5.1 As AFM measurement technology has matured and proliferated, the technique has been widely adopted by the nanotechnology research and development community to the extent that it is now considered an indispensible tool for visualizing and quantifying structures on the nanoscale. Whether used as a stand-alone method or to complement other dimensional measurement methods, AFM is now a firmly established component of the nanoparticle measurement tool box. International standards for AFM-based determination of nanoparticle size are nonexistent as of the drafting of this guide. Therefore, this standard aims to provide practical and metrological guidance for the application of AFM to measure the size of substrate-supported nanoparticles based on maximum displacement as the probe is rastered across the particle surface to create a line profile.1.1 The purpose of this document is to provide guidance on the quantitative application of atomic force microscopy (AFM) to determine the size of nanoparticles2 deposited in dry form on flat substrates using height (z-displacement) measurement. Unlike electron microscopy, which provides a two-dimensional projection or a two-dimensional image of a sample, AFM provides a three-dimensional surface profile. While the lateral dimensions are influenced by the shape of the probe, displacement measurements can provide the height of nanoparticles with a high degree of accuracy and precision. If the particles are assumed to be spherical, the height measurement corresponds to the diameter of the particle. In this guide, procedures are described for dispersing gold nanoparticles on various surfaces such that they are suitable for imaging and height measurement via intermittent contact mode AFM. Generic procedures for AFM calibration and operation to make such measurements are then discussed. Finally, procedures for data analysis and reporting are addressed. The nanoparticles used to exemplify these procedures are National Institute of Standards and Technology (NIST) reference materials containing citrate-stabilized negatively charged gold nanoparticles in an aqueous solution.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 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.1.4 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.

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5.1 Exposures to high concentrations of aerosolized fine and ultrafine non-fibrous metal particles, including manganese (Mn), chromium (Cr), and nickel (Ni) generated during processes that involve high energy such as welding or smelting, may elicit deleterious health effects. Animal and epidemiological studies have associated welding and related work processes with a wide range of adverse health effects, including upper respiratory effects (rhinitis and laryngitis), pulmonary effects (pneumonitis, chronic bronchitis, decreased pulmonary function), potential neurological disorders (manganese-induced Parkinsonism), and high lung cancer and pneumoconiosis death rates. Manganese has been associated with neurological diseases.5.2 Nanoparticles produced from metals, or their oxides and chalcogenides, have found many industrial uses. Examples of nanometals include silver (Ag), gold (Au), iron (Fe), copper (Cu), cadmium (Cd), zinc (Zn), platinum (Pt), and lead (Pd); examples of nanometal oxides include aluminium oxide (Al2O3), magnesium oxide (MgO), zirconium dioxide (ZrO2), cerium(IV) oxide (CeO2), titanium dioxide (TiO2), zinc oxide (ZnO), iron(III) oxide (Fe2O3), and tin(II) oxide (SnO); examples of nanometal sulfides include copper monosulfide (CuS), cadmium sulfide (CdS), zinc sulfide (ZnS), silver sulfide (AgS), tin sulfide (SnS), and many sulfides of Ni and cobalt (Co); examples of nanometal selenides include zinc selenide (ZnSe), cadmium selenide (CdSe), and mercury selenide (HgSe). Both the manufacture and use of these nanoparticles can result in particle inhalation, and consequent ill-effects. A stronger association has often been found between adverse health and cellular effects and inhalation of nanoparticles compared to larger particles of the same composition.5.3 Aerosol sampling methods generally specify the collection of workplace air samples using inhalable and related samplers. These exposure assessment methods, as well as the use of respirable and thoracic samplers (ISO 7708), are inadequate for measurements of nanoparticle exposure when paired with gravimetric analysis. Large particles (>1 μm) weigh substantially more than nanoparticles typical of fumes and, consequently, obscure the ability to detect nanoparticles through gravimetric filter sampling. Additionally, most size-selective samplers collect all particles in the fraction of aerosol that can penetrate into the respiratory tract. Particle deposition, which is governed by the principles of impaction, interception, and diffusion (ISO 13138), is typically overestimated by these samplers.5.4 There is a need to measure nanoparticle airborne concentrations apart from larger particles. An NRD sampler selectively collects nanoparticles in