Nanotechnologies — Measurements of particle size and shape distributions by transmission electron microscopy

This document specifies how to capture, measure and analyse transmission electron microscopy images to obtain particle size and shape distributions in the nanoscale. This document broadly is applicable to nano-objects as well as to particles with sizes larger than 100 nm. The exact working range of the method depends on the required uncertainty and on the performance of the transmission electron microscope. These elements can be evaluated according to the requirements described in this document.

Nanotechnologies — Détermination de la distribution de taille et de forme des particules par microscopie électronique à transmission

Le présent document spécifie une méthode permettant d'acquérir, de mesurer et d'analyser des images de microscopie électronique à transmission afin d'obtenir des distributions de taille et de forme à l'échelle nanométrique. Le présent document s'applique de façon générale aux nano-objets ainsi qu'aux particules de dimensions supérieures à 100 nm. La plage de fonctionnement exacte de la méthode dépend de l'incertitude exigée et des performances du microscope électronique à transmission. Ces éléments peuvent être évalués conformément aux exigences décrites dans le présent document.

General Information

Status
Published
Publication Date
24-Jun-2020
Current Stage
6060 - International Standard published
Start Date
25-Jun-2020
Due Date
28-Oct-2019
Completion Date
25-Jun-2020
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INTERNATIONAL ISO
STANDARD 21363
First edition
2020-06
Nanotechnologies — Measurements of
particle size and shape distributions
by transmission electron microscopy
Nanotechnologies — Détermination de la distribution de taille et de
forme des particules par microscopie électronique à transmission
Reference number
ISO 21363:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21363:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 21363:2020(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms, definitions and symbols .......................................................................................................................................................... 1

3.1 Core terms — Particles .................................................................................................................................................................... 1

3.2 Core terms — Image capture and analysis ..................................................................................................................... 4

3.3 Core terms — Statistical symbols and definitions ................................................................................................... 5

3.4 Core terms — Measurands ........................................................................................................................................................... 7

3.5 Core terms — Metrology .............................................................................................................................................................10

3.6 Core terms — Transmission electron microscopy ................................................................................................13

3.7 Statistical symbols, measurands and descriptors ..................................................................................................14

3.7.1 Statistical symbols .......................................................................................................................................................14

3.7.2 Measurands and descriptors ..............................................................................................................................14

4 Stakeholder needs for TEM measurement procedures ............................................................................................15

5 Sample preparation ........................................................................................................................................................................................16

5.1 General ........................................................................................................................................................................................................16

5.2 Sample sources ....................................................................................................................................................................................17

5.3 Use a representative sample ....................................................................................................................................................17

5.3.1 General...................................................................................................................................................................................17

5.3.2 Powder samples ............................................................................................................................................................17

5.3.3 Nanoparticle dispersions in liquids .............................................................................................................17

5.4 Minimize particle agglomeration in the sample dispersion .........................................................................18

5.5 Selection of the mounting support .....................................................................................................................................18

6 Instrument factors ...........................................................................................................................................................................................18

6.1 Instrument set-up..............................................................................................................................................................................18

6.2 Calibration ...............................................................................................................................................................................................19

6.2.1 General...................................................................................................................................................................................19

6.2.2 Calibration standards ...............................................................................................................................................19

6.2.3 General calibration procedure ..........................................................................................................................19

6.3 Setting TEM operating conditions for calibration .................................................................................................21

7 Image capture .......................................................................................................................................................................................................22

7.1 General ........................................................................................................................................................................................................22

7.2 Setting a suitable operating magnification ..................................................................................................................22

7.3 Minimum particle area ..................................................................................................................................................................23

7.4 Number of particles to count for particle size and shape distributions .............................................23

7.5 Uniform background .......................................................................................................................................................................24

7.6 Measurement procedure .............................................................................................................................................................24

7.6.1 General...................................................................................................................................................................................24

7.6.2 Developing a test sample .......................................................................................................................................25

7.6.3 Effects of magnification ...........................................................................................................................................25

7.6.4 Frames (micrographs) .............................................................................................................................................25

7.7 Revision of image capture protocols .................................................................................................................................25

8 Particle analysis .................................................................................................................................................................................................25

8.1 General ........................................................................................................................................................................................................25

8.2 Individual particle analysis .......................................................................................................................................................25

8.3 Automated particle analysis .....................................................................................................................................................26

8.4 Example — Automated particle analysis procedure ...........................................................................................26

9 Data analysis ..........................................................................................................................................................................................................27

9.1 General ........................................................................................................................................................................................................27

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ISO 21363:2020(E)

9.2 Raw data triage — Detecting touching particles, unselected particles, artefacts and

contaminants .........................................................................................................................................................................................27

9.3 Data quality assessment — Repeatability, intermediate precision and reproducibility ......28

9.4 Fitting distributions to data ......................................................................................................................................................30

9.5 Assessing measur ement uncertainty for samples under repeatability, intermediate

precision or reproducibility conditions..........................................................................................................................31

9.5.1 Grand statistics for fitted parameters — Three or more datasets .....................................31

9.5.2 Measurement uncertainty of fitted parameters ................................................................................31

9.5.3 Example — Measurement uncertainty for a size descriptor ..................................................32

9.6 Bivariate analysis ...............................................................................................................................................................................32

10 Reporting ...................................................................................................................................................................................................................33

Annex A (informative) Case studies overview .........................................................................................................................................36

Annex B (informative) Discrete spheroidal nanoparticles .........................................................................................................38

Annex C (informative) Size mixture ....................................................................................................................................................................41

Annex D (informative) Shape mixture .............................................................................................................................................................53

Annex E (informative) Amorphous aggregates .......................................................................................................................................58

Annex F (informative) Nanocrystalline aggregates ............................................................................................................................62

Annex G (informative) Nanofibres with irregular cross-sections .......................................................................................66

Annex H (informative) Nanoparticles with specific crystal habits .....................................................................................73

Bibliography .............................................................................................................................................................................................................................80

iv © ISO 2020 – All rights reserved
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ISO 21363:2020(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/

iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
© ISO 2020 – All rights reserved v
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ISO 21363:2020(E)
Introduction

Characterization procedures for nanoparticles often include, but are not limited to, size, shape, surface

structure (or texture), and surface chemistry. These measurements, combined with phase information,

such as crystalline phase, constitute the morphology of the material. This document focuses on two

attributes of morphology, size and shape distributions, for discrete, agglomerated and aggregated nano-

objects (materials with at least one dimension in the nanoscale, 1 nm < a length dimension < 100 nm).

Transmission electron microscopy, a standard tool for measurements on the nanoscale, provides

two-dimensional images of particle projections. This generic workflow for measuring and evaluating

particle size and shape distributions on the nanoscale includes sample preparation, instrument factors,

image capture, particle analysis, data analysis, and reporting. Seven case studies have been included to

illustrate how the generic protocol can be applied to different particle morphologies and sample types.

