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

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.