5.1 This test method provides for accurate and reproducible enumeration of particles and fibers released from a wiper immersed in a cleaning solution with moderate mechanical stress applied. When performed correctly, this counting test method is sensitive enough to quantify very low levels of total particle and fiber burden. The results are accurate and not influenced by artifact or particle size limitations. A further advantage to this technique is that it allows for morphological as well as X-ray analysis of individual particles.1.1 This test method covers testing all wipers used in cleanrooms and other controlled environments for characteristics related to particulate cleanliness.1.2 This test method includes the use of computer-based image analysis and counting hardware and software for the counting of densely particle-laden filters (see 7.7 – 7.9). While the use of this equipment is not absolutely necessary, it is strongly recommended to enhance the accuracy, speed, and consistency of counting.1.3 The values stated in SI units are to be regarded as the standard.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.

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5.1 This wipe sampling and indirect analysis test method is used for the general testing of surfaces for asbestos. It is used to assist in the evaluation of surfaces in buildings, such as ceiling tiles, shelving, electrical components, duct work, and so forth. This test method provides an index of the concentration of asbestos structures per unit area sampled as derived from a quantitative measure of the number of asbestos structures detected during analysis.5.1.1 This test method does not describe procedures or techniques required for the evaluation of the safety or habitability of buildings with asbestos-containing materials, or compliance with federal, state, or local regulations or statutes. It is the user's responsibility to make these determinations.5.1.2 At present, a single direct relationship between asbestos sampled from a surface and potential human exposure does not exist. Accordingly, the user should consider these data in relationship to other available information (for example, air sampling data) in their evaluation.5.2 One or more large asbestos-containing particles dispersed during sample preparation may result in large asbestos surface loading results in the TEM analyses of that sample. It is, therefore, recommended that multiple replicate independent samples be secured in the same area, and that a minimum of three such samples be analyzed by the entire procedure.1.1 This test method covers a procedure to identify asbestos in samples wiped from surfaces and to provide an estimate of the concentration of asbestos reported as the number of asbestos structures per unit area of sampled surface. The procedure outlined in this test method employs an indirect sample preparation technique. It is intended to disperse aggregated asbestos into fundamental fibrils, fiber bundles, clusters, or matrices. However, as with all indirect sample preparation techniques, the asbestos observed for quantification may not represent the physical form of the asbestos as sampled. More specifically, the procedure described neither creates nor destroys asbestos, but it may alter the physical form of the mineral fiber aggregates.1.2 This test method describes the equipment and procedures necessary for wipe sampling of surfaces for levels of asbestos structures. The sample is collected onto a particle-free wipe material (wipe) from the surface of a sampling area that may contain asbestos.1.2.1 The collection efficiency of this wipe sampling technique is unknown and will vary among substrates. Properties influencing collection efficiency include surface texture, adhesiveness, and other factors.1.2.2 This test method is generally applicable for an estimate of the surface loading of asbestos structures starting from approximately 1000 asbestos structures per square centimetre.1.3 Asbestos identification by transmission electron microscopy (TEM) is based on morphology, electron diffraction (ED), and energy dispersive X-ray analysis (EDXA).1.4 This test method allows determination of the type(s) of asbestos fibers present.1.4.1 This test method cannot always discriminate between individual fibers of the asbestos and nonasbestos analogues of the same amphibole mineral.1.4.2 There is no lower limit to the dimensions of asbestos fibers that can be detected. However, in practice, the lower limit to the dimensions of asbestos fibers, that can be detected, is variable and dependent on individual microscopists. Therefore, a minimum length of 0.5 μm has been defined as the shortest fiber to be incorporated in the reported results.1.5 The values stated in SI units are to be regarded as 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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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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4.1 Carbon black morphology significantly affects the transient and end-use properties of carbon black loaded polymer systems. A carbon black’s particle size distribution is its single most important property, and it relates to degree of blackness, rubber reinforcement, and ability to impart UV protection. For a given loading of carbon black, blackness, reinforcement, and UV protection increase with smaller particle size. Aggregate size and shape (structure) also affect a carbon black's end-use performance, as higher carbon black structure increases viscosity and improves dispersion. The stiffness (modulus) of elastomer systems becomes significantly higher with increasing structure. The preferred method for measuring carbon black morphology (for