Three discrete particle test samples are reported: spheroidal (gold nanospheres), a bimodal mixture of

particle sizes (colloidal silicas), and a mixture of particle shapes (gold nanorods and gold nanocubes).

Two aggregate test samples are reported: amorphous aciniform aggregates (carbon black) and

aggregates of primary crystallites (titania). Measurements methods are also presented for low aspect

ratio samples and nanoparticles with specific crystal habits. Several of the case studies are supported

by interlaboratory collaborations conducted under the guidelines of the Versailles Project on Advanced

[42]
Materials and Standards (VAMAS) for interlaboratory comparisons (ILCs) .

Three types of size and shape descriptors are considered. Size descriptors include those determined by

linear or areal measurements. Shape descriptors include elongational descriptors, such as ratios of two

length descriptors, and ruggedness descriptors, which represent surface irregularities.

The protocol emphasizes qualitative and quantitative analysis of data quality by the user. Qualitative

comparisons of datasets include determining the similarity or differences between single descriptor

means or multivariate means. Quantitative comparisons of datasets are based on difference or

similarities between the parameters of reference models fitted to descriptor distributions. At least two

parameters (mean and spread) and their uncertainties are needed to define a descriptor distribution.

In some cases, these two quantitative parameters and their uncertainties may not be sufficient for

characterization of particle size and shape distributions. Data visualization techniques, such as residual

deviation and quantile plots, and data correlations, such as pairs of size and shape descriptors or

fractal analysis, can provide additional ways to evaluate and differentiate test samples. Taken together,

qualitative and quantitative quality metrics plus visualization and correlation tools permit users to

tailor the protocol to their qualitative and quantitative quality targets.
vi © ISO 2020 – All rights reserved
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INTERNATIONAL STANDARD ISO 21363:2020(E)
Nanotechnologies — Measurements of particle size and
shape distributions by transmission electron microscopy
1 Scope

This document specifies how to capture, measure and analyse transmission electron microscopy

images to obtain particle size and shape distributions in the nanoscale.

This document broadly is applicable to nano-objects as well as to particles with sizes larger than

100 nm. The exact working range of the method depends on the required uncertainty and on the

performance of the transmission electron microscope. These elements can be evaluated according to

the requirements described in this document.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 9276-3, Representation of results of particle size analysis — Part 3: Adjustment of an experimental

curve to a reference model

ISO 9276-6:2008, Representation of results of particle size analysis — Part 6: Descriptive and quantitative

representation of particle shape and morphology

ISO 29301, Microbeam analysis — Analytical electron microscopy — Methods for calibrating image

magnification by using reference materials with periodic structures
3 Terms, definitions and symbols
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Core terms — Particles
3.1.1
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (3.1.2)

[SOURCE: ISO/TS 80004-2:2015, 2.2]
3.1.2
nanoscale
length range approximately from 1 nm to 100 nm
[SOURCE: ISO/TS 80004-1:2015, 2.1, modified — Note 1 to entry has been deleted.]
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ISO 21363:2020(E)
3.1.3
particle
minute piece of matter with defined physical boundaries

[SOURCE: ISO 26824:2013, 1.1, modified — Notes 1, 2 and 3 to entry have been deleted.]

3.1.4
constituent particle
identifiable, integral component of a larger particle (3.1.3)
[SOURCE: ISO/TS 80004-2:2015, 3.3, modified — Note 1 to entry has been deleted.]
3.1.5
agglomerate

collection of weakly or medium strongly bound particles (3.1.3) where the resulting external surface

area is similar to the sum of the surface areas of the individual components

Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or

simple physical entanglement.

Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed

primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
3.1.6
aggregate

particle (3.1.3) comprising strongly bonded or fused particles where the resulting external surface area

may be significantly smaller than the sum of calculated surface areas of the individual components

Note 1 to entry: The forces holding an aggregate together are strong forces (for example, covalent bonds) or

those resulting from sintering or complex physical entanglement.

Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed

primary particles.

Note 3 to entry: Entries 3.1.6 to 3.1.10 define elements of agglomerates and aggregates, some of which are

illustrated in Figure 1. Constituent particles in an aggregate are tightly fused into a discrete entity (the

aggregate), while the constituent particles in an agglomerate are weakly bound and generally easily dispersed

under shear or mechanical stress.
2 © ISO 2020 – All rights reserved
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ISO 21363:2020(E)

a) Primary particles in an b) Primary particles in an c) Agglomerate of aggregates

agglomerate aggregate
d) Nano-object (if less than 100 nm) e) Agglomerate of both primary
or particle particles and aggregates
Figure 1 — Schematic showing elements of agglomerates and aggregates

[SOURCE: ISO/TS 80004-2:2015, 3.5, modified — In the definition, “may be significantly smaller” has

replaced “is significantly smaller” and “calculated” has been added before “surface areas”. In Note 1

to entry, “ionic bonds” in the example and the final phrase “or otherwise combined former primary

particles” have been deleted. Note 3 to entry and Figure 1 have been added.]
3.1.7
nanoparticle

nano-object (3.1.1) with all three external dimensions in the nanoscale (3.1.2) where the lengths of the

longest and shortest axes of the nano-object do not differ significantly

[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — “three” has been added and Note 1 to entry has been

deleted.]
3.1.8
nanorod
solid nanofibre (3.1.9)
[SOURCE: ISO/TS 80004-2:2015, 4.7]
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ISO 21363:2020(E)
3.1.9
nanofibre

nano-object (3.1.1) with two similar external dimensions in the nanoscale (3.1.2) and the third

dimension significantly larger

[SOURCE: ISO/TS 80004-2:2015, 4.5, modified — “similar” has been added and Notes 1, 2 and 3 to entry

have been deleted.]
3.1.10
nanophase

physically or chemically distinct region or collective term for physically distinct regions of the same

kind in a material with the discrete regions having one, two or three dimensions in the nanoscale (3.1.2)

Note 1 to entry: Nano-objects (3.1.1) embedded in another phase constitute a nanophase.

3.1.11
nanodispersion

material in which nano-objects (3.1.1) or a nanophase (3.1.10) are dispersed in a continuous phase of a

different composition
[SOURCE: ISO/TS 80004-4:2011, 2.14]
3.1.12
particle size

dimension of a particle (3.1.3) determined by a specified measurement method and under specified

measurement conditions

Note 1 to entry: Different methods of analysis are based on the measurement of different physical properties.

Independent of the particle property actually measured, the particle size can be reported as a linear dimension,

an area or a volume.

