Characterization of Nanoparticles: Measurement Processes for Nanoparticles

Denmark

Chapter 1

Introduction

Alexander G. Sharda; Vasile-Dan Hodoroabab; Wolfgang E.S. Ungerb    a National Physical Laboratory, Teddington, United Kingdom

b Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin, Germany

Abstract

The purpose of this book is to provide a comprehensive collection of analytical methods that are commonly used to measure nanoparticles, providing information on one, or more, property of importance. The chapters provide up-to-date information and guidance on the use of these techniques, detailing the manner in which they may be reliably employed. Within this chapter, we detail the rationale and context of the whole book, which is driven by the observation of a low level of reproducibility in nanoparticle research. The aim of the book is to encourage awareness of both the strengths and weaknesses of the various methods used to measure nanoparticles and raise awareness of the range of methods that are available. The editors of the book have, for many years, been engaged in European projects and standardization activities concerned with nanoparticle analysis and have identified authors who are experts in the various methods included within the book. This has produced a book that can be used as a definitive guide to current best practice in nanoparticle measurement.

Keywords

Nanoparticle; Size; Size distribution; Shape; Chemistry; Coating; Concentration; Standards; Charge; Characterization

Abbreviations

ACS 

American Chemical Society

CCQM 

Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology

DLS 

dynamic light scattering

ISO 

International Organization for Standardization

OECD 

The Organisation for Economic Co-operation and Development

REACH 

Registration, Evaluation, Authorisation and Restriction of Chemicals

VAMAS 

Versailles Project on Advanced Materials and Standards

Acknowledgements

We are deeply indebted to the contributors to this book for their considerable efforts in writing the chapters for this book and for their patience with the compilation and editing process. We are indebted to Gabriela Capile and her colleagues from Elsevier for assisting us in the collation and organization of this work.

Rationale

The purpose of this book is to provide a succinct collection of measurement methods that may be applied to nanoparticles to determine their most important physical and chemical properties. Nanoparticles are increasingly used in innovative products manufactured by advanced industries and provide enhanced, unique properties of great commercial and societal value. The demand for high-performance materials places increasingly stringent tolerances on the properties of nanoparticles. Additionally, the risk assessment of new nanoparticles requires fit-for-purpose measurements by physical and chemical methods to ensure that valid toxicity and ecotoxicity conclusions are reached.

The major purpose of this work is to summarize the most useful and widely available methods to measure important physical and chemical properties of high-performance nanoparticles. The aim is to support the drive for reproducibility and confidence in the nanoparticle supply chain and to help manufacturers to produce the correct physical and chemical property data needed to prepare registration dossiers under current and future regulation. In Europe, for instance, this binding registration process is regulated under REACH. Europe's manufacturers and importers of chemicals have a general obligation to register each nanoform substance manufactured or imported in relevant quantities. In the registration dossier, they must identify the risks linked to the substances they produce and market based on valid measurement procedures. There are similar obligations in other economies also.

Context

In 2016, the editors of ACS Nano stated ‘It is now time for the nanomaterials community to consolidate and to agree on methods of characterization and minimum levels of analysis of materials’ [1], and this book, in part, is aimed at addressing this challenge. In that editorial, they provided an example of toxic surfactants adsorbed on gold nanoparticles: the unknown presence of which could skew the assessment of gold nanoparticle toxicity [2]. There are many examples of the influence of nanoparticle coatings on biological effects, and coatings are present on virtually all particles whether they are intended or not. Size measurement alone is insufficient and, whilst the editorial suggested a range of ‘standard’ measurement techniques, none of the listed methods would have identified the presence of the toxic adsorbate.

Furthermore, the OECD council recommended in 2013 that ‘…the approaches for the testing and assessment of traditional chemicals are in general appropriate for assessing the safety of nanomaterials but may have to be adapted to the specificities of nanomaterials’ [3]. As a consequence, the OECD Working Party on Manufactured Nanomaterials (WPMN) launched projects to further explore the need for revisions of existing test guidelines and the preparation of new ones. Projects of the Working Party within the remit of this book are addressing (i) the determination of the (volume) specific surface area of manufactured nanomaterials, (ii) particle size and size distribution of manufactured nanomaterials, (iii) an identification and quantification of the surface chemistry and coatings of nano- and microscale materials, and (iv) environmental transformations of nanomaterials. The last project is rather challenging for nanoforms that are neither soluble nor have high dissolution rate but morphological, chemical transformation, and other abiotic degradation has to be carefully measured.

The obligation to register new nanoforms, there are hundreds of them every year, will lead to a big workload in testing. A possible workaround can be the development of grouping and read-across approaches that are based on the use of several sets of information: data obtained by measurement methods as described in this book, information on fundamental behaviour and reactivity of specified nanomaterials, and the toxicological data of relevant REACH endpoints or assays. The first step in the read-across workflow is the identification of appropriate characterization methods for the nanoforms in the substance. Currently, it is assumed that the minimum parameters that should be measured are composition (including impurities), particle size (in one or more dimensions), particle shape, and surface chemistry (chemical identity).

Recent research activities have been attempting the effective prediction of nanomaterial's health and environmental risks using existing or newly generated data and newly developed predictive hazard assessment models based on refined hazard-correlated endpoints. Achieving this goal requires the development of multiscale algorithms in nanoinformatics, which link existing and emerging data and advance the current state-of-the-art in silico modelling and predictive toxicology approaches.

