成明显的两簇(图7小提琴图中J2-dilu50组膨胀的区域)。This experiment is to use the data collected from proteins with known aggregation/dispersion modes to verify the application of the ANNA method to the quantitative analysis of the degree of aggregation of colloidal gold labels in electron microscopy of the present invention. Select vesicle-related (with the property of aggregation) or irrelevant (without the property of aggregation) for gold labeling, and use the method for quantitative analysis of the degree of aggregation of colloidal gold markers in electron microscopy of the present invention to calculate and obtain the quantification of the degree of aggregation The data and experimental strategy are shown in Figure 4. Vesicle-related targets include: endoplasmic reticulum localization signal four-amino acid short peptide (named HDEL), protein sterol methyltransferase (named SMT1) that controls membrane bending to form vesicles (results of colloidal gold labeling by electron microscopy are shown in Figure 5 , gold particles are aggregated and distributed), and biofilm-associated cytoskeleton actin (named actin). Non-vesicle-related targets include: tomato spotted wilt virus TSWV inclusion body protein (named Nss) (the results of colloidal gold labeling by electron microscopy are shown in Figure 6, and the gold particles are in a discrete distribution state). The target double-stranded RNA (named J2), which is related to both vesicles and non-vesicles, has both vesicle localization (TSWV is an RNA virus, and its nucleic acid replication needs to be completed in vesicles, so it exists in vesicles Viruses produce double-stranded RNA intermediates) and there is a non-vesicular localization (double-stranded RNA of the host). In addition, two negative controls with no specificity were set up: TSWV encoded proteins, named Gc and Gn (its localization has nothing to do with vesicles, nor with fibrous inclusions, and there is no positive label on the target area of fibrous substances , only non-specific false positive markers) is used to verify whether the ANNA method is indistinguishable from positive markers when the number of non-specific marker gold particles is rare. Double-stranded RNA (J2) was labeled at two concentrations to achieve significantly different areal densities. One mark is called J2-dilute30, and the surface density of the electron microscope colloidal gold mark is 220±120; the other mark is called J2-dilute50, and the surface density of the electron microscope colloidal gold mark is 100±40. A total of 7 sets of data, 50 pictures were repeatedly taken in each group, and the method for quantitative analysis of the aggregation degree of colloidal gold markers of the electron microscope of the present invention was used to analyze the aggregation degree of gold particles, and correspondingly obtained HDEL, SMT1, Nss, J2-dilute30, The aggregation degree data of 7 groups of gold particles including J2-dilute50, Gc and Gn are plotted as a violin diagram, as shown in Figure 7, in which SMT1’s electron microscope colloidal gold is marked as the typical gold particle aggregation distribution data; and Nss’ electron microscope colloidal gold Gold is marked as data of a typical discrete distribution of gold particles, as shown in Figures 7 and 8. The ANNA value of HDEL protein and SMT1 protein distributed in vesicles was significantly less than 1, indicating that they had a strong tendency to aggregate. The degree of aggregation of SMT1 protein is more severe than that of HDEL protein. Moreover, the ANNA value of the gold particles representing the position of the SMT1 protein showed a multi-cluster phenomenon, indicating the diversity of its aggregation degree, which can be divided into three obvious categories. The ANNA value of Nss distributed on the fiber is significantly greater than 1, indicating that it has a strong dispersion tendency. The double-stranded RNA (J2-dilu50) theoretically has two distribution modes. The double-stranded RNA derived from the virus may be distributed in the vesicles, and the double-stranded RNA derived from the host is not distributed in the vesicles. The ANNA results show that its value spans 1, showing two distinct clusters, both clustered and discrete, implying that it is composed of two types with different distribution patterns (Figure 7). Colloidal gold-labeled false-positive proteins Gn and Gc on the fibrous substance of the target have an average median value of ANNA higher than 1.8, and a strange peak shape in the violin plot (Fig. Positive results are highly differentiated and can be easily screened out and discarded. This is the unique treatment of the application of the ANNA method in the electron microscope colloidal gold labeling compared to the application of the ANNA method in other fields. Among them, the ANNA value of HDEL and SMT1 distributed in vesicles is significantly less than 1, indicating that they have a strong tendency to aggregate; the ANNA value of Nss distributed on fibers is significantly greater than 1, indicating that they have a strong tendency to disperse; different sources The double-stranded RNA (J2-dilu50) had two distribution patterns with ANNA values spanning 1, forming distinct two clusters in aggregated and discrete regions (Fig.
利用不同的J2双链RNA抗体稀释浓度，对电镜胶体金标记颗粒不同面密度对本发明的ANNA分析的影响做了研究。J2-dilu50是抗体稀释50倍，达成金颗粒面密度100±40，其ANNA中位数略微小于1.0。而出了抗体浓度，其他完全一致的标记J2-dilu30(抗体稀释30倍)，其金颗粒面密度220±120，ANNA中位数略微大于1.0。表明随着抗体变浓金标阳性率增加，金颗粒的面密度增加，会使ANNA数值往离散方向偏移(图7，J2-dilu50组和J2-dilu30组)。电镜胶体金标记所达成的阳性率在金颗粒面密度100±40时较为适宜用于本发明的ANNA分析。Using different dilution concentrations of the J2 double-stranded RNA antibody, the influence of different surface densities of electron microscope colloidal gold-labeled particles on the ANNA analysis of the present invention was studied. J2-dilu50 is a 50-fold dilution of the antibody to achieve a surface density of gold particles of 100±40, and its median ANNA is slightly less than 1.0. In addition to the antibody concentration, the other completely consistent marker J2-dilu30 (antibody diluted 30 times), its surface density of gold particles is 220±120, and the median of ANNA is slightly greater than 1.0. It shows that as the concentration of the antibody increases, the positive rate of the gold label increases, and the surface density of the gold particles increases, which will shift the ANNA value to the discrete direction (Figure 7, J2-dilu50 group and J2-dilu30 group). The positive rate achieved by electron microscope colloidal gold labeling is more suitable for the ANNA analysis of the present invention when the surface density of gold particles is 100±40.
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.
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