Note 2 to entry: The symbol x is used denote linear particle size. However, it is recognized that the symbol d is

also widely used. Therefore, the symbol x may be replaced by d.

[SOURCE: ISO 9276-1:1998, 4.2, modified — Converted into a term and definition entry.]

3.1.13
particle size distribution
distribution of particles (3.1.3) as a function of particle size (3.1.12)

[SOURCE: ISO/TS 80004-6:2013, 3.1.2, modified — Note 1 to entry has been deleted.]

3.1.14
particle shape
external geometric form of a particle (3.1.3)

Note 1 to entry: Shape description requires two scalar descriptors, i.e. length and spread.

[SOURCE: ISO/TS 80004-6:2013, 3.1.3, modified — Note 1 to entry has been added.]
3.1.15
particle shape distribution

distribution of a specific particle shape (3.1.14) descriptor for a sample population

3.2 Core terms — Image capture and analysis
3.2.1
field of view
field that is viewed by the viewing device
[SOURCE: ISO 13322-1:2014, 3.1.6, modified — Note 1 to entry has been deleted.]
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ISO 21363:2020(E)
3.2.2
measurement frame

selected area from the field of view (3.2.1) in which particles (3.1.3) are sized and counted for image

analysis
[SOURCE: ISO 13322-1:2014, 3.1.10]
3.2.3
binary image

digitized image consisting of an array of pixels (3.2.4), each of which has a value of 0 or 1, whose

values are normally represented by dark and bright regions on the display screen or by the use of two

distinct colours
[SOURCE: ISO 13322-1:2014, 3.1.2]
3.2.4
pixel

smallest element of an image that can be uniquely processed, and is defined by its spatial coordinates

and encoded with colour values
[SOURCE: ISO 12640-2:2004, 3.6, modified — Note 1 to entry has been deleted.]
3.2.5
pixel-resolution
number of imaging pixels (3.2.4) per unit distance of the detector
[SOURCE: ISO 29301:2017, 3.24, modified — Note 1 to entry has been deleted.]
3.2.6
pixel count

total number of pixels (3.2.4) per file, length, or area depending on the unit used

[SOURCE: ISO 19262:2015, 3.191]
3.2.7
micrograph
record of an image formed by a microscope
[SOURCE: ISO 10934-1:2002, 2.94]
3.2.8
artefact
artifact

unwanted distortion or added feature in measured data arising from lack of idealness of equipment

[SOURCE: ISO 18115-2: 2013, 5.6]
3.3 Core terms — Statistical symbols and definitions
3.3.1
coefficient of variation
ratio of the standard deviation to the arithmetic mean
Note 1 to entry: It is commonly reported as a percentage.

Note 2 to entry: For example, the coefficient of variation for a sample mean may be represented by:

s⋅100
c =
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ISO 21363:2020(E)

where x is the descriptor’s mean and s is the descriptor’s standard deviation for several datasets. These “grand

statistics” are used to evaluate descriptor data for interlaboratory comparisons.

[SOURCE: ISO 27448:2009, 3.11, modified — Notes 1 and 2 to entry have been added.]

3.3.2
standard error of estimation
est

measure of dispersion of the dependent variable (output) about the least-squares line obtained by curve

fitting or regression analysis
Note 1 to entry: The standard error of estimation may be determined by:
yy−
∑ i
i=1
σ =
est
nk−
where
n is the number of data points;
k is the number of coefficients in the equation.
Note 2 to entry: The standard error of the mean may be determined by:
σ =
est,x

Note 3 to entry: The standard error is the standard deviation of the sampling distribution of a statistic. The

example is for a sample mean. Standard error of the mean is an estimate of how close the sample mean is to the

population mean. This value decreases as the sample size increases.

[SOURCE: ISO 772:2011, 7.31, modified — The admitted term “residual standard deviation” has been

deleted. Notes 1, 2 and 3 to entry have replaced the original Notes 1 and 2 to entry.]

3.3.3
relative standard error
RSE
standard error divided by its statistic
Note 1 to entry: It is expressed as a percentage.
Note 2 to entry: For example,
...

NORME ISO
INTERNATIONALE 21363
Première édition
2020-06
Nanotechnologies — Détermination
de la distribution de taille et de
forme des particules par microscopie
électronique à transmission
Nanotechnologies — Measurements of particle size and shape
distributions by transmission electron microscopy
Numéro de référence
ISO 21363:2020(F)
ISO 2020
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ISO 21363:2020(F)
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ISO 21363:2020(F)
Sommaire Page

Avant-propos ................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ................................................................................................................................................................................... 1

3 Termes, définitions et symboles .......................................................................................................................................................... 1

3.1 Termes «cœur» — Particules...................................................................................................................................................... 1

3.2 Termes «cœur» — Acquisition et analyse d’image .................................................................................................. 5

3.3 Termes «cœur» — Définitions et symboles statistiques ..................................................................................... 6

3.4 Termes «cœur» — Mesurandes ................................................................................................................................................ 7

3.5 Termes «cœur» — Métrologie ................................................................................................................................................11

3.6 Termes «cœur» — Microscopie électronique à transmission .....................................................................13

3.7 Symboles statistiques, mesurandes et descripteurs ...........................................................................................14

3.7.1 Symboles statistiques ...............................................................................................................................................14

3.7.2 Mesurandes et descripteurs ................................................................................................................................15

4 Besoins des parties prenantes en termes de modes opératoires de mesure par MET ............16

5 Préparation des échantillons ...............................................................................................................................................................17

5.1 Généralités ...............................................................................................................................................................................................17

5.2 Sources d’échantillons ...................................................................................................................................................................17

5.3 Emploi d’un échantillon représentatif .............................................................................................................................18

5.3.1 Généralités .........................................................................................................................................................................18

5.3.2 Échantillons en poudre ...........................................................................................................................................18

5.3.3 Dispersions de nanoparticules dans des liquides ............................................................................18

5.4 Minimisation de l’agglomération des particules dans la dispersion d’échantillon ...................19

5.5 Choix du support d’échantillon ..............................................................................................................................................19

6 Facteurs instrumentaux .............................................................................................................................................................................20

6.1 Réglage de l’instrument ................................................................................................................................................................20

6.2 Étalonnage ...............................................................................................................................................................................................20

6.2.1 Généralités .........................................................................................................................................................................20

6.2.2 Étalons ...................................................................................................................................................................................20

6.2.3 Mode opératoire d’étalonnage général ......................................................................................................20

6.3 Réglage des conditions de fonctionnement du MET pour l’étalonnage..............................................22

7 Acquisition d’images .....................................................................................................................................................................................23

7.1 Généralités ...............................................................................................................................................................................................23

7.2 Réglage d’un grandissement de fonctionnement adapté ................................................................................24

