1.1 本指导文件旨在促进使用落射荧光显微镜收集显微镜图像，该显微镜允许从图像中提取定量荧光测量值。该文件是为经常使用荧光染色技术来可视化基于细胞的实验系统的组件的细胞生物学家量身定制的。只有当图像是基于合理的实验设计和数字阵列检测器(诸如电荷耦合器件(CCD)或科学互补金属氧化物半导体(sCMOS)或类似相机)的适当操作而定量的情况下，才可能对这些图像中可用的强度数据进行定量比较。涉及阵列探测器和控制器软件设置的问题，包括收集暗计数图像以估计偏移、平面-考虑了场校正、背景校正、激发灯的基准和荧光收集光学器件。 1.2 本文档是围绕落射荧光显微术开发的，但是这里讨论的许多问题很可能适用于其他荧光显微术系统(例如荧光共聚焦显微术)中的定量成像。本指南是围绕单色荧光显微镜成像或多色成像开发的，其中测量的荧光在光谱上被很好地分离。 1.3 荧光强度是相对测量值，其本身不具有相关的SI单位。本文档确实讨论了与相对测量和实验设计相关的计量问题，这些问题可能需要确保在改变显微镜、样品和灯配置后定量荧光测量具有可比性。1.4 本标准并不旨在解决与其使用相关的所有安全性问题（如果有）。本标准的使用者有责任在使用前建立适当的安全、健康和环境实践并确定法规限制的适用性。 1.5 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认的标准化原则制定的。 ======意义和用途====== 5.1 测量系统概述- 通过宽视场落射荧光显微镜进行的相对强度测量被用作基于细胞的测定的一部分，以定量诸如探针分子的丰度等属性（参见实践 F2997 )、荧光标记的抗体或荧光蛋白报告分子。用于量化相对强度的一般过程是获取数字图像，然后执行图像分析以分割对象并计算强度。由于中列出的因素，通过落射荧光显微镜获取的原始数字图像通常不适合相对强度定量 4.2 本指南提供了荧光显微镜图像中经常存在的潜在偏差源的清单，并建议了存储和归一化原始图像数据以确保计算无偏差的方法。 5.2 应用领域- 宽视野荧光显微镜经常用于测量细胞内或细胞之间荧光探针分子的位置和丰度。在感兴趣区域(ROI)和另一ROI之间进行边缘比较的情况下，准确的归一化过程对于测量过程是必不可少的，以最小化有偏差的结果。本指导文件可能适用的示例用例包括: 5.2.1 通过定量单个细胞中DNA丰度表征细胞周期分布 ( 1 ) . 8 5.2.2 测量细胞培养物中阳性染色矿化沉积物的面积（实践 F2997 ). 5.2.3 定量固定细胞的扩散面积（指南 F2998 ). 5.2.4 使用彗星试验测定真核细胞中的DNA损伤（指南 E2186 ). 5.2.5 提供与集落或细胞的基因型、表型、生物活性或生化特征相关信息的次级荧光标记的定量（实践 F2944 ).

1.1 This guidance document has been developed to facilitate the collection of microscopy images with an epifluorescence microscope that allows quantitative fluorescence measurements to be extracted from the images. The document is tailored to cell biologists that often use fluorescent staining techniques to visualize components of a cell-based experimental system. Quantitative comparison of the intensity data available in these images is only possible if the images are quantitative based on sound experimental design and appropriate operation of the digital array detector, such as a charge coupled device (CCD) or a scientific complementary metal oxide semiconductor (sCMOS) or similar camera. Issues involving the array detector and controller software settings including collection of dark count images to estimate the offset, flat-field correction, background correction, benchmarking of the excitation lamp, and the fluorescent collection optics are considered. 1.2 This document is developed around epifluorescence microscopy, but it is likely that many of the issues discussed here are applicable to quantitative imaging in other fluorescence microscopy systems such as fluorescence confocal microscopy. This guide is developed around single-color fluorescence microscopy imaging or multi-color imaging where the measured fluorescence is spectrally well separated. 1.3 Fluorescence intensity is a relative measurement and does not in itself have an associated SI unit. This document does discuss metrology issues related to relative measurements and experimental designs that may be required to ensure quantitative fluorescence measurements are comparable after changing microscope, sample, and lamp configurations. 1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 5.1 Overview of Measurement System— Relative intensity measurements made by widefield epifluorescence microscopy are used as part of cell-based assays to quantify attributes such as the abundance of probe molecules (see Practice F2997 ), fluorescently labeled antibodies, or fluorescence protein reporter molecules. The general procedure for quantifying relative intensities is to acquire digital images, then to perform image analysis to segment objects and compute intensities. The raw digital images acquired by epifluorescence microscopy are not typically amenable to relative intensity quantification because of the factors listed in 4.2 . This guide offers a checklist of potential sources of bias that are often present in fluorescent microscopy images and suggests approaches for storing and normalizing raw image data to ensure that computations are unbiased. 5.2 Areas of Application— Widefield fluorescence microscopy is frequently used to measure the location and abundance of fluorescent probe molecules within or between cells. In instances where RIM comparisons are made between a region of interest (ROI) and another ROI, accurate normalization procedures are essential to the measurement process to minimize biased results. Example use cases where this guidance document may be applicable include: 5.2.1 Characterization of cell cycle distribution by quantifying the abundance of DNA in individual cells ( 1 ) . 8 5.2.2 Measuring the area of positively stained mineralized deposits in cell cultures (Practice F2997 ). 5.2.3 Quantifying the spread area of fixed cells (Guide F2998 ). 5.2.4 Determining DNA damage in eukaryotic cells using the comet assay (Guide E2186 ). 5.2.5 The quantitation of a secondary fluorescent marker that provides information related to the genotype, phenotype, biological activity, or biochemical features of a colony or cell (Practice F2944 ).