In summary, it is therefore essential to establish the key measurement methods to support the manufacture, performance, and reliability of nanomaterials and to enable safe-by-design concepts. Many types of innovative nanoparticles exist: metals used in catalysis, medical applications and conductive inks; metal oxides employed in fuel cells and ferrofluids and as contrast agents for magnetic resonance imaging; semiconductors used as quantum dots and rods for bioimaging, photonics, display, and lighting technologies; and organic particles used for electronic applications, drug delivery vehicles, fluorescent reporters, and advanced coatings. In these applications, one of the important properties defining material performance is the size of the particles and, related to this, the size distribution. For monodisperse nanomaterials, particularly those which are spherical, the measurement of size is not a contentious issue; the currently accepted uncertainty is typically of the order of 1 nm [4], with dynamic light scattering (DLS) producing somewhat inaccurate results. However, DLS is a convenient and precise method and therefore is useful and, indeed, highly used in academic and industrial contexts where accuracy is not a priority.

The measurement of size distribution is far more challenging than measuring the average size of a population. This may be appreciated by considering a normal distribution and the error in estimating a mean value compared with the error in estimating a standard deviation from the sampled data. A sample size of ~ 10 is more than sufficient to establish the mean, but a sample size of more than 100 is required to have confidence that the standard deviation of the sample is within 10% of the standard deviation of the population. Therefore, it involves considerable effort to measure accurate size distributions using microscopies.

Relevance

The production of nanoparticles is of growing economic importance, an example being the number of medicines which are now formulated as nanoparticles. It is hard to assess the size of the nanomedicine market because this often incorporates drugs conjugated to polyethylene glycol or proteins. If these are included, the market size is greater than 100 billion, to an extent that it does not matter whether the units are US dollars, Euros, or pound sterling. Although these are technically nanoparticles in terms of size and certain measurement techniques that could be commonly employed, chemical measurements have a far greater importance, and the nature of these materials means that, for example, advanced mass spectrometries provide a majority of the required information. The main nonconjugate forms of nanomedicine on the market, where nanoparticle measurement is essential, consist of liposomes, nanocrystals, polymers, emulsions, virosomes, and magnetic particles [5].

In 2012, the OECD [6] and ISO [7] both presented a list of nanoparticle properties, which should be measured in a standardized manner. A number of the OECD measurements relate to the properties of dry powders, which are not of major concern for high value nanomaterials; some others, such as ‘redox potential’, ‘radical formation potential’, and ‘photocatalytic activity’, are functional measurements, which are hard to define. Nevertheless, these lists contain properties that are appropriate and may be succinctly listed as size, size distribution, aggregation state, shape, surface area, composition, surface chemistry, and surface charge. It is interesting to note that the concentration of nanoparticles is not listed as a property to be measured. This is a surprising omission given that this parameter is expected to be important in a toxicological context. Those working in other areas, such as nanomedicine, require accurate concentrations to establish dose, and therefore, this property is considered within many of the chapters in this book.

Accurate measurement of the concentration of nanoparticles in suspension is required to optimize and reproduce formulations and products as well as dose and exposure. Within the framework of the EU Commission recommendation for the definition of nanomaterials (2011/696/EU), this is based upon the number concentrations of particles within size limits. This definition is part of upcoming regulations, and therefore, it is essential for the industry to have a means of measuring particle number concentration both below and above the 100-nm limit of the definition to ensure that they comply with EU regulation. In most cases, this number concentration is not known but is calculated upon the basis of assumptions and mass-balance considerations. However, there is currently a lack of validated methods that can be used to determine nanoparticle number concentrations. This has been highlighted in a review authored by experts from industry: DuPont, BASF, Dow, and Bayer [8]. In the review, the authors highlighted the need for an interlaboratory comparison: ‘To our knowledge, no rigorous concerted interlaboratory evaluations of the ability of various techniques to accurately count industrial nano-objects have been completed’. This situation is changing; a recent VAMAS interlaboratory study [9] and an associated study ongoing at the committee addressing metrology in chemistry at the international Metre Convention [10] have been completed involving more than 50 international laboratories. The results of these comparisons have not yet been published, but it appears that the most accurate methods are capable of better than 10% relative uncertainty for spherical gold particle colloidal concentrations. For other types of particle or measurement method, the situation is significantly worse, and errors of a factor of two or more are not only possible, but common.

The chemical composition of particles is often assumed rather than measured. This is an area that requires considerable attention and is exacerbated by the requirement to understand the distribution of chemistry in and on the particle. Hence there is, in practice, an intrinsic core–shell or core–multiple shell structure to the majority of nanoparticles. Sometimes, the coating is intentional and is required to guarantee specific performance, examples being the prevention of aggregation, the use of capping layers on quantum dots to improve photoluminescence quantum yields and reduce environmental reactivity [11], the prevention of photocatalytic activity of TiO2 nanoforms in sun blockers by using alumina or silica capping [12], surface functional groups to enable bioconjugation and subsequent biological activity [13], protein and polymer coatings in diagnostic and drug delivery applications [14], core–shell metallic systems to enhance catalyst performance [15], and fluorocarbon coatings on magnetic particles for ferrofluids. In other cases, the coating is unintentional, such as oxidation or contamination of the particle surface or a protein corona formed in biological systems. All of these surface layers affect nanoparticle performance, but controlling the surface chemistry of nanoparticles is not trivial. Any surface modification, whether intentional or not, needs to be measured to fully understand the particles and their behaviour.