example, size and shape) is transmission electron microscopy (TEM), but due to the semi-quantitative nature of TEM, it is not suited for mean particle size (MPS) certification. While useful morphological information can be obtained from TEM measurements within a laboratory, due to their inherent between-laboratory variability, TEM generated values should not be used for specification purposes.4.2 Certification of carbon blacks for UV protection (weatherability) in certain plastics applications has historically been performed using TEM generated mean particle size values. ASTM Committee D24 has demonstrated that due to challenges with obtaining quantitative primary particle size data, particularly between laboratories, a qualification test based on surface area has been implemented, as detailed in Test Method B.4.3 Carbon black aggregate dimensional and shape properties are dependent upon the nature of the system in which the sample is dispersed, as well as the mixing procedure.1.1 This test method covers (1) the morphological (for example, size and shape) characterization of carbon black from transmission electron microscope images which are used to derive the mean particle and aggregate size of carbon black in the dry (as manufactured) state, from CAB chip dispersion or removed from a rubber compound and (2) the certification of mean particle size using a correlation based on statistical thickness surface area measurements.1.2 The values stated in SI units are to be regarded as the standard. The values in parentheses are for information only.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 Chemical composition of water-formed deposits is a major indicator of proper or improper chemical treatment of process water, and is often an indicator of operational parameters as well, for example, temperature control. This practice allows for rapid determination of constituents present in these deposits, particularly those indications of improper water treatment, since they usually have very distinctive and easily recognized optical properties.5.2 This practice, where applicable, eliminates the need for detailed chemical analysis, which is time-consuming, and which does not always reveal how cations and anions are mutually bound.5.3 Qualitative use of this practice should be limited to those deposits whose control is generally known or predictable, based on treatment and feedwater mineral content, and whose constituents are crystalline, or in other ways optically or morphologically distinctive. If these criteria are not met, other techniques of analysis should be used, such as Practice D2332 or Test Methods D3483, or both.5.4 Quantitative use of this practice should be limited to estimates only. For more precise quantitative results, other methods should be used (see 5.3).1.1 This practice describes a procedure for the examination of water-formed deposits by means of chemical microscopy. This practice may be used to complement other methods of examination of water-formed deposits as recommended in Practices D2331 or it may be used alone when no other instrumentation is available or when the sample size is very small.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 and health 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 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layer of vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yielding a very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with air drying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designed electron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam induced structural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accurately measure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from an air-dried negatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.FIG. 1 Left—An Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetate for Contrast; Right—An Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEMNOTE 1: Both images are shown to the same scale; scale bar is 200 nm.5.1.1 Fig. 1 demonstrates that liposomes may become distorted and are difficult to measure and analyze when they are air-dried, while the same liposomal preparation is clearly easier to analyze when the specimen is near-instantly preserved by vitrification.5.1.2 Cryo-TEM involves applying a small volume of sample to a specially prepared holey, ultra-thin or continuous carbon grid suspended in a cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These grids can be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample has wet the surface of the grid, and sufficient time allowed for the solution to equilibrate with regard to liposome spreading over the grid surface, the excess is wicked off (blotted) with filter paper and the grid plunged into the liquid ethane, vitrifying the sample. Once frozen, the sample is maintained at a liquid nitrogen temperature while it is imaged in a cryo-TEM operating under low electron dose conditions. There are several limitations associated with implementing this technique to analyze liposomes:5.1.2.1 Thick Ice—The vitrified ice thickness is often determined by the sample or the cryo-TEM procedure itself. Large liposomes, defined to include larger structure and sizes with respect to this practice, are generally associated with thicker ice, while smaller liposomes (structure and sizes) are associated with thinner ice. Generally, thick ice occurs when either excess water forms a thicker ice layer or samples containing larger liposomes are fully covered with water making the ice thicker around the sample. Thicker ice tends to block the electron beam either completely or partially which compromises image quality.5.1.2.2 Larger liposomes (structure and sizes) are preferentially lost