1.1 本指南旨在使实验室科学家熟悉通过暗场显微镜/高光谱成像（DFM/HSI）方法在细胞中成像和识别工程纳米材料所需的背景信息和技术内容。 1.2 DFM/HSI是一种联用生物分析技术/工具，将光学显微镜与高分辨率光谱成像相结合，以在适当制备的测试样品中空间定位和识别ENM的分布。 1.2.1 在哺乳动物细胞的背景下，与亚细胞器和细胞结构特征相比，ENM将具有独特的光散射特性，从而可以区分ENM的光谱轮廓和细胞成分。 1.2.2 ENM在其他测试样本（如固定组织、植物、复杂药物制剂、过滤介质等）中的光散射特性也将不同于这些其他类型样本固有的天然基质成分散射信号，因此允许ENM可视化和识别。 1.3 本指南适用于使用DFM/HSI识别上述矩阵中的ENM。 1.4 本指南描述并讨论了设置和使用DFM/HSI仪器、样品成像技术、光学注意事项、图像分析和参考光谱库（RSL）的基本实践。DFM/HSI通常用于工业、学术界和政府部门，作为纳米技术不同领域的研发和质量控制工具。 1.5 以国际单位制表示的数值应视为标准。本标准不包括其他计量单位。 1.6 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.7 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》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 本指南中的信息和建议与细胞和其他生物（例如，固定组织、整株植物）和非生物（例如，药物制剂、过滤介质、土壤和废水）基质中的ENM的成像和识别有关，在执行适当的样品制备程序后 ( 3- 5. ) . DFM/HSI是最近开发的一种分析工具；然而，样品制备的相对简单性以及获取高对比度ENM图像和高含量ENM光谱响应的潜力促进了该工具在药物递送、毒理学、环境科学、生物学和医学中的多种应用。 5.2 例如，为了评估和理解ENMs对生命系统的生物效应，有必要验证ENMs在细胞中的摄取和空间分布。类似地，复杂药物制剂中ENMs空间分布的紧密性可以作为建立制剂之间物理化学相似性的重要标准 ( 6. ) . 最新版本的《非专利药物使用者费用法案》（GDUFA）重新授权承诺书中描述了复杂产品: ( 7. ) . 本指南涵盖了对含有ENM的生物和非生物来源的样品（例如，金属和金属氧化物纳米颗粒或碳纳米管，或两者）进行DFM/HSI分析的标准和一般注意事项。 本指南未涵盖或阐述细胞或其他基质中非工程（天然）纳米颗粒/纳米材料的成像或识别规定，也未描述或讨论DFM/HSI在确定ENM尺寸方面的应用。

1.1 This guide has been prepared to familiarize laboratory scientists with the background information and technical content necessary to image and identify engineered nanomaterials (ENMs) in cells via darkfield microscopy/hyperspectral imaging (DFM/HSI) methodology. 1.2 DFM/HSI is a hyphenated bioanalytical technique/tool that combines optical microscopy with high-resolution spectral imaging to both spatially localize the distribution of and identify ENMs within a suitably prepared test sample. 1.2.1 In the context of mammalian cells, ENMs will have distinctive light-scattering properties in comparison to subcellular organelles and cell structural features, which can allow one to discriminate between the spectral profiles of ENMs and cellular components. 1.2.2 The light-scattering properties of ENMs in other test samples, such as fixed tissues, plants, complex drug product formulations, filter media, and so forth, will also be different from the native matrix component scattering signals inherent to these other types of samples, thus allowing for ENM visualization and identification. 1.3 This guide is applicable to the use of DFM/HSI for identifying ENMs in the matrices mentioned. 1.4 This guide describes and discusses basic practices for setting up and using DFM/HSI instrumentation, sample imaging techniques, considerations for optics, image analysis, and the use of reference spectral libraries (RSLs). DFM/HSI is routinely used in industry, academia, and government as a research and development and quality control tool in diverse areas of nanotechnology. 1.5 The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard. 1.6 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. 1.7 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 The information and recommendations in this guide are relevant for imaging and identifying ENMs in cells and other biological (for example, fixed tissues, whole plants) and nonbiological (for example, drug formulations, filter media, soil, and wastewater) matrices after appropriate sample preparation procedures have been performed ( 3- 5 ) . DFM/HSI is a recently developed analytical tool; however, the relative simplicity of sample preparation combined with the potential to acquire high-contrast ENM images and high-content ENM spectral responses facilitates the increasing use of the tool for diverse applications in drug delivery, toxicology, environmental science, biology, and medicine. 5.2 Verification of the uptake and spatial distribution of ENMs in cells, for example, is necessary for evaluating and understanding the biological effects of ENMs on living systems. Similarly, the closeness of the spatial distribution of ENMs in complex drug formulations can be an important criterion in establishing physicochemical similarity between formulations ( 6 ) . Complex products are described in the most recent version of the Generic Drug User Fee Act (GDUFA) reauthorization commitment letter: ( 7 ) . This guide covers the criteria and general considerations for performing DFM/HSI analyses on samples of biological and nonbiological origins containing ENMs (for example, metal and metal oxide nanoparticles, or carbon nanotubes, or both). This guide does not cover or address provisions for imaging or identifying, or both, non-engineered (natural) nanoparticles/nanomaterials in cells or other matrices, nor does this guide describe or discuss the application of DFM/HSI for determining the dimensions of ENMs.