Scope

High-performance nanoparticles imply an intentional control of particle shape, size, and chemistry and a consequent homogeneity in these properties across the population. They have to be safe by design according to regulations. Consequently, such materials are amenable to measurement and characterization at a far higher level than high volume, low-cost nanoparticles such as those used in paints, composites, and consumer products such as toothpastes and sunscreens. There is, additionally, a greater impetus from manufacturers and users of these materials to apply appropriate and accurate measurement methods to confirm the quality of nanoparticle ingredients. This book covers both types of nanoparticles and many of the demonstrations of analytical capability are provided using high quality nanoparticles. In cases where the particle population has a high variability, the effect of this one the measurement is noted.

This book is concerned only with the measurement of nanoparticles and does not review or consider the potential applications and use of such materials. There is an emphasis in the book on nanoparticles in colloidal suspension or attached to solid surfaces in which high-performance particles are generally employed, and coverage of a number of methods applicable to nanoparticles in powder form is also described.

Structure

This book provides details of many measurement techniques that may be applied to nanoparticles to measure specific properties. The measurement of particle shape is best performed using microscopic imaging methods, and these are covered in Section 2. These methods also provide a measure of particle size and size distribution, although due attention must be paid to adequate sampling, statistical relevance and artefacts of the preparation, and imaging methods. The analysis of particles in colloidal suspension is dealt with in Section 3, which is organized into methods that measure individual particles (Section 3.1), ensembles of particles (Section 3.2), and separation or fractionation methods (Section 3.3). Dry particle analysis is covered in Section 4, which includes surface area measurements by gas adsorption and various methods of chemical analysis. Because the aim of this book is to establish best practice and consistency in nanoparticle measurement, a chapter covering the relevant existing and future metrology and standards is provided in Section 5.

References

[1] Mulvaney P., et al. Standardizing Nanomaterials. ACS Publications; 2016.

[2] Leonov A.P., et al. Detoxification of gold nanorods by treatment with polystyrenesulfonate. ACS Nano. 2008;2(12):2481–2488.

[3] OECD. Draft Recommendation of the Council on the Safety Testing and Assessment of Manufactured Nanomaterials. 2013.

[4] Meli F., et al. Traceable size determination of nanoparticles, a comparison among European metrology institutes. Meas. Sci. Technol. 2012;23(12):125005.

[5] Weissig V., Pettinger T.K., Murdock N. Nanopharmaceuticals (part 1): products on the market. Int. J. Nanomed. 2014;9:4357.

[6] OECD. Guidance on Sample Preparation and Dosimetry for the Safety Testing of Manufactured Nanomaterials. 2012.

[7] ISO. ISO/TR 13014 Nanotechnologies—Guidance on Physico-Chemical Characterization of Engineered Nanoscale Materials for Toxicological Assessment. 2012.

[8] Brown S.C., et al. Toward advancing nano-object count metrology: a best practice framework. Environ. Health Perspect. 2013;121(11–12):1282–1291.

[9] TWA 34 Nanoparticle Populations. Available from: http://www.vamas.org/twa34/index.html.

[10] Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM). Available from: https://www.bipm.org/en/committees/cc/ccqm/.

[11] Shirasaki Y., et al. Emergence of colloidal quantum-dot light-emitting technologies. Nat. Photonics. 2013;7(1):13.

[12] Smijs T.G., Pavel S. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness. Nanotechnol. Sci. Appl. 2011;4:95.

[13] Medintz I.L., et al. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 2005;4(6):435.

[14] Byrne J.D., Betancourt T., Brannon-Peppas L. Active targeting schemes for nanoparticle systems in cancer therapeutics. Adv. Drug Deliv. Rev. 2008;60(15):1615–1626.

[15] Serpell C.J., et al. Core@ shell bimetallic nanoparticle synthesis via anion coordination. Nat. Chem. 2011;3(6):478.

Chapter 2.1.1

Characterization of nanoparticles by scanning electron microscopy

András E. Vladára; Vasile-Dan Hodoroabab    a National Institute of Standards and Technology (NIST), Gaithersburg, MD, United States

b Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin, Germany

Abstract

In this chapter, sample preparation, image acquisition, and nanoparticle size and shape characterization methods using the scanning electron microscope (SEM) in reflective and transmitted working modes are described. These help in obtaining reliable, highly repeatable results. The best solutions vary case by case and depend on the raw (powdered or suspension) nanoparticle material, on the required measurement uncertainty, and on the performance of the SEM.

Keywords

Nanoparticle; Sample preparation; Scanning; Electron microscope; SEM; Characterization; Size; Shape; Circular equivalent; Threshold

Introduction

The high-resolution scanning electron microscope (SEM) is excellent for nanoparticle size and shape characterization, because the necessary sample preparation and image acquisition are relatively quick and simple. Although the SEM image is a two-dimensional (2D) representation of the three-dimensional (3D) objects from a certain viewing angle, the SEM image does contain a certain amount of 3D information that with model-based measurement can be used to reconstruct the shape with sub-nm accuracy of a simple structure [1].