7.3 Surface de particule minimale ................................................................................................................................................24

7.4 Nombre de particules à compter pour les distributions de taille et de forme des

particules ..................................................................................................................................................................................................24

7.5 Fond uniforme ......................................................................................................................................................................................25

7.6 Mode opératoire de mesure ......................................................................................................................................................26

7.6.1 Généralités .........................................................................................................................................................................26

7.6.2 Élaboration d’un échantillon d’essai............................................................................................................26

7.6.3 Effets du grandissement .........................................................................................................................................26

7.6.4 Images (micrographies) .........................................................................................................................................26

7.7 Révision des protocoles d’acquisition d’images ......................................................................................................26

8 Analyse des particules .................................................................................................................................................................................27

8.1 Généralités ...............................................................................................................................................................................................27

8.2 Analyse de particules individuelle ......................................................................................................................................27

8.3 Analyse de particules automatisée .....................................................................................................................................27

8.4 Exemple de mode opératoire d’analyse de particules automatisée .......................................................27

9 Traitement des données ............................................................................................................................................................................28

9.1 Généralités ...............................................................................................................................................................................................28

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ISO 21363:2020(F)

9.2 Tri des données brutes — Détections des particules en contact, des particules non

sélectionnées, des artefacts et des contaminants ..................................................................................................29

9.3 Évaluation de la qualité des données — Répétabilité, fidélité intermédiaire et

reproductibilité ....................................................................................................................................................................................30

9.4 Ajustement des distributions aux données .................................................................................................................32

9.5 Évaluation de l’incertitude de mesure pour les échantillons dans des conditions de

répétabilité, de fidélité intermédiaire ou de reproductibilité .....................................................................33

9.5.1 Statistiques générales des paramètres ajustés — Trois ensembles de

données ou plus .............................................................................................................................................................33

9.5.2 Incertitude de mesure des paramètres ajustés ..................................................................................34

9.5.3 Exemple — Incertitude de mesure pour un descripteur de taille ......................................34

9.6 Analyse à deux variables .............................................................................................................................................................34

10 Rapport........................................................................................................................................................................................................................35

Annexe A (informative) Présentation d’études de cas ....................................................................................................................38

Annexe B (informative) Nanoparticules sphéroïdales discrètes ..........................................................................................40

Annexe C (informative) Mélange de tailles ..................................................................................................................................................43

Annexe D (informative) Mélange de formes ..............................................................................................................................................56

Annexe E (informative) Agrégats amorphes ..............................................................................................................................................61

Annexe F (informative) Agrégats nanocristallins.................................................................................................................................65

Annexe G (informative) Nanofibres à sections transverses irrégulières .....................................................................69

Annexe H (informative) Nanoparticules à caractéristiques cristallines spécifiques ......................................76

Bibliographie ...........................................................................................................................................................................................................................83

iv © ISO 2020 – Tous droits réservés
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ISO 21363:2020(F)
Avant-propos

L’ISO (Organisation internationale de normalisation) est une fédération mondiale d’organismes

nationaux de normalisation (comités membres de l’ISO). L’élaboration des Normes internationales est

en général confiée aux comités techniques de l’ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l’ISO participent également aux travaux.

L’ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents

critères d’approbation requis pour les différents types de documents ISO. Le présent document a été

rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www

.iso .org/ directives).

L’attention est attirée sur le fait que certains des éléments du présent document peuvent faire l’objet de

droits de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l’élaboration du document sont indiqués dans l’Introduction et/ou dans la liste des déclarations de

brevets reçues par l’ISO (voir www .iso .org/ brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la nature volontaire des normes, la signification des termes et expressions

spécifiques de l’ISO liés à l’évaluation de la conformité, ou pour toute information au sujet de l’adhésion

de l’ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles

techniques au commerce (OTC), voir www .iso .org/ avant -propos.

Le présent document a été élaboré par le comité technique ISO/TC 229, Nanotechnologies.

Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent

document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l’adresse www .iso .org/ fr/ members .html.
© ISO 2020 – Tous droits réservés v
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ISO 21363:2020(F)
Introduction

Les modes opératoires de caractérisation des nanoparticules couvrent souvent, mais sans s’y limiter, la

taille, la forme, la structure de surface (ou la texture) et la chimie de surface. Ces mesurages, associés à

des informations sur les phases, telles que la phase cristalline, constituent la morphologie du matériau.

Le présent document porte sur deux attributs de morphologie, les distributions de taille et de forme,

des nano-objets discrets, agglomérés et agrégés (matériaux comportant au moins une dimension à

l’échelle nanométrique, 1 nm < une dimension < 100 nm). La microscopie électronique à transmission,

un outil classique de mesure à l’échelle nanométrique, fournit des images bidimensionnelles de

projections de particules. Ce flux d’opérations générique permettant de mesurer et d’évaluer des

distributions de taille et de forme à l’échelle nanométrique comprend la préparation des échantillons,

les facteurs instrumentaux, l’acquisition d’images, l’analyse des particules, le traitement des données

et la communication des résultats au travers d’un rapport. Sept études de cas ont été incluses pour

illustrer la façon dont le protocole générique peut être appliqué à différentes morphologies particulaires

et à différents types d’échantillons. Trois échantillons de particules discrètes sont présentés: un type

sphéroïdal (nanosphères d’or), un mélange de particules avec une granulométrie bimodale (silices

colloïdales) et un mélange de formes de particules (nanotiges d’or et nanocubes d’or). Deux échantillons

agrégés sont mentionnés: des agrégats amorphes en grappes (noir de carbone) et des agrégats de

cristallites primaires (dioxyde de titane). Des méthodes de mesure sont également présentées pour les

échantillons à faible rapport d’aspect et les nanoparticules à caractéristiques cristallines spécifiques.

Plusieurs des études de cas s’appuient sur des collaborations interlaboratoires menées conformément

aux lignes directrices du VAMAS (Versailles Project on Advanced Materials and Standards) concernant

[42]
les comparaisons interlaboratoires (CIL) .

Trois types de descripteurs de taille et de forme sont pris en compte. Les descripteurs de taille incluent

ceux déterminés par des mesurages linéaires ou surfaciques. Les descripteurs de forme comprennent

des descripteurs d’allongement, tels que les rapports entre deux descripteurs de longueur, et des

descripteurs de rugosité, représentant les irrégularités de surface.

Le protocole met l’accent sur l’analyse qualitative et quantitative de la qualité des données par

l’utilisateur. Les comparaisons qualitatives d’ensembles de données incluent la détermination de la

similitude ou des différences entre des moyennes de descripteur unique ou des moyennes à plusieurs

variables. Les comparaisons quantitatives d’ensembles de données s’appuient sur la différence ou les

similitudes entre les paramètres des modèles de référence ajustés aux distributions des descripteurs.