during sample preparation.Larger liposomes, defined to include larger structures and sizes with respect to this practice, are more difficult to image for two reasons. The first is the cryo-TEM procedure itself. This procedure requires the use of filter paper to blot away excess aqueous solution from the EM grid just prior to vitrification. The larger liposomes suspended within the sample preferentially wash away from the grid and into the filter paper, ending up in the filter paper. This is perhaps because the larger liposomes have larger surface areas that expose them to relatively larger forces during the rapid flow of the water to the filter paper. This makes them difficult to find and measure in electron micrographs when their relative concentration in the specimen is low, meaning that few are left behind after blotting. The second reason is that larger liposomes that are left behind on the EM grid, are often embedded in thicker ice that is too thick for the electron beam to either penetrate or, if it does, results in images that are too low in quality to provide adequate signal for image processing.5.1.2.3 Liposomal Distortion—Because liposomes are essentially loose membrane bounded fluid compartments, freezing them within a layer of vitrified ice that is thinner than their diameter may cause the surface tension on both sides of the specimen to compress some of the liposomes leading to various levels of flattening distortions. Accurate size measurements of such distorted liposomes would require volumetric measurements of all the liposomes within a field of view through a three-dimensional analysis using electron tomography.1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmission electron microscope (cryo-TEM) for the purpose of evaluating their shape, size distribution and lamellarity for quality assessment. The sample is vitrified in liquid ethane onto specially prepared holey, ultra-thin, or continuous carbon TEM grids, and imaged in a cryo-holder placed in a cryo-TEM.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 This microvacuum sampling and indirect analysis method is used for the general testing of non-airborne dust samples for asbestos. It is used to assist in the evaluation of dust that may be found on surfaces in buildings such as ceiling tiles, shelving, electrical components, duct work, carpet, etc. This test method provides an index of the surface loading of asbestos structures in the dust per unit area analyzed as derived from a quantitative TEM analysis.5.1.1 This test method does not describe procedures or techniques required to evaluate the safety or habitability of buildings with asbestos-containing materials, or compliance with federal, state, or local regulations or statutes. It is the user’s responsibility to make these determinations.5.1.2 At present, no relationship has been established between asbestos-containing dust as measured by this test method and potential human exposure to airborne asbestos. Accordingly, the users should consider other available information in their interpretation of the data obtained from this test method.5.2 This definition of dust accepts all particles small enough to pass through a 1-mm (No. 18) screen. Thus, a single, large asbestos containing particle(s) (from the large end of the particle size distribution) dispersed during sample preparation may result in anomalously large asbestos surface loading results in the TEM analyses of that sample. It is, therefore, recommended that multiple independent samples are secured from the same area, and that a minimum of three samples be analyzed by the entire procedure.1.1 This test method covers a procedure to (a) identify asbestos in dust and (b) provide an estimate of the surface loading of asbestos in the sampled dust reported as the number of asbestos structures per unit area of sampled surface.1.1.1 If an estimate of the asbestos mass is to be determined, the user is referred to Test Method D5756.1.2 This test method describes the equipment and procedures necessary for sampling, by a microvacuum technique, non-airborne dust for levels of asbestos structures. The non-airborne sample is collected inside a standard filter membrane cassette from the sampling of a surface area for dust which may contain asbestos.1.2.1 This procedure uses a microvacuuming sampling technique. The collection efficiency of this technique is unknown and will vary among substrates. Properties influencing collection efficiency include surface texture, adhesiveness, electrostatic properties and other factors.1.3 Asbestos identified by transmission electron microscopy (TEM) is based on morphology, selected area electron diffraction (SAED), and energy dispersive X-ray analysis (EDXA). Some information about structure size is also determined.1.4 This test method is generally applicable for an estimate of the surface loading of asbestos structures starting from approximately 1000 asbestos structures per square centimetre.1.4.1 The procedure outlined in this test method employs an indirect sample preparation technique. It is intended to disperse aggregated asbestos into fundamental fibrils, fiber bundles, clusters, or matrices that can be more accurately quantified by transmission electron microscopy. However, as with all indirect sample preparation techniques, the asbestos observed for quantification may not represent the physical form of the asbestos as sampled. More specifically, the procedure described neither creates nor destroys asbestos, but it may alter the physical form of the mineral fibers.1.5 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.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 and health practices and determine the applicability of regulatory limitations prior to use.

