Nanotechnologies -- Measurements of particle size and shape distributions by scanning electron microscopy

This document specifies methods of determining nanoparticle size and shape distributions by acquiring and evaluating scanning electron microscope images and by obtaining and reporting accurate results. NOTEÂ 1Â Â Â This document applies to particles with a lower size limit that depends on the required uncertainty and on the suitable performance of the SEM, which is to be proven first -according to the requirements described in this document. NOTEÂ 2Â Â Â This document applies also to SEM-based size and shape measurements of larger than nanoscale particles.

Nanotechnologies -- Détermination de la distribution de taille et de forme des particules par microscopie électronique à balayage

Le présent document spécifie des méthodes permettant de déterminer les distributions de taille et de forme des nanoparticules, par l’acquisition et l’évaluation d’images obtenues avec un microscope électronique à balayage, puis l’obtention de résultats exacts et la rédaction de rapports. NOTE 1       Le présent document s’applique aux particules dont la limite de taille inférieure dépend de l’incertitude exigée et des performances appropriées du MEB, après démonstration de sa conformité aux exigences décrites dans le présent document. NOTE 2       Le présent document s’applique également aux mesurages par MEB de taille et de forme des particules de taille supérieure à l’échelle nanométrique.

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Published
Publication Date
04-Jul-2021
Current Stage
5060 - Close of voting Proof returned by Secretariat
Start Date
19-Apr-2021
Completion Date
19-Apr-2021
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INTERNATIONAL ISO
STANDARD 19749
First edition
2021-07
Nanotechnologies — Measurements of
particle size and shape distributions
by scanning electron microscopy
Nanotechnologies — Détermination de la distribution de taille et de
forme des particules par microscopie électronique à balayage
Reference number
ISO 19749:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 19749:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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
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Email: copyright@iso.org
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Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 19749:2021(E)
Contents Page

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

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

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

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

3 Terms and definitions ..................................................................................................................................................................................... 2

3.1 General terms ........................................................................................................................................................................................... 2

3.2 Core terms: image analysis ........................................................................................................................................................... 4

3.3 Core terms: statistical symbols and definitions ......................................................................................................... 4

3.4 Core terms: measurands and descriptors ........................................................................................................................ 6

3.5 Core terms: metrology ...................................................................................................................................................................... 8

3.6 Core terms: scanning electron microscopy .................................................................................................................10

4 General principles ............................................................................................................................................................................................11

4.1 SEM imaging ...........................................................................................................................................................................................11

4.2 SEM image-based particle size measurements ........................................................................................................12

4.3 SEM image-based particle shape measurements ...................................................................................................13

5 Sample preparation ........................................................................................................................................................................................13

5.1 Sample preparation fundamental information ........................................................................................................13

5.2 General recommendations.........................................................................................................................................................14

5.3 Ensuring good sampling of powder or dispersion-in-liquid raw materials ....................................14

5.3.1 Powders ................................................................................................................................................................................14

5.3.2 Nanoparticle dispersions in liquids ............................................................................................................15

5.4 Ensuring representative dispersion ..................................................................................................................................15

5.5 Nanoparticle deposition on a substrate .........................................................................................................................15

5.5.1 General...................................................................................................................................................................................15

5.5.2 Nanoparticle deposition on wafers and chips of silicon or other materials .............16

5.5.3 Nanoparticle deposition on TEM grids ......................................................................................................17

5.6 Number of samples to be prepared ....................................................................................................................................18

5.7 Number of particles to be measured for particle size determination ..................................................18

5.8 Number of particles to be measured for particle shape determination .............................................19

6 Qualification of the SEM for nanoparticle measurements .....................................................................................19

7 Image acquisition .............................................................................................................................................................................................19

7.1 General ........................................................................................................................................................................................................19

7.2 Setting suitable image magnification and pixel resolution ............................................................................23

8 Particle analysis .................................................................................................................................................................................................24

8.1 Particle analysis fundamental information .................................................................................................................24

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

8.3 Automated particle analysis .....................................................................................................................................................25

8.4 Automated particle analysis procedure example ...................................................................................................26