The signal responsible for the creation of a SEM image is given by the secondary electrons (SEs) and/or backscatter electrons (BSE) created when the sample is bombarded by the primary electron (PE) beam. The typical maximum accelerating voltage of a SEM is 30 kV. Whilst the SE due to their low energy below 50 eV have a small escape depth in the range of a few nm, the BSE possess significantly higher kinetic energies (up to the excitation energy) and come from about a half of the depth of the interaction volume in the sample, see Fig. 1A. Thus, the SEs are capable of producing high-resolution SEM micrographs of superior surface morphology contrast, whilst BSE micrographs reveal superior compositional contrast but with poorer spatial resolution. Further, the SE electrons can be categorized into SE1, that is, those generated at the point of impact of the primary electros with the sample; SE2, that is, those generated by the high-energy electrons within the sample; and SE3, that is, those generated by the BSE electrons hitting the inner surfaces of the microscope specimen chamber. Detectors mounted inside the electron column, such as so-called in lens or through the lens, are able to collect secondary electrons with very good efficiency at an optimal working (final lens sample) distance and provide the highest spatial resolution possible.

Fig. 1 Schematic illustration of the types of electrons generated by the primary electron beam of the SEM and the typical reflected electron detectors, the conventional E–T detector and optional in-lens detector (A); and the STEM detection modes, the conventional type (B); and transmission sample holder with converter and E–T detector (C).

In the recent years, the SEM version of scanning transmission electron microscopy (STEM) working mode has gained popularity, especially in the analysis of nanoparticles due to the excellent, better than 1 nm spatial resolution of modern high-resolution SEMs. The main advantage of operating the SEM in the transmission mode is the enhanced mass–thickness contrast, which enables more accurate determination of the nanoparticle boundaries and, hence, enables more accurate determination of size and size distribution of nanoparticles. This STEM working mode is STEM-in-SEM (also ‘TSEM’ or ‘low-voltage TEM’); operation mode is similar to the conventional TEM (see Chapter 2.1.2), which needs preparation of the electron-transparent sample on thin foils (TEM grids). The lower accelerating voltages of the SEM result in less scattering events in the thin sample and thus lead to improved contrast for nanoparticles. For low-atomic number materials consisting of different structures with low electron density differences, the SEM transmission contrast is particularly superior to conventional TEM. Modern SEMs come with STEM detectors as part of their standard configuration, see Fig. 1B. STEM signals can be also detected by using special sample holders such as shown in Fig. 1C. Some of the primary electrons generate transmitted electrons (TE), which are converted into secondary electrons by a thin gold layer. These SEs are then detected by the regular Everhart–Thornley (E–T) detector as a transmitted electron signal. Other SEs are excluded from detection by simple shielding and or by electric fields. Both STEM versions can be operated in bright-field (BF) mode and in the dark-field (DF) mode. The BF images are generated by inelastically, forward scattered electrons, whilst the DF images are generated by elastically scattered electrons, which are deflected stronger, at higher angles. It should be noted that the relatively simple modelling of the transmission signal STEM-in-SEM (physical modelling or by Monte Carlo simulation) is straightforward and recently reported in the literature, for example, Ref. [2]. This background is of crucial importance towards accurate measurement of the nanoparticle size and makes STEM one of the very few analytical techniques capable of providing traceable results for nanoparticle size and size distribution.

A representative example of exploiting the complementary analysis top-view SEM and STEM imaging of the same field of view is given in Fig. 2. Thus, nanometre-scale details of the morphology of the particle surface provided by a high-resolution in-lens detector can be complemented with the information derived from a transmission micrograph. Further practical examples of nanoparticle analysis with the complementary SEM and STEM-in-SEM modes can be found, for example, in Refs. [3,4]. Even more additional information on elemental composition can be obtained by energy dispersive X-ray analysis (EDX). Many SEMs are equipped with an energy dispersive spectrometer (EDS), so that elemental maps can be added to SE or STEM images (see Chapter 4.4). The spatial resolution of the elemental information itself is worse than of that SE or STEM images, as its information volume is significantly larger. Still, compositional information can be indispensable in the analysis of nanoparticles.

Fig. 2 SEM micrographs of SiO 2 particles obtained with the high-resolution in-lens SE detector (A) and with converted transmitted electrons detected by the E–T detector (B). Note nanometre-scale details on particle surface morphology in the SE image (arrows) and sharp particle boundaries in the STEM image. (From V.-D. Hodoroaba, et al., Surf. Interface Anal. 46 (2014) 945–948; Wiley & Sons, Ltd.)

For the sake of simplicity, this chapter deals only with two attributes of morphology, size and projected shape, for discrete and agglomerated or aggregated nanoobjects. Rigorous 3D characterization of nanoparticles would include size, shape, surface structure (e.g. texture), surface, and internal material composition, including their chemical state and locations in the investigated volume.

Suitable sample preparation is key to obtaining high-quality high-resolution electron microscope images and superb nanoparticle characterization. The preferred techniques often vary with the sample material.

It is equally important to make sure that the SEM itself is suitable to carry out the measurements with the required uncertainty.

The size of nanoobjects that can be measured well depends on the sample, on the required uncertainty, and on the performance of the SEM. The resolving power of some SEMs may not be sufficient to determine size and shape differences among small, for example, sub-10 nm nanoparticles. For example, if the size of the spot that the electron-optical column focuses the primary electron beam is 5 nm, the difference between the images of 3- and 4-nm size particles could be only in signal intensity at the locations of the particles, which could make the size measurement uncertainty of these particles large. In some cases, due to noise, the small particles cannot be recognized in the SEM images, and this makes size and shape measurements unfeasible.

The number of nanoobjects that shall be measured for good-quality results depends on the sample, on the required uncertainty, and on the performance of the SEM. Typically, several hundreds or thousands of particles need to be measured for statistically sound size and shape characterization. Optimization and automation of the image acquisition and data analysis can reduce cost and improve the quality of the results.