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This microvacuum sampling and indirect analysis method is used for the general testing of non-airborne dust samples for asbestos. It is used to assist in the evaluation of dust that may be found on surfaces in buildings, such as ceiling tiles, shelving, electrical components, duct work, carpet, etc. This test method provides an estimate of the mass surface loading of asbestos in the dust reported as either the mass of asbestos per unit area or as the mass of asbestos per mass of sampled dust as derived from a quantitative TEM analysis.This test method does not describe procedures or techniques required to evaluate the safety or habitability of buildings with asbestos-containing materials, or compliance with federal, state, or local regulations or statutes. It is the user's responsibility to make these determinations.At present, no relationship has been established between asbestos-containing dust as measured by this test method and potential human exposure to airborne asbestos. Accordingly, the users should consider other available information in their interpretation of the data obtained from this test method.This definition of dust accepts all particles small enough to pass through a 1 mm screen. Thus, a single, large asbestos-containing particle(s) (from the large end of the particle size distribution) disassembled during sample preparation may result in anomalously large asbestos surface loading results in the TEM analyses of that sample. Conversely, failure to disaggregate large particles may result in anomalously low asbestos mass surface loadings. It is, therefore, recommended that multiple independent samples be secured from the same area, and that a minimum of three samples be analyzed by the entire procedure.1.1 This test method covers a procedure to (a) identify asbestos in dust and (b) provide an estimate of the surface loading of asbestos in the sampled dust, reported as either the mass of asbestos per unit area of sampled surface or as the mass of asbestos per mass of sampled dust.1.1.1 If an estimate of asbestos structure counts is to be determined, the user is referred to Test Method D 5755.1.2 This test method describes the equipment and procedures necessary for sampling, by a microvacuum technique, non-airborne dust for levels of asbestos. The non-airborne sample is collected inside a standard filter membrane cassette from the sampling of a surface area for dust which may contain asbestos.1.2.1 This procedure uses a microvacuuming sampling technique. The collection efficiency of this technique is unknown. Variability of collection efficiency for any particular substrate and across different types of substrates is also unknown. The effects of sampling efficiency differences and variability on the interpretation of dust sampling measurements have not been determined.1.3 Asbestos identified by transmission electron microscopy (TEM) is based on morphology, selected area electron diffraction (SAED), and energy dispersive X-ray analysis (EDXA). Some information about structure size is also determined.1.4 This test method is generally applicable for an estimate of the surface loading of asbestos starting from approximately 0.24 pg of asbestos per square centimetre (assuming a minimum fiber dimension of 0.5 μm by 0.025 μm, see 17.8), but will vary with the analytical parameters noted in 17.8.1.4.1 The procedure outlined in this test method employs an indirect sample preparation technique. It is intended to disaggregate and disperse asbestos into fibrils and fiber bundles that can be more accurately identified, counted, and sized by transmission electron microscopy. However, as with all indirect sample preparation techniques, the asbestos observed for quantitation may not represent the physical form of the asbestos as sampled. More specifically, the procedure described neither creates not destroys asbestos, but it may alter the physical form of the mineral fibers.1.5 The values stated in SI units are to be regarded as the 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 and health practices and determine the applicability of regulatory limitations prior to use.

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This specification describes the required properties and corresponding test methods for glass covers and slides for use in routine microscopy. The covers and slides comply to specified requirements as to dimension, planeness and parallelism, corrosion resistance, and workmanship. They shall also be tested for their conformance to other properties such as index of refraction, clarity, resistance to boiling, solubility, and wettability.1.1 This specification describes glass covers and slides for use in routine microscopy.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 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 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.1.1 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.