a manner similar to their typical deposition in the human respiratory tract. The constant motion of nanoparticles causes them to diffuse and potentially deposit in all regions of the respiratory tract, from the head airways to the deep alveolar region, as described by the ICRP (2). NRD samplers are designed to follow a nanoparticulate matter (NPM) deposition curve based on the ICRP model for deposition of particles smaller than 300 nm (the minimum in deposition for submicrometre particles) while removing the larger particles (1). Size-selective samplers (respirable, thoracic, and inhalable) mimic particle penetration rather than particle deposition. Many studies of welding fume have noted that size distribution of welding fume particles brackets the airways deposition minimum so that a substantial proportion of the fume is not deposited in the airways following inhalation (3-7). The use of an NRD sampler, however, approaches exposure assessment from a deposition estimation perspective (8) and provides a more relevant and physiological procedure for measuring actual hazards to workers (such as welders) posed by nanoparticle exposure. This knowledge is critical to the development of toxicological studies aimed at finding links between deposition of metal-containing nanoparticles and adverse health effects.5.5 Welding fumes are dominated by incidental nanoparticles (particles with any external dimension in the nanoscale), but also include larger particles generated by splatter. Current animal and epidemiological studies investigate exposure to welding fumes without differentiating between nanoparticles and larger particles. Welding fume nanoparticles have been found to induce more toxic effects at the cellular level and to generate more reactive oxygen species (ROS) activity when compared to larger particles.5.6 An NRD sampler was initially designed with nylon screens as the diffusion stage for the collection of nanoparticles (1), including welding fume (8, 9), although it was noted at the time that laboratory tests of this embodiment had not also included agglomerated particles, such as those which characterize welding fume. An additional collection mechanism, interception, was later found to play an important role as the sample collection of agglomerated nanoparticles progressed to higher loadings. Performance of the nylon screens for agglomerated particles was found to be affected by accumulated nanoparticle fraction loadings greater than 1 mg. The change in performance was accompanied by an increase in pressure drop across the screens to 14.3 kPa (57 in. of water) (5), which would cause many sampling pumps to fault. At the American Conference of Governmental Hygienists (ACGIH) Threshold Limit Value (TLV)5 for welding fume of 5 mg/m3, a one-hour sample at 2.5 L/min will collect 0.75 mg. Since the nanoparticle fraction of welding fume is typically less than half the total mass in air (3), the nylon screens are effective in sampling welding fume for one-hour or less as was borne out in field studies (9).5.7 A new diffusion stage substrate, polyurethane foam, has characteristics more closely resembling human airways (example, Ref (10)) and may be preferable for collecting agglomerated materials in higher loading scenarios (11). In addition, polyurethane foam does not contain titanium dioxide allowing this sampler to be used to assess nanoparticle titanium dioxide.5.8 The sampler with polyurethane foam has been shown to mimic the ICRP deposition curve closely when sampling spherical nanoparticles up to 100 nm diameter. Agglomerated particles collected in foam begin to show significant deviations from the simple curve as their size and shape factor increase (11). In Figure 3 of Ref (11), the curve modeling behavior of particles through foam is adjusted according to the dynamic shape factor of the aerosol and the sampler collection is shown to continue to match the modified curve at larger particle sizes. Since foam has proven to be a useful surrogate for lung deposition at larger particle sizes, it can be hypothesized that the adjusted foam model also will mimic the behavior of nanoparticle agglomerates in the lung. Enhanced deposition of larger agglomerates has been observed for agglomerated silica particles in human lung-casts (12) demonstrating that it may be necessary for an adjustment to the ICRP curve for agglomerates in this size range. However, until future research has identified a more precise adjustment to the ICRP deposition curve for agglomerated particles in the human airways the relationship of foam collection to human airways deposition remains a hypothesis.5.9 An accurate measurement of flow rate through an NRD sampler is required for experiments where sampling devices and filter materials are to be compared as to the size distribution aerosol they capture. Air flow rate affects the efficiency with which a sampler will capture a particular aerodynamic size of particles. Furthermore, air flow rate through a sampler may affect the distribution of aerosol particles captured on the filters and deposited