Au moins deux paramètres (moyenne et dispersion) ainsi que leurs incertitudes sont nécessaires pour

définir une distribution de descripteur. Dans certains cas, ces deux paramètres quantitatifs et leurs

incertitudes peuvent ne pas suffire à caractériser les distributions de taille et de forme. Les techniques

de visualisation des données, telles que les diagrammes quantiles et d’écart résiduel, et les corrélations

de données, telles que les paires de descripteurs de taille et de forme ou l’analyse fractale, peuvent

fournir d’autres méthodes pour évaluer et différencier des échantillons d’essai. L’association de mesures

de qualité quantitatives et d’outils de visualisation et de corrélation permet aux utilisateurs d’adapter

le protocole à leurs objectifs de qualité qualitatifs et quantitatifs.
vi © ISO 2020 – Tous droits réservés
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NORME INTERNATIONALE ISO 21363:2020(F)
Nanotechnologies — Détermination de la distribution
de taille et de forme des particules par microscopie
électronique à transmission
1 Domaine d’application

Le présent document spécifie une méthode permettant d’acquérir, de mesurer et d’analyser des images

de microscopie électronique à transmission afin d’obtenir des distributions de taille et de forme à

l’échelle nanométrique.

Le présent document s’applique de façon générale aux nano-objets ainsi qu’aux particules de dimensions

supérieures à 100 nm. La plage de fonctionnement exacte de la méthode dépend de l’incertitude exigée

et des performances du microscope électronique à transmission. Ces éléments peuvent être évalués

conformément aux exigences décrites dans le présent document.
2 Références normatives

Les documents suivants sont cités dans le texte de sorte qu’ils constituent, pour tout ou partie de leur

contenu, des exigences du présent document. Pour les références datées, seule l’édition citée s’applique.

Pour les références non datées, la dernière édition du document de référence s’applique (y compris les

éventuels amendements).

ISO 9276-3, Représentation de données obtenues par analyse granulométrique — Partie 3: Ajustement

d’une courbe expérimentale à un modèle de référence

ISO 9276-6:2008, Représentation de données obtenues par analyse granulométrique — Partie 6:

Description et représentation quantitative de la forme et de la morphologie des particules

ISO 29301, Analyse par microfaisceaux — Microscopie électronique analytique — Méthodes d’étalonnage

du grandissement d’image au moyen de matériaux de référence de structures périodiques

3 Termes, définitions et symboles

Pour les besoins du présent document, les termes et définitions suivants s’appliquent.

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp

— IEC Electropedia: disponible à l’adresse http:// www .electropedia .org/
3.1 Termes «cœur» — Particules
3.1.1
nano-objet

portion discrète de matériau dont une, deux ou les trois dimensions externes sont à l’échelle

nanométrique (3.1.2)
[SOURCE: ISO/TS 80004-2:2015, 2.2]
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ISO 21363:2020(F)
3.1.2
échelle nanométrique
échelle de longueur s’étendant approximativement de 1 nm à 100 nm

[SOURCE: ISO/TS 80004-1:2015, 2.1, modifiée — La Note 1 à l’article a été supprimée.]

3.1.3
particule
élément de matière isolé possédant des limites physiques définies

[SOURCE: ISO 26824:2013, 1.1, modifiée — Les Notes 1, 2 et 3 à l’article ont été supprimées.]

3.1.4
particule constituante

composante identifiable faisant partie intégrante d’une particule (3.1.3) plus grande

[SOURCE: ISO/TS 80004-2:2015, 3.3, modifiée — La Note 1 à l’article a été supprimée.]

3.1.5
agglomérat

ensemble de particules (3.1.3) faiblement ou moyennement liées, dont l’aire de la surface externe

résultante est similaire à la somme des aires de surface de chacun des composants

Note 1 à l'article: Les forces assurant la cohésion d’un agglomérat sont faibles, par exemple des forces de Van der

Waals ou des forces résultant d’un simple enchevêtrement physique.

Note 2 à l'article: Les agglomérats sont également appelés particules secondaires et les particules sources

initiales sont appelées particules primaires.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
3.1.6
agrégat

particule (3.1.3) composée de particules fortement liées ou fusionnées, dont l’aire de la surface externe

résultante peut être significativement plus petite que la somme des aires de surface calculées de chacun

des composants

Note 1 à l'article: Les forces assurant la cohésion d’un agrégat sont puissantes, par exemple des liaisons covalentes,

ou des forces résultant d’un frittage ou d’un enchevêtrement physique complexe.

Note 2 à l'article: Les agrégats sont également appelés particules secondaires et les particules sources initiales

sont appelées particules primaires.

Note 3 à l'article: Les entrées 3.1.6 à 3.1.10 définissent des éléments des agglomérats et des agrégats, dont

certains sont illustrés à la Figure 1. Les particules constituantes d’un agrégat sont étroitement fusionnées en

une entité discrète (l’agrégat), tandis que les particules constituantes d’un agglomérat sont faiblement liées et se

dispersent généralement facilement sous l’effet d’un cisaillement ou d’une contrainte mécanique.

2 © ISO 2020 – Tous droits réservés
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ISO 21363:2020(F)

a) Particules primaires dans b) Particules primaires dans c) Agglomérat d’agrégats

un agglomérat un agrégat

d) Nano-objet (si inférieur à 100 nm) ou e) Agglomérat de particules primaires et

particule d’agrégats

Figure 1 — Représentation schématique des éléments des agglomérats et des agrégats

[SOURCE: ISO/TS 80004-2:2015, 3.5, modifiée — Dans la définition, «peut être significativement

plus petite» a remplacé «est significativement plus petite» et «calculées» a été ajouté après «aires de

surface». Dans la Note 1 à l’article, «ou ioniques» dans l’exemple et la fin de phrase «ou sinon d’anciennes

particules primaires combinées» ont été supprimés. La Note 3 à l’article et la Figure 1 ont été ajoutées.]

3.1.7
nanoparticule

nano-objet (3.1.1) dont les trois dimensions externes sont à l’échelle nanométrique (3.1.2) et dont les

longueurs du plus grand et du plus petit axes ne diffèrent pas de façon significative

[SOURCE: ISO/TS 80004-2:2015, 4.4, modifiée — «trois» a été ajouté et la Note 1 à l’article a été

supprimée.]
© ISO 2020 – Tous droits réservés 3
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ISO 21363:2020(F)
3.1.8
nanobâtonnet
nanotige
nanofibre (3.1.9) solide
[SOURCE: ISO/TS 80004-2:2015, 4.7]
3.1.9
nanofibre

nano-objet (3.1.1) ayant deux dimensions externes similaires à l’échelle nanométrique (3.1.2) et la

troisième dimension externe significativement plus grande

[SOURCE: ISO/TS 80004-2:2015, 4.5, modifiée — «similaires» a été ajouté et les Notes 1, 2 et 3 à l’article

a été supprimées.]
3.1.10
nanophase

région physiquement ou chimiquement distincte, ou terme collectif désignant un ensemble de régions

de même nature et physiquement distinctes dans un matériau, cette ou ces régions discrètes ayant une,

deux ou trois dimensions à l’échelle nanométrique (3.1.2)

Note 1 à l'article: Les nano-objets (3.1.1) incorporés dans une autre phase constituent une nanophase.

