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    ASTM E3275 - 21

    Standard Guide for Visualization and Identification of Nanomaterials in Biological and Nonbiological Matrices Using Darkfield Microscopy/Hyperspectral Imaging (DFM/HSI) Analysis

    Active Standard ASTM E3275 | Developed by Subcommittee: E56.08

    Book of Standards Volume: 14.02


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    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.

    1. Scope

    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.


    2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.

    ASTM Standards

    E2456 Terminology Relating to Nanotechnology

    E3255 Practice for Quality Assurance of Forensic Science Service Providers Performing Forensic Chemical Analysis

    ISO Standards

    ISO 20473 Optics and photonicsSpectral bands

    ISO/TS 80004-1 NanotechnologiesVocabularyPart 1: Core terms


    Referencing This Standard
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    DOI: 10.1520/E3275-21

    Citation Format

    ASTM E3275-21, Standard Guide for Visualization and Identification of Nanomaterials in Biological and Nonbiological Matrices Using Darkfield Microscopy/Hyperspectral Imaging (DFM/HSI) Analysis, ASTM International, West Conshohocken, PA, 2021, www.astm.org

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