Sample preparation

The quality of SEM nanoparticle size and shape measurement results strongly depends on sample preparation. There is no single best preparation method for all nanoparticles, rather more or less excellent methods determined by the characteristics of nanoparticles and the measurand, the quantity or property to be measured. It is indispensable to ensure that the test or analytical samples are representative and relevant to the measurand. The tools used for preparation and handling nanoparticles must be kept clean and stored dry in a clean enclosure or bench, preferably in high-efficiency particulate air (HEPA) filtered air where the sample preparations take place.

The information on the health effects of various nanoparticles is not yet complete. Some nanoparticles are known to be hazardous to health, so it is important to always wear appropriate personal protective equipment, including disposable gloves, safety glasses, laboratory coats, and filter respirators, and take appropriate precautions when handling nanomaterial.

Nanoparticle samples are typically powders or suspensions, which often need to be diluted to image discrete particles or to minimize particle pile up. If the presence of smaller particles on larger particles can be visualized with high-resolution SEM, smaller particles ‘hidden’ beyond larger particles cannot be detected. Measuring touching particles is possible, but it may require operator-assisted separation of particles to avoid unacceptably high measurement uncertainty. The goal is to separate and deposit on a substrate surface a smaller number of nanoparticles from the raw nanoparticle material so that uniform, representative test or analytical samples are prepared. The contrast between particles and substrate should be high and the particles not far from each other, so that a large number of nanoparticles can be measured across a relatively small number of images. Charge-based capturing of nanoparticles on functionalized substrate surfaces has shown superior results compared with simple dispersion [5]. All TEM/STEM samples are suitable for SEM imaging, so nanoparticle preparation methods published for TEM/STEM imaging and measurements apply to samples prepared for SEM, even when using nonelectron-transparent substrates, for example, Si chips [5–9]. For SEM and STEM imaging, powder samples are often dispersed in a liquid and then deposited on a substrate (e.g. a 10 mm by 10 mm or smaller conductive Si chip) or a 3-mm electron-transparent grid.

Besides many preparation methods found in pertinent literature, ISO 14703:2008(en) (Fine ceramics (advanced ceramics, advanced technical ceramics)—Sample preparation for the determination of particle size distribution of ceramic powders), ISO 14887:2000(en) (Sample preparation—Dispersing procedures for powders in liquids), and ISO 14488:2007(en) (Particulate materials—Sampling and sample splitting for the determination of particulate properties) provide guidance on powder sample preparation [10,11].

Many nanoparticles can be in liquid colloidal phase, that is, suspended within another substance where electrostatic or steric stabilization methods prevent agglomeration. To minimize the number of touching nanoparticles on the measurement substrate or grid, these must be diluted. Charge-based capturing of nanoparticles and careful rinsing away the medium of suspension have produced superior results over simple deposition, see Fig. 3. Note the lack of residual material, cleaner background, and better contrast on the right that allows for easier and more accurate size and shape measurements.

Fig. 3 SE images of colloidal Au particles deposited on Si chips with thin SiO 2 on top; 80-nm nominal size, deposited after dilution only, left; and 60-nm nominal size, diluted, poly- l -lysine captured and rinsed particles, right. HFW 429 and 600 nm, respectively.

There are commercially available TEM grids useful for nanoparticle sample preparation. These may have surface coatings or functionalized surfaces, designed to ensure good particle dispersion and capturing. Adding dilution liquids and surfactants to the raw liquid nanoparticle material may help the preparation of useful test samples. Depending on imaging parameters, both of these may alter the apparent size of the nanoparticles, so proper care must be taken, and if necessary, compensation of the results for these effects must be applied.

Nonspherical particles may appear very different in size and shape, depending on the viewpoint. Quasi-spherical and nonspherical nanoparticles may get deposited preferentially and laid down on their largest surface area. Some elongated shape nanoparticles, for example, TiO2 nanorods, may not always adhere to the sample surface at the same angle, so their apparent size and shape vary substantially, see Fig. 4.

Fig. 4 TiO 2 nanorod sample prepared on Si chip. The boundaries of 11 horizontally deposited particles were determined manually. 635-nm HFW.

Image pairs taken from two angles can help in the determination whether a nanorod is indeed deposited parallel to the flat substrate.

In the determination of their size and shape, to minimize measurement errors and the uncertainty of the results occurring due to this, it is indispensable to assess and use the information about how the 3D particles were deposited on the 2D sample surface. For this, operator-assisted determination of the particle boundaries is preferred.

An important objective of sample preparation is to generate a uniform distribution of particles across the entire measurement substrate. Several methods for depositing nanoparticles on measurement substrates are described, for example, in ISO 19749:2019(en) Nanoparticle size and shape distribution measurement using scanning electron microscopy and ISO 21363:2019(en) Measurements of Particle Size and Shape Distributions by Transmission Electron Microscopy. These also provide a lot of useful information and help in nanoparticle size and shape characterization including also relevant examples.

The knowledge about and the number of methods of nanoparticle deposition are growing rapidly; it is advisable to explore and use the most suitable methods in pertinent literature, specific to the nanoparticle measurand and material type.