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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 ASTM 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 assure 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).75.2.2 Measuring the area of positively stained mineralized deposits in cell cultures (ASTM F2997).5.2.3 Quantifying the spread area of fixed cells (ASTM F2998).5.2.4 Determining DNA damage in eukaryotic cells using the comet assay (ASTM 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 (ASTM F2944).1.1 This guidance document has been developed to facilitate the collection of microscopy images with an epifluorescence microscope that allow 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.

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5.1 Under well-controlled conditions, the quantitative evaluation of morphological features of a cell population can be used to identify changes in cellular behavior or state. Cell morphology changes may be expected when, for example, there is a response to changes in cellular cytoskeleton organization (1), a response of cells to toxic compounds, changes in differentiation state, and changes in adhesion properties of cells to a substrate by either chemical or mechanical-induced extracellular matrix-based (ECM-based) signaling pathways (2, 3). Typically, populations of cells exhibit a range of morphologies even when the cells are genetically identical and are in a homogeneous environment (4). This biological variation in cell response is due to both cell-cycle variations and stochasticity in the cellular reactions that control adhesion and spreading in cells. By using cell-by-cell, microscopy-based measurements and appropriate statistical sampling procedures, the distribution of cell morphologies such as cell spreading area per cell can be measured. This distribution is highly characteristic of the culture and conditions being examined.5.2 It is important to note that the use of this technique for cells on or in a 3-D scaffold materials can complicate the interpretation of the data. The topographic transforms of the cells on a 3-D material may require full volumetric imaging and not just wide-field fluorescence imaging as described here.5.3 the following are several examples of how this measurement can be used in a laboratory:—5.3.1 Quantify Cellular Response to a Biomaterial—The measurement of cell spread area can be used to characterize the response of cells to biomaterials. For example, spreading of most cell types is extremely sensitive to the stiffness of the culture substrate (5), (6). It is important to note that cell response to an ECM may be dependent on the preparation of the matrix. For example, the same ECM proteins prepared in a fibrillar or non-fibrillar surface coating can result in different morphology response5.3.2 Quality Control Metric for General Cell Culture Conditions—Cell spread area may be a useful metric for monitoring a change in cell culture conditions (that is, due to a serum component, pH, passage number, confluence, etc.). Cell morphology is often altered when cells are stressed and proceeding through cell-death related processes (that is, apotoposis).5.3.3 Quality Control Metric for Biomaterial Fabrication—Cell spread area measurements may be useful for generating specifications for a biomaterial. These specifications may stipulate how a particular cell line responds to a fabricated biomaterial.5.3.4 Quality Control Metric for Cell Line Integrity and Morphology Benchmarking—The morphology characteristic of a cell line grown under specified conditions should ideally be the same over time and in different laboratories. Thus, cell spread area measurements may be useful for validating that no significant changes in the cell cultures have occurred. This measurement provides a benchmark that is useful for establishing the current state of the cell culture and a metric that can be charted to increased confidence for within and between laboratory comparisons of cellular measurements (7).1.1 This guide describes several measurement and technical issues involved in quantifying the spread area of fixed cells. Cell spreading and the distribution of cell spread areas of a population of cells are the result of a biological response that is dependent on intracellular signaling mechanisms and the characteristics of cell adhesion to a surface. Cell spread area is a morphological feature that can be responsive to alteration in the metabolic state or the state of stress of the cells. Changes in cell spread area can also indicate an alteration in the adhesion substrate that may be due to differences in manufacturing of the substrate material or be in response to extracellular matrix secretions. High quality measurement of cell spread area can serve as a useful metric for benchmarking and detecting changes cell behavior under experimental conditions.1.2 The measurement described in this document is based on the use of fluorescence microscopy imaging of fixed cells and the use of image analysis algorithms to extract relevant data from the images. To produce robust cell spread area measurements, technical details involved in sample preparation, cell staining, microscopy imaging, image analysis and statistical analysis should be considered. Several of these issues are discussed within this document.1.3 This standard is meant to serve as a guide for developing methods to reliably measure the area to which cells spread at a surface. This surface can be conventional tissue culture polystyrene or sophisticated engineered biomaterial surfaces. An example of a detailed procedure to measure the spreading area of cells on a tissue culture polystyrene surface is provided in the appendix section.1.4 Cell morphology features such as cell spreading area and perimeter are generally reported in units of length. For example, spreading area per cell (that is, cell spread area) is likely reported in units of µm2. A spatial calibration standard is required to convert between numbers of pixels in a CCD camera image to µm2 as an SI unit.1.5 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 and health practices and determine the applicability of regulatory limitations prior to use.1.5.1 Sodium azide is used as a anti-bacterial reagent in the slide mounting media. This preserves the integrity of the mounting media. The toxicity of this reagent (for example, MSDS) should be considered before use of this reagent in large scale slide mounting procedures.