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

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

9.2 Raw data screening: detecting touching particles, artefacts and contaminants .........................27

9.3 Fitting models to data ....................................................................................................................................................................27

9.4 Assessment of measurement uncertainty ....................................................................................................................27

9.4.1 General...................................................................................................................................................................................27

9.4.2 Example: Measurement uncertainty for particle size measurements ............................28

9.4.3 Bivariate analysis ..........................................................................................................................................................29

10 Reporting the results ....................................................................................................................................................................................29

Annex A (normative) Qualification of the SEM for nanoparticle measurements ................................................31

Annex B (informative) Cross-sectional titanium dioxide samples preparation ...................................................36

© ISO 2021 – All rights reserved iii
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ISO 19749:2021(E)

Annex C (informative) Case study on well-dispersed 60 nm size silicon dioxide nanoparticles ........38

Annex D (informative) Case study on 40 nm size titanium dioxide nanoparticles ...........................................46

Annex E (informative) Example for extracting particle size results of SEM-based

nanoparticle measurements using ImageJ .............................................................................................................................55

Annex F (informative) Effects of some image acquisition parameters and thresholding

methods on SEM particle size measurements ....................................................................................................................57

Annex G (informative) Example for reporting results of SEM-based nanoparticle measurements 61

Bibliography .............................................................................................................................................................................................................................71

iv © ISO 2021 – All rights reserved
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ISO 19749:2021(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 2021 – All rights reserved v
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ISO 19749:2021(E)
Introduction

This document provides guidance for measuring and reporting the size and shape distributions of

nanometer-scale particles using images acquired by the scanning electron microscope (SEM). This

document applies to the SEM-based measurement of larger particles also. Nanoparticles are three-

dimensional (3D) objects, but the SEM image is only a two-dimensional (2D) representation of the 3D

shape from a certain viewing angle. The SEM image carries valuable information about the size and

shape of particles. While the SEM image does contain a certain amount of 3D information, for sake of

simplicity, this document does not deal with reconstructing 3D information. Rigorous three-dimensional

characterization of nanoparticles would include size, shape, surface structure (e.g. texture), surface

and internal material composition, and their locations in the investigated 3D volume. This document

deals with two attributes of morphology, size and shape, for discrete and aggregated nano-objects

(materials with at least one dimension in the nanometer-scale, i.e. within 1 nm to 100 nm). Suitable

sample preparation is essential to obtaining high-quality electron microscope images and 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. Typical guidance suggests that

a large number, several hundreds or thousands of particles need to be measured for statistically sound

size and shape distribution results. The actual number of nano-objects needed to be measured depends

on the sample, the required uncertainty and on the performance of the SEM. Statistical evaluation of

the data and the evaluation of uncertainty of the measurands are included as part of the measurement

and reporting procedures.

This document contains measurement procedures, particle and data analysis and reporting clauses. In

the Annexes, there are specific examples for measurements and guidance for the qualification of the

SEM for reliable quantitative measurements. Automation of the image acquisition and data analysis can

reduce cost and improve the quality of the results. Measurements of samples of discrete nanoparticles

are generally easier to carry out with automated image acquisition and particle analysis systems.

Measurements of complex discrete nanoparticles, and aggregates or agglomerates of nanoparticles

may require operator-assisted image acquisition and analysis. Evaluation of particle shape is facilitated

by many pertinent analysis software solutions that allow for automatic selection of various shape

attributes as well.
vi © ISO 2021 – All rights reserved
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INTERNATIONAL STANDARD ISO 19749:2021(E)
Nanotechnologies — Measurements of particle size and
shape distributions by scanning electron microscopy
1 Scope

This document specifies methods of determining nanoparticle size and shape distributions by acquiring

and evaluating scanning electron microscope images and by obtaining and reporting accurate results.

NOTE 1 This document applies to particles with a lower size limit that depends on the required uncertainty

and on the suitable performance of the SEM, which is to be proven first -according to the requirements described

in this document.