3.1.11
nanodispersion

matériau dans lequel des nano-objets (3.1.1) ou une nanophase (3.1.10) sont dispersés dans une phase

continue de composition différente
[SOURCE: ISO/TS 80004-4:2011, 2.14]
3.1.12
taille d’une particule

dimension d’une particule (3.1.3) déterminée par une méthode de mesure spécifiée dans des conditions

de mesure spécifiées

Note 1 à l'article: Différentes méthodes d’analyse sont fondées sur le mesurage de différentes propriétés

physiques. Indépendamment de la propriété de particule réellement mesurée, la taille de la particule peut être

consignée comme une dimension linéaire, une surface ou un volume.

Note 2 à l'article: Le symbole x est utilisé pour indiquer la taille linéaire d’une particule. Cependant, il est reconnu

que le symbole d est également couramment utilisé. Le symbole x peut donc être remplacé par d.

[SOURCE: ISO 9276-1:1998, 4.2, modifiée — Un terme et sa définition ont été créés à partir de ce

paragraphe.]
3.1.13
distribution de taille de particules
distribution de particules (3.1.3) en fonction de leur taille (3.1.12)

[SOURCE: ISO/TS 80004-6:2013, 3.1.2, modifiée — La Note 1 à l’article a été supprimée.]

3.1.14
forme d’une particule
forme géométrique externe d’une particule (3.1.3)

Note 1 à l'article: La description de la forme exige deux descripteurs scalaires, la longueur et la largeur.

[SOURCE: ISO/TS 80004-6:2013, 3.1.3, modifiée — La Note 1 à l’article a été ajoutée.]

4 © ISO 2020 – Tous droits réservés
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ISO 21363:2020(F)
3.1.15
distribution de forme de particules

distribution d’un descripteur de forme de particule (3.1.14) spécifique pour une population d’échantillons

3.2 Termes «cœur» — Acquisition et analyse d’image
3.2.1
champ de vision
champ qui est perçu par un dispositif d’observation

[SOURCE: ISO 13322-1:2014, 3.1.6, modifiée — La Note 1 à l’article a été supprimée.]

3.2.2
cadre de mesure

surface sélectionnée d’un champ de vision (3.2.1) dans laquelle des particules (3.1.3) sont dimensionnées

et comptabilisées pour l’analyse d’images
[SOURCE: ISO 13322-1:2014, 3.1.10]
3.2.3
image binaire

image numérisée constituée d’une matrice de pixels (3.2.4), possédant chacun une valeur 0 ou 1, dont

les valeurs sont normalement représentées par des régions sombres et claires sur l’écran d’affichage ou

par l’utilisation de deux couleurs distinctes
[SOURCE: ISO 13322-1:2014, 3.1.2]
3.2.4
pixel

plus petit élément d’une image pouvant être traité de façon unique, qui est défini par ses coordonnées

spatiales et codé avec des valeurs de couleurs

[SOURCE: ISO 12640-2:2004, 3.6, modifiée — La Note 1 à l’article a été supprimée.]

3.2.5
résolution de pixels
nombre de pixels (3.2.4) par unité de distance d’un détecteur

[SOURCE: ISO 29301:2017, 3.24, modifiée — La Note 1 à l’article a été supprimée.]

3.2.6
comptage de pixels
nombre total de pixel
...

INTERNATIONAL ISO
STANDARD 21363
First edition
Nanotechnologies — Measurements of
particle size and shape distributions
by transmission electron microscopy
Nanotechnologies — Détermination de la distribution de taille et de
forme des particules par microscopie électronique à transmission
PROOF/ÉPREUVE
Reference number
ISO 21363:2020(E)
ISO 2020
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ISO/FDIS 21363:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH­1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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ISO/FDIS 21363:2020(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms, definitions and symbols .......................................................................................................................................................... 1

3.1 Core terms — Particles .................................................................................................................................................................... 1

3.2 Core terms — Image capture and analysis ..................................................................................................................... 4

3.3 Core terms — Statistical symbols and definitions ................................................................................................... 5

3.4 Core terms — Measurands ........................................................................................................................................................... 7

3.5 Core terms — Metrology .............................................................................................................................................................10

3.6 Core terms — Transmission electron microscopy ................................................................................................13

3.7 Statistical symbols, measurands and descriptors ..................................................................................................14

3.7.1 Statistical symbols .......................................................................................................................................................14

3.7.2 Measurands and descriptors ..............................................................................................................................14

4 Stakeholder needs for TEM measurement procedures ............................................................................................15

5 Sample preparation ........................................................................................................................................................................................16

5.1 General ........................................................................................................................................................................................................16

5.2 Sample sources ....................................................................................................................................................................................17

5.3 Use a representative sample ....................................................................................................................................................17

5.3.1 General...................................................................................................................................................................................17

5.3.2 Powder samples ............................................................................................................................................................17

5.3.3 Nanoparticle dispersions in liquids .............................................................................................................17

5.4 Minimize particle agglomeration in the sample dispersion .........................................................................18

5.5 Selection of the mounting support .....................................................................................................................................18

6 Instrument factors ...........................................................................................................................................................................................18

6.1 Instrument set­up..............................................................................................................................................................................18

6.2 Calibration ...............................................................................................................................................................................................19

6.2.1 General...................................................................................................................................................................................19

6.2.2 Calibration standards ...............................................................................................................................................19

6.2.3 General calibration procedure ..........................................................................................................................19

6.3 Setting TEM operating conditions for calibration .................................................................................................21

7 Image capture .......................................................................................................................................................................................................22

7.1 General ........................................................................................................................................................................................................22

7.2 Setting a suitable operating magnification ..................................................................................................................22

7.3 Minimum particle area ..................................................................................................................................................................23

7.4 Number of particles to count for particle size and shape distributions .............................................23

7.5 Uniform background .......................................................................................................................................................................24

7.6 Measurement procedure .............................................................................................................................................................24

7.6.1 General...................................................................................................................................................................................24

7.6.2 Developing a test sample .......................................................................................................................................25

7.6.3 Effects of magnification ...........................................................................................................................................25