SEM image acquisition

The first thing before starting the acquisition of the images for nanoparticle characterization is to make sure that the SEM is working suitably. ISO TD 21383:2019(en) ‘Microbeam analysis, Qualification of the scanning electron microscope for quantitative measurements’ and ISO/TS 24597:2011(en) ‘Microbeam analysis—Scanning electron microscopy—Methods of evaluating image sharpness’ provide guidance in this. Its various performance evaluation procedures and a useful spreadsheet help in proving that the SEM is indeed capable of measuring nanoparticles reliably.

SEMs are easy to operate and provide results quickly, but a number of notorious problems hinder operating them always at their best performance and are reasons for lack of excellent repeatability in imaging and measurements. The most bothersome are unintended motions, drifts of the sample stage and the primary electron beam, electron beam-induced contamination, image blur (lack of sharp electron beam focus), geometry distortions, wrong scale, charging, and noise. Quantification of these essential performance parameters is necessary to ensure that the SEMs perform at or better than its manufacturer's specification; it also helps in the calculation of measurement uncertainties and in maintenance and repairs.

Electron beam-induced contamination can easily make nanoparticle measurements erroneous or downright impossible. Contamination can arise from carbonaceous (mostly alkane) molecules that cover the surfaces of the sample or the instrument or both, which have high mobility under vacuum conditions, so they may stream in from far but get deposited when secondary electrons brake their chemical bonds. In some cases, the energetic electrons of the primary beam may overwhelm the deposition, and the centre of the image may be cleaner than the surroundings of the image frame.

Fig. 5 shows a SE and bright-field STEM image pair of a TiO2 sample where the deposited contamination makes it impossible to discern the particles in SE mode, whilst the STEM image shows them better. Contamination makes size measurement impossible with SEM and prone to errors with the transmitted image.

Fig. 5 A severe case of contamination: secondary electron (left) and dark-field STEM (right) images of TiO2 nanoparticles. 15-keV landing energy, 635-nm HFW.

In vacuum with low-energy oxygen or hydrogen plasma or ultraviolet light cleaning, it is possible to make the source molecules volatile and pumped out so that essentially contamination-free operation becomes achievable [12].

For good-quality SEM-based characterization of nanoparticles, it is indispensable to acquire good-quality images. For this, besides making sure that the SEM meets its performance specification, after setting the focus, astigmatism, it is necessary to use most of the grey levels without under- or oversaturating the image by setting the contrast and brightness appropriately, see Fig. 6 for an example.

Fig. 6 Properly set contrast and brightness, with no under- or oversaturation, as shown in the histogram at the lower left corner. SE image of evaporated tin spheres, varying in diameter from several micrometre to a few nanometre.

In the SEM, both reflected SE and BSE and transmitted electron imaging modes are possible, see Fig. 7. There is advantage in acquiring simultaneously more than one signal's images, as they present additional information useful for nanoparticle characterization and there is no time burden, only extra file storage that is a negligible added expense. Simultaneous acquisition eliminates the distortion difference between the image pair as well.

Fig. 7 Simultaneously acquired SE image (left) and bright-field STEM image (right) of TiO 2 nanoparticles at 30-keV landing energy. 508-nm HFW.

In the SEM in scanning transmission working mode, the landing energy is much lower than of the transmission electron microscopes (TEMs), which is in most instances advantageous, that is, with respect to enhanced contrast for close Z elements. Measurements of samples of discrete nanoparticles are generally easier to carry out with automated image acquisition and particle analysis. Overlapping, aggregate, or agglomerate particles and highly varying particle/background samples may require operator-assisted image acquisition and analysis.

SEM image-based particle size measurements

Size measurements determine a set of parameters related to the size or size distribution of 3D particles, for example, the circular equivalent diameters in a projected view image. The first step consists of the acquisition of high-quality image of the nanoparticles; the second is the use of a suitable processing, criteria, and algorithms that generate a binary (black and white) version of the greyscale image, in which the particles are segregated, that is, their boundaries from the background and from each other are determined, and finally carry out the size measurement with the binary image, see Fig. 8 for illustration.

Fig. 8 Determination of circular equivalent diameter: SEM SE image of a carbon black aggregate nanoparticle (left), its binary version (middle), and its area-equivalent circle, A 1  =  A 2 . 424 nm HFW.

Depending on the resolving power of the SEM, it is possible to measure the size and size distribution of sub-10 nm particles. The smallest measurable nanoparticles start at 1-nm size for the highest resolution SEMs that have a resolution of 0.4 nm or better. Other instruments, especially those that are equipped with conventional tungsten filament electron source have a spatial resolution of a few nanometres, may not be able to suitably image the smallest nanoparticles. The actual minimum value of the measurable nanoparticle size depends on the instrument, its environment, and the sample as well. When reliable sizing of the nanoparticles—due to lack of sufficiently high resolution—is not possible, particle counting still might be feasible. For this, it might be sufficient to detect the presence of nanoparticles. The limit of the characterization of very small size nanoparticles with a given SEM can be determined with comparisons of the results of higher resolution and more sensitive other measurements, using suitable samples.

The measurement uncertainty also depends on the resolving power of the SEM. Generally, sharper, more focused electron beam will result in images that show more details of particles that appear smaller, whilst lack of focus or resolving power makes them appear larger with less detail and sharpness. This may not necessarily mean that good-quality size and shape distribution measurements cannot be carried out. Proper calibration using relevant reference samples with known particle sizes [3,5–7] could help to significantly reduce measurement uncertainty.

SEM image-based particle shape measurements

SEM shape measurements determine more information than simpler size measurements. The 2D projection, a view from a certain angle, may be a poor account for the 3D shape of a particle, so one should explore the errors arising from this and choose the viewing angles and the number of images appropriately to characterize the nanoparticles according to the requirements.