NOTE 2 This document applies also to SEM-based size and shape measurements of larger than nanoscale

particles.
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/IEC Guide 99, International vocabulary of metrology — Basic and general concepts and associated

terms (VIM)

ISO 9276-1, Representation of results of particle size analysis — Part 1: Graphical representation

ISO 9276-2, Representation of results of particle size analysis — Part 2: Calculation of average particle

sizes/diameters and moments from particle size distributions

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

curve to a reference model

ISO 9276-5, Representation of results of particle size analysis — Part 5: Methods of calculation relating to

particle size analyses using logarithmic normal probability distribution

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

representation of particle shape and morphology

ISO 13322-1, Particle size analysis — Image analysis methods — Part 1: Static image analysis methods

ISO 16700, Microbeam analysis — Scanning electron microscopy — Guidelines for calibrating image

magnification

ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories

ISO/TS 24597:2011, Microbeam analysis — Scanning electron microscopy — Methods of evaluating image

sharpness
ISO 26824, Particle characterization of particulate systems — Vocabulary
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-3, Nanotechnologies — Vocabulary — Part 3: Carbon nano-objects
ISO/TS 80004-4, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials
© ISO 2021 – All rights reserved 1
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ISO 19749:2021(E)

ISO/TS 80004-6, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/IEC Guide 99, ISO 9276-6,

ISO 26824, ISO/TS 80004-1, ISO/TS 80004-2, ISO/TS 80004-3, ISO/TS 80004-4, ISO/TS 80004-6, and

the following 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 General terms
3.1.1
nanoscale
length range from approximately 1 nm to 100 nm

Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this

length range.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.1.2
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (3.1.1)

[SOURCE: ISO/TS 80004-1:2015, 2.5, modified — Note 1 to entry and the source have been deleted.]

3.1.3
particle
minute piece of matter with defined physical boundaries

[SOURCE: ISO/TR 16197:2014, 3.10, modified — Notes 1, 2 and 3 to entry and the source have been

deleted.]
3.1.4
primary particle

original source particle (3.1.3) of agglomerates (3.1.5) or aggregates (3.1.6) or mixtures of the two

[SOURCE: ISO 26824:2013, 1.4, modified — Notes 1, 2 and 3 to entry have 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: Agglomerate originates from the Latin “agglomerare” meaning “to form into a ball”.

Note 2 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or

simple physical entanglement.

Note 3 to entry: Agglomerates are also termed secondary particles and the original source particles are termed

primary particles (3.1.4).
[SOURCE: ISO 26824:2013, 1.2, modified — Note 1 to entry has been added.]
2 © ISO 2021 – All rights reserved
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ISO 19749:2021(E)
3.1.6
aggregate

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

is significantly smaller than the sum of 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, or otherwise combined former primary particles

(3.1.4).

Note 2 to entry: Aggregate comes from the Latin “aggregat” meaning “herded together”.

Note 3 to entry: Figure 1 shows examples of individual, aggregate and agglomerate (3.1.5) particles.

NOTE The images are projected views from certain angles of the 3D objects. Depending on the viewing

angle, the observable size of particles can vary substantially.

Figure 1 — SEM images of individual gold (left) and carbon black aggregate (middle) and

corundum agglomerate (right) particles

[SOURCE: ISO 26824:2013, 1.3, modified — Notes 2 and 3 to entry have been added.]

3.1.7
nanoparticle

nano-object (3.1.2) with all external dimensions in the nanoscale (3.1.1) 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 — Note 1 to entry has been deleted.]
3.1.8
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 (3.1.3) size. However, it is recognized that the symbol

d is also widely used. Therefore, the symbol x may be replaced by d.
3.1.9
particle size distribution

distribution of the quantity of particles (3.1.3) as a function of particle size (3.1.8)

[SOURCE: ISO/TS 80004-6:2021, 4.1.2, modified — Notes 1 and 2 to entry have been deleted.]

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ISO 19749:2021(E)
3.1.10
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 breadth.