7.6.4 Frames (micrographs) .............................................................................................................................................25

7.7 Revision of image capture protocols .................................................................................................................................25

8 Particle analysis .................................................................................................................................................................................................25

8.1 General ........................................................................................................................................................................................................25

8.2 Individual particle analysis .......................................................................................................................................................25

8.3 Automated particle analysis .....................................................................................................................................................26

8.4 Example — Automated particle analysis procedure ...........................................................................................26

9 Data analysis ..........................................................................................................................................................................................................27

9.1 General ........................................................................................................................................................................................................27

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ISO/FDIS 21363:2020(E)

9.2 Raw data triage — Detecting touching particles, unselected particles, artefacts and

contaminants .........................................................................................................................................................................................27

9.3 Data quality assessment — Repeatability, intermediate precision and reproducibility ......28

9.4 Fitting distributions to data ......................................................................................................................................................30

9.5 Assessing measur ement uncertainty for samples under repeatability, intermediate

precision or reproducibility conditions..........................................................................................................................31

9.5.1 Grand statistics for fitted parameters — Three or more datasets .....................................31

9.5.2 Measurement uncertainty of fitted parameters ................................................................................31

9.5.3 Example — Measurement uncertainty for a size descriptor ..................................................32

9.6 Bivariate analysis ...............................................................................................................................................................................32

10 Reporting ...................................................................................................................................................................................................................33

Annex A (informative) Case studies overview .........................................................................................................................................36

Annex B (informative) Discrete spheroidal nanoparticles .........................................................................................................38

Annex C (informative) Size mixture ....................................................................................................................................................................41

Annex D (informative) Shape mixture .............................................................................................................................................................53

Annex E (informative) Amorphous aggregates .......................................................................................................................................58

Annex F (informative) Nanocrystalline aggregates ............................................................................................................................62

Annex G (informative) Nanofibres with irregular cross-sections .......................................................................................66

Annex H (informative) Nanoparticles with specific crystal habits .....................................................................................73

Bibliography .............................................................................................................................................................................................................................80

iv © ISO 2020 – All rights reserved
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ISO/FDIS 21363:2020(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non­governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/

iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
© ISO 2020 – All rights reserved v
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ISO/FDIS 21363:2020(E)
Introduction

Characterization procedures for nanoparticles often include, but are not limited to, size, shape, surface

structure (or texture), and surface chemistry. These measurements, combined with phase information,

such as crystalline phase, constitute the morphology of the material. This document focuses on two

attributes of morphology, size and shape distributions, for discrete, agglomerated and aggregated nano-

objects (materials with at least one dimension in the nanoscale, 1 nm < a length dimension < 100 nm).

Transmission electron microscopy, a standard tool for measurements on the nanoscale, provides

two-dimensional images of particle projections. This generic workflow for measuring and evaluating

particle size and shape distributions on the nanoscale includes sample preparation, instrument factors,

image capture, particle analysis, data analysis, and reporting. Seven case studies have been included to

illustrate how the generic protocol can be applied to different particle morphologies and sample types.

Three discrete particle test samples are reported: spheroidal (gold nanospheres), a bimodal mixture of

particle sizes (colloidal silicas), and a mixture of particle shapes (gold nanorods and gold nanocubes).

Two aggregate test samples are reported: amorphous aciniform aggregates (carbon black) and

aggregates of primary crystallites (titania). Measurements methods are also presented for low aspect

ratio samples and nanoparticles with specific crystal habits. Several of the case studies are supported

by interlaboratory collaborations conducted under the guidelines of the Versailles Project on Advanced

[42]
Materials and Standards (VAMAS) for interlaboratory comparisons (ILCs) .

Three types of size and shape descriptors are considered. Size descriptors include those determined by

linear or areal measurements. Shape descriptors include elongational descriptors, such as ratios of two

length descriptors, and ruggedness descriptors, which represent surface irregularities.

The protocol emphasizes qualitative and quantitative analysis of data quality by the user. Qualitative

comparisons of datasets include determining the similarity or differences between single descriptor

means or multivariate means. Quantitative comparisons of datasets are based on difference or

similarities between the parameters of reference models fitted to descriptor distributions. At least two

parameters (mean and spread) and their uncertainties are needed to define a descriptor distribution.

In some cases, these two quantitative parameters and their uncertainties may not be sufficient for

characterization of particle size and shape distributions. Data visualization techniques, such as residual

deviation and quantile plots, and data correlations, such as pairs of size and shape descriptors or

fractal analysis, can provide additional ways to evaluate and differentiate test samples. Taken together,

qualitative and quantitative quality metrics plus visualization and correlation tools permit users to

tailor the protocol to their qualitative and quantitative quality targets.
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 21363:2020(E)
Nanotechnologies — Measurements of particle size and
shape distributions by transmission electron microscopy
1 Scope

This document specifies how to capture, measure and analyse transmission electron microscopy

images to obtain particle size and shape distributions in the nanoscale.

This document broadly is applicable to nano-objects as well as to particles with sizes larger than

100 nm. The exact working range of the method depends on the required uncertainty and on the

performance of the transmission electron microscope. These elements can be evaluated according to

the requirements described in this document.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 9276­3, Representation of results of particle size analysis — Part 3: Adjustment of an experimental

curve to a reference model

ISO 9276­6:2017, Representation of results of particle size analysis — Part 6: Descriptive and quantitative

representation of particle shape and morphology

ISO 29301, Microbeam analysis — Analytical electron microscopy — Methods for calibrating image

magnification by using reference materials with periodic structures
3 Terms, definitions and symbols
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Core terms — Particles
3.1.1
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (3.1.2)

[SOURCE: ISO/TS 80004­2:2015, 2.2]
3.1.2
nanoscale
length range approximately from 1 nm to 100 nm
[SOURCE: ISO/TS 80004-1:2015, 2.1, modified — Note 1 to entry has been deleted.]
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ISO/FDIS 21363:2020(E)
3.1.3
particle
minute piece of matter with defined physical boundaries

[SOURCE: ISO 26824:2013, 1.1, modified — Notes 1, 2 and 3 to entry have been deleted.]

3.1.4
constituent particle
identifiable, integral component of a larger particle (3.1.3)
[SOURCE: ISO/TS 80004-2:2015, 3.3, modified — Note 1 to entry has been deleted.]
3.1.5
agglomerate

collection of weakly or medium strongly bound particles (3.1.3) where the resulting external surface

area is similar to the sum of the surface areas of the individual components

Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or

simple physical entanglement.

Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed

primary particles.
[SOURCE: ISO/TS 80004­2:2015, 3.4]
3.1.6
aggregate

particle (3.1.3) comprising strongly bonded or fused particles where the resulting external surface area

may be significantly smaller than the sum of calculated surface areas of the individual components

Note 1 to entry: The forces holding an aggregate together are strong forces (for example, covalent bonds) or

those resulting from sintering or complex physical entanglement.

Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed

primary particles.

Note 3 to entry: Entries 3.1.6 to 3.1.10 define elements of agglomerates and aggregates, some of which are

illustrated in Figure 1. Constituent particles in an aggregate are tightly fused into a discrete entity (the

aggregate), while the constituent particles in an agglomerate are weakly bound and generally easily dispersed

under shear or mechanical stress.
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ISO/FDIS 21363:2020(E)

a) Primary particles in an b) Primary particles in an c) Agglomerate of aggregates

agglomerate aggregate
d) Nano-object (if less than 100 nm) e) Agglomerate of both primary
or particle particles and aggregates
Figure 1 — Schematic showing elements of agglomerates and aggregates

[SOURCE: ISO/TS 80004-2:2015, 3.5, modified — In the definition, “may be significantly smaller” has

replaced “is significantly smaller” and “calculated” has been added before “surface areas”. In Note 1

to entry, “ionic bonds” in the example and the final phrase “or otherwise combined former primary

particles” have been deleted. Note 3 to entry and Figure 1 have been added.]
3.1.7
nanoparticle

nano-object (3.1.1) with all three external dimensions in the nanoscale (3.1.2) where the lengths of the

longest and shortest axes of the nano-object do not differ significantly

[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — “three” has been added and Note 1 to entry has been

deleted.]
3.1.8
nanorod
solid nanofibre (3.1.9)
[SOURCE: ISO/TS 80004­2:2015, 4.7]
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ISO/FDIS 21363:2020(E)
3.1.9
nanofibre

nano-object (3.1.1) with two similar external dimensions in the nanoscale (3.1.2) and the third

dimension significantly larger
[SOURCE: ISO/TS 80004-2:2015, 4.5, modified — Note 1 to entry has been deleted.]
3.1.10
nanophase

physically or chemically distinct region or collective term for physically distinct regions of the same

kind in a material with the discrete regions having one, two or three dimensions in the nanoscale (3.1.2)

Note 1 to entry: Nano-objects (3.1.1) embedded in another phase constitute a nanophase.

3.1.11
nanodispersion

material in which nano-objects (3.1.1) or a nanophase (3.1.10) are dispersed in a continuous phase of a

different composition
[SOURCE: ISO/TS 80004­4:2011, 2.14]
3.1.12
particle size

dimension of a particle (3.1.3) determined by a specified measurement method and under specified

measurement conditions

Note 1 to entry: Different methods of analysis are based on the measurement of different physical properties.

Independent of the particle property actually measured, the particle size can be reported as a linear dimension,

an area or a volume.

Note 2 to entry: The symbol x is used denote linear particle size. However, it is recognized that the symbol d is

also widely used. Therefore, the symbol x may be replaced by d.

[SOURCE: ISO 9276-1:1998, 4.2, modified — Converted into a term and definition entry.]

3.1.13
particle size distribution
distribution of particles (3.1.3) as a function of particle size (3.1.12)

[SOURCE: ISO/TS 80004-6:2013, 3.1.2, modified — Note 1 to entry has been deleted.]

3.1.14
particle shape
external geometric form of a particle (3.1.3)

Note 1 to entry: Shape description requires two scalar descriptors, i.e. length and spread.

[SOURCE: ISO/TS 80004-6:2013, 3.1.3, modified — Note 1 to entry has been added.]
3.1.15
particle shape distribution

distribution of a specific particle shape (3.1.14) descriptor for a sample population

3.2 Core terms — Image capture and analysis
3.2.1
field of view
field that is viewed by the viewing device
[SOURCE: ISO 13322-1:2014, 3.1.6, modified — Note 1 to entry has been deleted.]
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ISO/FDIS 21363:2020(E)
3.2.2
measurement frame

selected area from the field of view (3.2.1) in which particles (3.1.3) are sized and counted for image

analysis
[SOURCE: ISO 13322­1:2014, 3.1.10]
3.2.3
binary image

digitized image consisting of an array of pixels (3.2.4), each of which has a value of 0 or 1, whose

values are normally represented by dark and bright regions on the display screen or by the use of two

distinct colours
[SOURCE: ISO 13322­1:2014, 3.1.2]
3.2.4
pixel

smallest element of an image that can be uniquely processed, and is defined by its spatial coordinates

and encoded with colour values
[SOURCE: ISO 12640-2:2004, 3.6, modified — Note 1 to entry has been deleted.]
3.2.5
pixel-resolution
number of imaging pixels (3.2.4) per unit distance of the detector
[SOURCE: ISO 29301:2017, 3.24, modified — Note 1 to entry has been deleted.]
3.2.6
pixel count

total number of pixels (3.2.4) per file, length, or area depending on the unit used

[SOURCE: ISO 19262:2015, 3.191]
3.2.7
micrograph
record of an image formed by a microscope
[SOURCE: ISO 10934­1:2002, 2.94]
3.2.8
artefact
artifact

unwanted distortion or added feature in measured data arising from lack of idealness of equipment

[SOURCE: ISO 18115­2: 2013, 5.6]
3.3 Core terms — Statistical symbols and definitions
3.3.1
coefficient of variation
ratio of the standard deviation to the arithmetic mean
Note 1 to entry: It is commonly reported as a percentage.

Note 2 to entry: For example, the coefficient of variation for a sample mean may be represented by:

s⋅100
c =
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ISO/FDIS 21363:2020(E)

where x is the descriptor’s mean and s is the descriptor’s standard deviation for several datasets. These “grand

statistics” are used to evaluate descriptor data for interlaboratory comparisons.

[SOURCE: ISO 27448:2009, 3.11, modified — Notes 1 and 2 to entry have been added.]

3.3.2
standard error of estimation
est

measure of dispersion of the dependent variable (output) about the least-squares line obtained by curve

fitting or regression analysis
Note 1 to entry: The standard error of estimation may be determined by:
yy−
∑ i
i=1
σ =
est
nk−
where
n is the number of data points;
k is the number of coefficients in the equation.
Note 2 to entry: The standard error of the mean may be determined by:
σ =
est,x

Note 3 to entry: The standard error is the standard deviation of the sampling distribution of a statistic. The

example is for a sample mean. Standard error of the mean is an estimate of how close the sample mean is to the

population mean. This value decreases as the sample size increases.

[SOURCE: ISO 772:2011, 7.31, modified — The admitted term “residual standard deviation” has been

deleted. Notes 1, 2 and 3 to entry have replaced the original Notes 1 and 2 to entry.]

3.3.3
relative standard error
RSE
standard error divided by its statistic
Note 1 to entry: It is ex
...

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