on the sampler collection substrates and walls. To determine aerosol concentration from a mass of captured particles it is necessary to set and measure flow rates accurately.NOTE 2: Refer to Guide E1370 for guidance on the development of appropriate exposure assessment and measurement strategies.1.1 This practice describes specified apparatus and procedures for collection of non-fibrous airborne metal nanoparticles generated during work activities.1.2 Nanoparticle respiratory deposition (NRD) samplers are designed to follow a nanoparticulate matter (NPM) deposition curve based on the International Commission on Radiological Protection (ICRP) model for deposition of particles smaller than 300 nm (the minimum deposition for submicrometre particles) while removing the larger particles (1).21.3 This practice is applicable to personal and area sampling during work processes and situations where metal nanoparticles may be generated (for example, welding, smelting, shooting ranges).1.4 This practice is intended for use by professionals experienced in the use of devices for occupational air sampling (such as cyclone samplers).1.5 This practice is not applicable to the sampling of fibrous nanoparticles such as carbon nanotubes.1.6 Detailed operating instructions are not provided owing to differences among various makes and models of suitable devices and instruments. The user is expected to follow specific instructions provided by the manufacturers of particular items of equipment. This practice does not address comparative accuracy of different devices nor the precision between instruments of the same make and model.1.7 This practice contains notes that are explanatory and are not part of the mandatory requirements of the method.1.8 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.9 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.1.10 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.

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5.1 Particle size is a key property of manufactured or engineered nanoparticles used in a wide range of applications. For purposes relevant to evaluations of safety, effectiveness, performance, quality, public health impact, or regulatory status of products, the correct measurement and uniform reporting of size and related parameters under use conditions, or during the manufacturing process, are critical to suppliers, analysts, regulators and other stakeholders.5.2 This test method is intended principally for the analysis of nanoparticles in aqueous suspension with dimensions between about 1 nm and 100 nm, but may be applied to diffusive colloidal particles even if their dimensions fall outside the nanoscale range (up to 1000 nm).5.3 For more detailed guidance on DLS measurements, including operational aspects, refer to Appendix X2 of this test method.NOTE 1: The user is also referred to Guide E2490, which provides broad guidance for the application of DLS to nanomaterials. Guide E2490 is not required for the implementation of this test method.1.1 This test method addresses the determination of nanoparticle size (equivalent sphere hydrodynamic diameter) using batch-mode (off-line) dynamic light scattering (DLS) in aqueous suspensions and establishes general procedures that are applicable to many commercial DLS instruments. This test method specifies best practices, including sample preparation, performance verification, data analysis and interpretation, and reporting of results. The document includes additional general information for the analyst, such as recommended settings for specific media, potential interferences, and method limitations. Issues specific to the use of DLS data for regulatory submissions are addressed.1.2 The procedures and practices described in this test method, in principle, may be applied to any particles that exhibit Brownian motion and are kinetically stable during the course of a typical experimental time frame. In practice, this includes particles up to about 1000 nm in diameter, subject to limitations as described in the test method.1.3 This test method does not provide test specimen preparation procedures for all possible materials and applications, nor does it address synthesis or processing prior to sampling. The test specimen (suspension) preparation procedures should provide acceptable results for a wide range of materials and conditions. The analyst must validate the appropriateness for their particular application.1.4 This test method is applicable to DLS instruments that implement correlation spectroscopy. Analysts using instruments based on frequency analysis may still find useful information relevant to many aspects of the measurement process, including limits of applicability and best practices. On-line (flow-mode) DLS measurements are not treated here specifically and may have additional limitations or issues relative to batch-mode operation.1.5 Units—The values stated in SI units are to be regarded as standard. Where appropriate, c.g.s. units are given in addition to SI.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.