[SOURCE: ISO/TS 80004-6:2021, 4.1.3, modified — Note 1 to entry has been added.]
3.1.11
analytical sample

portion of material, resulting from the original sample or composite sample by means of an appropriate

method of sample pretreatment and having the size (volume/mass) necessary for the desired testing or

analysis

Note 1 to entry: The sample in analytical chemistry is a portion of material selected from a larger quantity of

material. The term needs to be qualified, for example, bulk sample, representative sample, primary sample,

bulked sample, test sample. The term 'sample' implies the existence of a sampling error, i.e. the results obtained

on the portions taken are only estimates of the concentration of a constituent or the quantity of a property present

in the parent material. If there is no or negligible sampling error, the portion removed is a test portion, aliquot

or specimen. The term 'specimen' is used to denote a portion taken under conditions such that the sampling

variability cannot be assessed (usually because the population is changing), and is assumed, for convenience, to

be zero. The manner of selection of the sample should be prescribed in a sampling plan.

[SOURCE: ISO 11074:2015, 4.1.3, modified — Note 1 to entry has been added.]
3.2 Core terms: image analysis
3.2.1
binary image

digitized image consisting of an array of pixels (3.2.2), 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

colors
[SOURCE: ISO 13322-1:2014, 3.1.2]
3.2.2
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.]
3.2.3
pixel resolution
number of imaging pixels (3.2.2) per unit distance of the detector
Note 1 to entry: The typical unit is sometimes expressed as dots per inch (dpi).

[SOURCE: ISO 29301:2017, 3.24, modified — the hyphen has been deleted in this term.]

3.3 Core terms: statistical symbols and definitions
3.3.1
arithmetic mean
sum of values divided by the number of values

Note 1 to entry: See ISO 9276-1:1998 for other quantity measures and types of distributions.

4 © ISO 2021 – All rights reserved
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ISO 19749:2021(E)
3.3.2
standard deviation

measure of the dispersion of a series of results around their mean, equal to the positive square root of

the variance and estimated by the positive square root of the mean square
[SOURCE: ISO 4259-1:2017, 3.21]
3.3.3
coefficient of variation
ratio of the standard deviation (3.3.2) to the arithmetic mean (3.3.1)
[SOURCE: ISO 27448:2009, 3.11]
3.3.4
relative standard error
standard error (SE ) divided by the mean (x ) and expressed as a percentage
3.3.5
analysis of variance
ANOVA

technique which subdivides the total variation of a response variable into components associated with

defined sources of variation
3.3.6
p-value

probability of observing the observed test statistic value or any other value at least as unfavorable to

the null hypothesis

Note 1 to entry: If the null hypothesis were true and if the experiment were repeated many times, a p-value is the

probability that a value at least as extreme as the computed test statistic would be observed.

Note 2 to entry: In hypothesis testing, a statement claiming that the null parameter is the true parameter is

called the null hypothesis. The purpose of a hypothesis test is to determine whether the data provide evidence

against the null hypothesis. When a statistic is obtained that is very different from the null parameter, the null

hypothesis can be rejected. An alternative, or research hypothesis, is a hypothesis that states that the true

parameter is not (or is less than or is greater than) the null parameter; it is the hypothesis that corresponds

to the research question. The goal of a hypothesis test is to reject the null hypothesis in favour of the research

hypothesis.

[SOURCE: ISO/TR 14468:2010, 3.13, modified — Note 1 to entry has been modified and Note 2 to entry

has been added.]
3.3.7
residual deviation

difference between the observed value of the response variable and the estimated value of the response

variable
3.3.8
residual standard deviation
scatter of the information values about the calculated regression line

Note 1 to entry: It is a figure of merit, describing the precision (3.5.3) of the calibration.

Note 2 to entry: For this document, the standard deviation (3.3.2) of the method means the standard of deviation

of the calibration procedure.

[SOURCE: ISO 8466-1:1990, 2.5, modified — the symbol has been deleted and the entire entry has been

editorially revised.]
© ISO 2021 – All rights reserved 5
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ISO 19749:2021(E)
3.3.9
quantile plot

graphical method of comparing two distributions where the quantiles of the empirical (data)

distribution are plotted on the y-axis while the quantiles of the theoretical (reference) distribution with

the same mean and variance as the empirical distribution are plotted on the x-axis

Note 1 to entry: The quantile-quantile (q-q) plot is a probability plot, a graphical technique for determining if two

data sets come from populations with a common distribution. A q-q plot is a plot of the quantiles of the first data

set against the quantiles of the second data set. See ISO/TS 80004-6.
3.4 Core terms: measurands and descriptors
3.4.1
measurand
quantity intended to be measured
[SOURCE: ISO/IEC Guide 98-4:2012, 3.2.4]
3.4.2
Feret diameter

distance between two parallel lines which are tangent to the perimeter (3.4.5) of a particle (3.1.3)