Accurate determination of the shape nanoparticles requires higher resolving power images than simpler size measurements. ISO 9276-6:2008(en) Representation of results of particle size analysis—Part 6: Descriptive and quantitative representation of particle shape and morphology states: ‘The optimum resolution shall not and cannot be stated absolutely but shall rather be related to the size of the element features to be determined (e.g. agglomerate branches, roughness scale). Analysis of image parameters is generally based on a digitized image. The process of digitizing an image can result in information loss because of the transformation of the continuous features innate to the particle into discrete elements of finite size — the finite resolution. For pixel errors, smaller than 5 % for a circle, the necessary pixel numbers per particle range from 100 to 200; for robust parameters, like projection area and ellipse ratio, up to 5,000 for parameters using the perimeter’. For a large number of particles measured, fewer pixels per particle might be sufficient.

Fig. 9 shows some of the particle shape measurement capabilities of ImageJ, an excellent public domain, high-performance image processing, and analysis software [13]. There are many image processing and analysis software available today; their quality, capabilities, and costs vary substantially.

Fig. 9 Schematic illustration of ImageJ particle shape measurement capabilities.

The selection and learning the use of suitable image processing and analysis software may not be easy, especially when custom solution is necessary for high-accuracy and/or high-speed nanoparticle characterization.

ISO 19749:2019(en) Nanoparticle size and shape distribution measurement using scanning electron microscopy and ISO 21363:2019(en) Measurements of Particle Size and Shape Distributions by Transmission Electron Microscopy provide a lot of useful information to help nanoparticle size and shape characterization.

ISO 13322-1:2014(en) Particle size analysis—Image analysis methods—Part 1. Static image analysis methods

The choice of various imaging parameters in the acquisition of SEM images of nanoparticles and the choice of evaluation or analysis procedures, especially thresholding algorithms, are essential in obtaining good results in particle size and shape measurements. Certain instrument parameters and or thresholding algorithms—depending on the sample—may have more or less impact on the quality and the uncertainty of the results. The use of local or adaptive thresholding algorithms may be necessary on backgrounds with varying grey levels as global thresholding algorithms will adversely impact the size and shape measurements. It is up to the operators of the SEM and the analysis solution to select suitable instrument parameters and algorithms and to evaluate the effects of these and select appropriate measurement conditions.

The effect of slight electron beam-induced contamination

Depending on the choice of thresholding algorithm used to turn the greyscale image into binary to determine the boundaries of nanoparticles, even relatively small amount of contamination that does not result in obvious growth of the particle size can change the measured size of nanoparticles. This happens because the contamination changes the secondary electron intensity and therefore the particle and background grey levels. Fig. 10 shows the binary version of the same area of nominally 60-nm size Au nanoparticles on a Si chip without and with small amount of contamination. The maximum entropy (ME) thresholding [14] resulted in a 1.7% decrease in particle size in the contaminated area in the centre, whilst Otsu thresholding [15] on the same image did not produce measurable difference in particle size there.

Fig. 10 Binary version of the same area of Au nanoparticles on a Si chip without (left) and with small amount of contamination (right) in the centre. 2.54-μm HFW.

Here are a few parameters that may impact particle size measurements:

•Linearity: generally, in SEMs, the most linear part of an image is the centre, as more nonlinearity likely to occur at around the edges of the acquired image.

•Scale or magnification of the SEM is often set in ranges, so even small change may result in a jump in actual magnification.

•Imaging working distance change introduces some image rotation and change in magnification, unless the SEM is well compensated.

•Imaging focus can have large effect on particle size; out-of-focus structures appear larger. Fig. 11 shows the same portions of the images of nominally 60-nm size Au nanoparticles. Depending on the thresholding procedure, the circular equivalent size of the particles measured with out-of-focus images can be larger by 7% with ME and 1.2% with Otsu algorithms than with in-focus images 64.9-nm ME and 60.5-nm Otsu.

Fig. 11 In-focus (top) and out-of-focus SE images (bottom) of the same nominally 60-nm size Au particles.

Using the sample, images were taken in focus (a), out of focus (k), in focus (l), and out of focus (v), back in focus (w) again at the same area to assess the size variation due to focus changes.

The particle size results using ME and Otsu algorithms are given in Table 1. ME results in larger sizes, and it is more sensitive to focus variation than Otsu. The more out of focus the particles are, the less pronounced the transition is between background and particles at the baseline, and the larger the effect is on ME thresholding.

Table 1

The relative difference is calculated with respect to the first (a) in-focus data.

Using all particles in all in-focus images, the standard deviation of the focusing uncertainty was estimated to be subnanometre.

The effect of landing energy

To assess the effect of changing the primary electron beam landing energy (accelerating voltage), secondary electron images were taken at 5 and 15 keV on a nominally 60-nm size Au nanoparticle sample. Fig. 12 shows the same portions of the images to make it easy to see the difference due to landing energy change.

Fig. 12 The same portions of the images of the same particles taken at 5 keV (left) and 15 keV (right) landing energy.

Measurable difference was found due to landing energy change with ME algorithm. One reason for this is the change in spatial resolution (beam focus), which was measured by FEI Image software (Version 1.05) to be 1.4 nm at 5 keV and 1 nm at 15 keV landing energy. Another reason is the electron beam–sample (sample including here particle and substrate) interaction that leads to different edge effect, which broadens the signal transition at the particle edge, shown in Fig. 13A. There, at the baseline, ME thresholding yields higher values, but almost no effect was found at the middle where the Otsu greyscale thresholding value is computed. See Table 2.