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5.1 This test method is one of a series of tests listed in Practice F748 and ISO 10993-4 to assess the biocompatibility of materials contacting blood in medical applications.5.2 This test method is similar to Practice F756 but modified to accommodate nanoparticulate materials.1.1 This test method covers assessing the effect of nanoparticulate materials on the integrity of red blood cells.1.2 This test method uses diluted whole blood incubated with nanoparticulate material and the hemoglobin released from damaged red blood cells is determined.1.3 This test method is similar to Practice F756 with the volumes reduced to accommodate nanoparticulate material.1.4 This test method is part of the in-vitro preclinical characterization and is important for nanoparticulate material that will contact the blood in medical applications.1.5 The values stated in SI units are to be regarded as 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.

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4.1 In this guide, the conditions, measurement apparatus, and procedures for measuring several characteristics of nanoparticle properties on three different instrument platforms using laser-amplified detection/power spectrum analysis (LAD/PSA) technology are described. This is a more recently developed technology, commercialized in 1990, than the older technology known as either photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QLS)—those titles are interchangeable—developed first in 1961. Nanoparticle tracking analysis (NTA) is the most recent DLS technology to be commercialized. All three of these technologies fall under the broader category of DLS, based on the “dynamic” movement of the measured nanoparticles under Brownian motion.4.2 DLS in the lower end of the nanometre size range becomes progressively more difficult as the particle optical scattering coefficients drop sharply, reducing the scattered light intensity. The advantage of the heterodyne detection mode over the homodyne detection mode, especially at the low end of the nanometre range, will be explained.4.3 The LAD/PSA technology will be described and the major differences between it and the PCS-QLS and NTA technologies will be made clear. For thorough discussions of PCS-QLS, refer to Guide E2490, Test Method E3247, and ISO 22412 Annex Section A.1. For a thorough discussion of nanoparticle tracking analysis (NTA), refer to Guide E2834. For detailed information on laser-amplified detection/frequency power spectrum (LAD/FPS) technology, refer to ISO 22412 Annex Section A.2. General information on particle characterization practices can be found in Practice E1817, and nanotechnology terminology is given in Terminology E2456. Detailed information on sampling for particle characterization can be found in ISO 14488.1.1 The technology, laser-amplified detection/power spectrum analysis (LAD/PSA), is available in three different platforms, which will be designated as Platforms A, B, and C.1.1.1 Platform A—This is a solid-state probe configuration that serves as the optical bench in each of the platforms. It consists of an optical fiber coupler with a y-beam splitter that directs the scattered light signal from the nanoparticles at 180° back to a photodiode detector. The sensing end of the probe can be immersed in a suspension or positioned to measure one drop of a sample on top of the sensing surface.1.1.2 Platform B—The same probe is mounted in a case, positioned horizontally, to detect the signal from either a disposable or permanent cuvette.1.1.3 Platform C—Two probes are mounted in a case, horizontally, at opposite sides of a permanent sample cell. Both size distribution and zeta potential can be measured in this configuration.1.2 The laser beam travelling through the probe measuring the scattered light from the sample of nanoparticles, in all three platforms, is partially reflected back to the same photodiode detector, and the high optical power of the laser is added to the low optical power of the scattered light signal. The interference (mixing or beating) of those two signals is known as heterodyne beating. The resulting high-power detected signal provides the highest signal-to-noise ratio among dynamic light-scattering (DLS) technologies.1.3 This combined, amplified, optical signal is converted with a Fast Fourier transform (FFT) into a frequency power spectrum, then into a logarithmic power spectrum that is deconvolved into number and volume size distributions. The mean intensity, polydispersity, number and volume size distributions, concentration, and molecular weight can be reported in all platforms, plus zeta potential on Platform C.1.4 This technology is capable of measuring nanoparticles in a size range from 2.0 nanometres (nm) to 10 micrometres (µm), at concentrations in a suspending liquid medium up to 40 % cc/mL for all parameters given in 1.3.1.5 Units—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.

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