[SOURCE: ISO 10788:2014, 2.1.4, modified — Note 1 to entry has been deleted.]
3.4.3
maximum Feret diameter
maximum length of an object whatever its orientation

[SOURCE: ISO/TR 945-2:2011, 2.1, modified — the word "Féret" in the term has been changed to "Feret"

and Note 1 to entry has been deleted.]
3.4.4
minimum Feret diameter
minimum length of an object whatever its orientation

Note 1 to entry: The Feret diameter (3.4.2) or Feret's diameter is a measure of an object size along a specified

direction; it is applied to projections of a three-dimensional object on a two-dimensional plane, see Figure 2. It is

also called the caliper diameter.
Key
1 vertical Feret diameter 3 breadth
2 horizontal Feret diameter 4 length
Figure 2 — Horizontal Feret diameter (88 nm) and vertical Feret diamet
...

NORME ISO
INTERNATIONALE 19749
Première édition
2021-07
Nanotechnologies — Détermination
de la distribution de taille et de
forme des particules par microscopie
électronique à balayage
Nanotechnologies — Measurements of particle size and shape
distributions by scanning electron microscopy
Numéro de référence
ISO 19749:2021(F)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 19749:2021(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
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Publié en Suisse
ii © ISO 2021 – Tous droits réservés
---------------------- Page: 2 ----------------------
ISO 19749:2021(F)
Sommaire Page

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

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

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

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

3 Termes et définitions ....................................................................................................................................................................................... 2

4 Principes généraux .........................................................................................................................................................................................12

4.1 Imagerie MEB ........................................................................................................................................................................................12

4.2 Mesurages de taille d’une particule fondés sur une image MEB...............................................................12

4.3 Mesurages de forme d’une particule fondés sur une image MEB ............................................................14

5 Préparation des échantillons ...............................................................................................................................................................14

5.1 Renseignements fondamentaux concernant la préparation des échantillons ..............................14

5.2 Recommandations générales...................................................................................................................................................14

5.3 Garantie d’une bonne préparation d’échantillon de poudre ou de matériaux bruts

dispersés dans un liquide ...........................................................................................................................................................15

5.3.1 Poudres ...............................................................................................................................................................................15

5.3.2 Dispersions de nanoparticules dans des liquides ............................................................................15

5.4 Garantie d’une dispersion représentative ....................................................................................................................15

5.5 Dépôt de nanoparticules sur un substrat ......................................................................................................................16

5.5.1 Généralités .........................................................................................................................................................................16

5.5.2 Dépôt de nanoparticules sur des plaquettes de silicium ou d’autres matériaux ..16

5.5.3 Dépôts de nanoparticules sur des grilles MET ...................................................................................18

5.6 Nombre d’échantillons à préparer ......................................................................................................................................19

5.7 Nombre de particules à mesurer pour en déterminer la taille ...................................................................19

5.8 Nombre de particules à mesurer pour en déterminer la forme ................................................................20

6 Qualification du MEB pour les mesurages des nanoparticules.........................................................................20

7 Acquisition d’images .....................................................................................................................................................................................21

7.1 Généralités ...............................................................................................................................................................................................21

7.2 Réglage d’une résolution et d’un grandissement corrects de l’image ..................................................25

8 Analyse de particules ....................................................................................................................................................................................26

8.1 Renseignements fondamentaux concernant l’analyse de particules ....................................................26

8.2 Analyse de particules individuelles ....................................................................................................................................27

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

8.4 Exemple de mode opératoire d’analyse automatisée des particules ....................................................28

9 Analyse des données......................................................................................................................................................................................29

9.1 Généralités ...............................................................................................................................................................................................29

9.2 Tri des données brutes: détections des particules en contact, des artefacts et

des contaminants ...............................................................................................................................................................................29

9.3 Ajustement des modèles aux données ............................................................................................................................30

9.4 Évaluation de l’incertitude de mesure .............................................................................................................................30