Fig. 13 Secondary electron line scans of the same gold nanoparticles obtained at 15 and 5 keV, both at 340 pA (A). Note the slightly different shape of the line scan. Secondary electron line scans of the same gold nanoparticles obtained at 340 and 21 pA, both at 15 keV (B). Note the difference in noise of the line scans.

Table 2

The relative difference was calculated with respect to the 15-keV data.

The effect of primary electron beam current

SEMs can focus smaller primary electron beam currents better than higher ones, so some effect due to a change in the primary electron beam current is expected. See Table 3 for measured results with ME and Otsu thresholding algorithms. Larger currents generate stronger signals, so the signal-to-noise ratio improves.

Table 3

The relative difference is calculated with respect to the 340-pA data.

The spatial resolution (focus and spot size), as measured by the SEM manufacturer's software, was found to be 1.1 nm at 340 pA, 1 nm at 85 pA, and 1 nm at 21 pA. Fig. 13B illustrates the difference in noise. Since the ME thresholding is more sensitive to noise than the Otsu, the difference in particle size between high- and low-current conditions is slightly larger for ME thresholding algorithm.

The effect of sample tilt

The sample tilt should have no effect on the size of spherical particles, but significant effect on nonspherical ones. It is known that even if the beam and sample stage are set to be perpendicular, some stray tilt is likely occurring. Generally, this does not exceed more than a couple of degrees. There might be imaging and thresholding differences also that might lead to size differences. These might be large on particles that are much different in height than in width.

To explore this matter, images of several hundred gold nanoparticles were taken at 0- and at 52-degree sample tilts (Fig. 14). Area-equivalent diameter distributions were obtained and compared at the two tilts using both the ME and Otsu thresholding algorithms in Figs. 15 and 16, respectively.

Fig. 14 Illustration of 52-degree sample tilt, when the working distance remains the same (A). Representative SEM images of 60-nm gold nanoparticles at 0- (B) and 52-degree (C) tilts at same working distance. 15-keV, 340-pA, 1.27-μm HFW images.

Fig. 15 Comparison of the area-equivalent diameter distributions of several hundred gold nanoparticles taken at 0- and 52-degree tilts. Image analysis was done using ME thresholding (A). Same data except for a 1.9-nm shift of the 52-degree distribution to larger sizes (B).

Fig. 16 Comparison of the area-equivalent diameter distributions of several hundred gold nanoparticles taken at 0- and 52-degree tilts. Image analysis was done using Otsu thresholding (A). Same data except for a 1.3-nm shift of the 52-degree distribution to larger sizes (B).

The comparisons yield a 1.9-nm (ME) and a 1.3-nm (Otsu) difference between the 0- and 52-degree tilt conditions, although this difference from 3% to 2% is still relatively small as it is showing that the particles are not completely spherical.

Reasons for the difference observed between the two tilts may include particle geometry, that is, the height of the particles is systematically lower than their width, and imaging/thresholding artifacts. To investigate the latter in the case of the ME thresholding, histograms and background uniformity of the images are compared in Fig. 17. The background is very uneven for the 52-degree images, which has significant effect on the ME thresholding. Specifically, the greyscale histogram of the background is significantly broader at 52-degree tilt leading to an increased thresholding value and to a consequential decrease in the diameter measurement.

Fig. 17 Representative SEM images—thresholded at around the average background signal level—of 60-nm gold nanoparticles imaged at 0- (A) and 52-degree (B) tilts. (C) Respective greyscale histograms and ME thresholding values for the 0- and 52-degree images. 15-keV, 340-pA, 1.27-μm HFW images.

In the case of the Otsu thresholding at 0-degree tilt, the generated signal may produce a few missing pixels at the centre of particles, see Fig. 18A. This can be easily remedied using the ‘fill hole’ function of the ImageJ software, which completes the particles without altering their size.

Fig. 18 Representative SEM image taken at 0-degree tilt—thresholded with the Otsu algorithm—of 60-nm gold nanoparticles (A). Representative SEM image taken at 52-degree tilt (B). Same image thresholded with the Otsu algorithm (C). Arrows show examples of missing pixels. 15-keV, 340-pA, 1.27-μm HFW images.

The asymmetry of the SE signal on and around the particles may result in missing pixels where the particles are attached to the substrate (Fig. 18C). The correction in the case of 52-degree images is not trivial. When Otsu thresholding is used at 52-degree tilt, undercounting of particle pixels leads to a decrease in the average circular equivalent particle size measurement results.

The effect of the number of pixels representing particles

In addition to 100 thousand times magnification (1.27-μm HFW) and 50 thousand times magnification (2.54-μm HFW), the investigation of the effect of magnification on particle size measurements was carried out with 25 thousand times magnification (5.08-μm HFW), 13 thousand times magnification (9.77-μm HFW), 7 thousand times magnification (18.1-μm HFW), and 4 thousand times magnification (31.7-μm HFW), see Table 4. All images were taken at 1024-pixel by 884-pixel resolution. The average size of the same nanoparticles is obtained from images taken at different magnifications, and the relative size difference is calculated with respect to the 100 thousand times magnification (1.27-μm HFW) data. The results for four sets of images are shown as a function of HFW and the number of pixels across a 60-nm particle in Fig. 19A and B,