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5.1 This document will be of use to forensic laboratory personnel who are involved in the analysis of GSR samples by SEM/EDS (5).5.2 SEM/EDS analysis of GSR is a non-destructive method that provides (6, 7) both morphological information and the constituent elements detected in individual particles.5.3 Particle analysis contrasts with bulk sample methods, such as atomic absorption spectrophotometry (AAS) (8), neutron activation analysis (NAA) (9), inductively coupled plasma atomic emission spectrometry (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS), where the sampled material is dissolved or extracted prior to the determination of total element concentrations, thereby sacrificing size, shape, and individual particle identification.1.1 This practice covers the analysis of gunshot residue (GSR) by scanning electron microscopy/energy-dispersive X-ray spectrometry (SEM/EDS). The analysis is performed using automated software control of both the SEM and EDS systems, to screen the sample for candidate particles that could be associated with GSR. Manual control of the instrument is then used to perform confirmatory analysis and classification of the candidate particles. This practice refers solely to the analysis of electron microscopy stubs (1).21.2 Since software and hardware formats vary among commercial systems, guidelines will be offered in the most general terms possible. For proper terminology and operation, consult the SEM/EDS system manuals for each instrument.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard cannot replace knowledge, skills, or abilities acquired through education, training, and experience (Practice E2917), and is to be used in conjunction with professional judgment by individuals with such discipline-specific knowledge, skills, and abilities.1.5 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.1.6 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 practice is intended to be used by subject matter experts in the field of forensic pGSR analysis who have met their laboratory’s technical requirements to be assigned to the role of trainer in the category of testing that deals with the detection, analysis, and classification of pGSR particles.5.2 This practice is intended to be used in conjunction with Practice E2917, as well as the laboratory’s existing generalized training protocols, standard operating procedures, and quality practices, to develop a complete training-to-competency program in pGSR analysis by SEM/EDS. This practice provides the required additional, discipline-specific elements for pGSR analysis by SEM/EDS, in accordance with 5.3.2 of Practice E2917; it does not include the core specific elements covered in 5.3.1 of Practice E2917.5.3 The topics and procedures outlined in this practice are grounded in the body of scientific literature that exists in the field of pGSR examination.5.3.1 Additional sources of information on pGSR examination, not specifically mentioned in this document, should be considered, added, or substituted. A review of new sources of information on general forensic methods and pGSR examinations should be carried out on a regular basis (e.g. annually or biannually) to incorporate well-established current findings and methods into the training program and to replace any outdated methods.5.3.2 When possible, make additional training available to the trainee. Such training might include off-site short courses, short internships, and specialized training by experienced examiners.1.1 This practice describes the minimum requirements of a training program in primer gunshot residue (pGSR) analysis by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS). It describes lessons, practical exercises, and progress monitoring and evaluation that should be part of a laboratory’s training program.1.2 The primary purpose of this practice is to facilitate the development and implementation of training programs in crime laboratories or other such analytical entities that participate in the detection, analysis, and classification of pGSR particles.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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