9.4.1 Généralités .........................................................................................................................................................................30

9.4.2 Exemple: Incertitude de mesure pour les mesurages de taille de particules ...........30

9.4.3 Analyse à deux variables ........................................................................................................................................31

10 Rapport de résultats ......................................................................................................................................................................................31

Annexe A (normative) Qualification du MEB pour les mesurages des nanoparticules ................................33

Annexe B (informative) Préparation d’échantillons transversaux de dioxyde de titane ............................38

Annexe C (informative) Étude de cas portant sur des nanoparticules bien dispersées

de dioxyde de silicium de 60 nm .......................................................................................................................................................40

© ISO 2021 – Tous droits réservés iii
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ISO 19749:2021(F)

Annexe D (informative) Étude de cas portant sur des nanoparticules de dioxyde de titane

de 40 nm .....................................................................................................................................................................................................................50

Annexe E (informative) Exemple d’extraction de résultats de taille des particules issus

de mesurages de nanoparticules par MEB à l’aide d’ImageJ ...............................................................................59

Annexe F (informative) Effets de certains paramètres d’acquisition d’images et

des méthodes de seuillage sur les mesurages de taille de particules par MEB ...............................61

Annexe G (informative) Exemple de rapport de résultats de mesurages de nanoparticules

par MEB .......................................................................................................................................................................................................................66

Bibliographie ...........................................................................................................................................................................................................................76

iv © ISO 2021 – Tous droits réservés
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ISO 19749:2021(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 le lien suivant: www .iso .org/ iso/ fr/ avant -propos.

Le présent document a été préparé 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 2021 – Tous droits réservés v
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ISO 19749:2021(F)
Introduction

Le présent document fournit des recommandations pour mesurer et établir des rapports sur les

distributions de taille et de forme des particules à l’échelle nanométrique, en utilisant les images

obtenues par un microscope électronique à balayage (MEB). Le présent document s’applique également

au mesurage par MEB des particules de plus grande taille. Les nanoparticules sont des objets

tridimensionnels (3D), mais l’image MEB n’est qu’une représentation bidimensionnelle (2D) de la forme

3D observée avec un angle de vue particulier. L’image MEB fournit de précieuses informations sur la

taille et la forme des particules. Bien que l’image MEB contienne une certaine quantité d’informations

3D, pour des raisons de simplicité, le présent document ne traite pas de la reconstruction des

informations 3D. Une caractérisation tridimensionnelle rigoureuse des nanoparticules inclurait la

taille, la forme, la structure de surface (par exemple, la texture), la composition de la surface et des

matériaux internes ainsi que leurs emplacements dans le volume 3D étudié. Le présent document

porte sur deux attributs de morphologie, à savoir la taille et la forme, pour des nano-objets discrets

et agrégés (matériaux ayant au moins une dimension à l’échelle nanométrique, c’est-à-dire entre 1 nm

et 100 nm). Une préparation appropriée de l’échantillon est essentielle pour obtenir des images de

qualité avec un microscope électronique et les techniques préférées varient souvent selon le matériau

de l’échantillon. Il est tout aussi important de s’assurer que le MEB lui-même est capable d’effectuer les

mesurages avec l’incertitude exigée. Les recommandations habituelles suggèrent de mesurer un grand

nombre de particules, c’est-à-dire plusieurs centaines ou plusieurs milliers, afin d’obtenir des résultats

statistiquement fiables pour les distributions de taille et de forme. Le nombre réel de nano-objets à

mesurer dépend de l’échantillon, de l’incertitude exigée et des performances du MEB. L’évaluation

statistique des données et l’évaluation de l’incertitude des mesurandes font partie intégrante des modes

opératoires de mesure et de rédaction de rapports.

Le présent document contient des articles sur les modes opératoires de mesure, l’analyse des particules

et des données et la rédaction de rapports. Les annexes fournissent des exemples spécifiques de

mesurages ainsi que des recommandations concernant la qualification du MEB pour des mesurages

quantitatifs fiables. L’automatisation de l’acquisition d’images et de l’analyse des données peut réduire

le coût et améliorer la qualité des résultats. Les mesurages d’échantillons de nanoparticules isolées sont

généralement plus faciles à effectuer avec des systèmes automatisés d’acquisition d’images et d’analyse

de particules. Les mesurages de nanoparticules isolées complexes et d’agrégats ou d’agglomérats de

nanoparticules peuvent nécessiter une acquisition d’images et une analyse assistées d’un opérateur.

L’évaluation de la forme des particules est facilitée par un grand nombre de solutions logicielles

d’analyse pertinentes qui permettent également la sélection automatique de divers attributs de forme.

vi © ISO 2021 – Tous droits réservés
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NORME INTERNATIONALE ISO 19749:2021(F)
Nanotechnologies — Détermination de la distribution
de taille et de forme des particules par microscopie
électronique à balayage
1 Domaine d’application

Le présent document spécifie des méthodes permettant de déterminer les distributions de taille et

de forme des nanoparticules, par l’acquisition et l’évaluation d’images obtenues avec un microscope

électronique à balayage, puis l’obtention de résultats exacts et la rédaction de rapports.

NOTE 1 Le présent document s’applique aux particules dont la limite de taille inférieure dépend de l’incertitude

exigée et des performances appropriées du MEB, après démonstration de sa conformité aux exigences décrites

dans le présent document.

NOTE 2 Le présent document s’applique également aux mesurages par MEB de taille et de forme des particules

de taille supérieure à l’échelle nanométrique.
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).

Guide ISO/IEC 99, Vocabulaire international de métrologie — Concepts fondamentaux et généraux et

termes associés (VIM)

ISO 9276-1, Représentation de données obtenues par analyse granulométrique — Partie 1: Représentation

graphique

ISO 9276-2, Représentation de données obtenues par analyse granulométrique — Partie 2: Calcul des

tailles/diamètres moyens des particules et des moments à partir de distributions granulométriques

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

d'une courbe expérimentale à un modèle de référence

ISO 9276-5, Représentation de données obtenues par analyse granulométrique — Partie 5: Méthodes de

calcul relatif à l'analyse granulométrique à l'aide de la distribution de probabilité logarithmique normale

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

représentation quantitative de la forme et de la morphologie des particules

ISO 13322-1, Analyse granulométrique — Méthodes par analyse d'images — Partie 1: Méthodes par analyse

d'images statiques

ISO 16700,Analyse par microfaisceaux — Microscopie électronique à balayage — Lignes directrices pour

l'étalonnage du grandissement d'image

ISO/IEC 17025,Exigences générales concernant la compétence des laboratoires d'étalonnages et d'essais

ISO/TS 24597:2011, Analyse par microfaisceaux — Microscopie électronique à balayage — Méthodes

d'évaluation de la netteté d'image

ISO 26824, Caractérisation des particules dans les systèmes particulaires — Vocabulaire

ISO/TS 80004-1, Nanotechnologies — Vocabulaire — Partie 1: Termes "coeur"
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ISO 19749:2021(F)
ISO/TS 80004-2, Nanotechnologies — Vocabulaire — Partie 2: Nano-objets
ISO/TS 80004-3, Nanotechnologies — Vocabulaire — Partie 3: Nano-objets carbonés

ISO/TS 80004-4, Nanotechnologies — Vocabulaire — Partie 4: Matériaux nanostructurés

ISO/TS 80004-6, Nanotechnologies — Vocabulaire — Partie 6: Caractérisation des nano-objets

3 Termes et définitions

Pour les besoins du présent document, les termes et définitions de le Guide ISO/IEC 99, l’ISO 9276-6,

l’ISO 26824, l’ISO/TS 80004-1, l’ISO/TS 80004-2, l’ISO/TS 80004-3, l’ISO/TS 80004-4, l’ISO/TS 80004-6,

ainsi que les 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 https:// www .electropedia .org/ .
3.1 Termes généraux
3.1.1
échelle nanométrique
échelle de longueur s’étendant approximativement de 1 nm à 100 nm

Note 1 à l'article: Les propriétés qui ne constituent pas des extrapolations par rapport à des dimensions plus

grandes sont principalement manifestes dans cette échelle de longueur.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.1.2
nano-objet

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

nanométrique (3.1.1)

[SOURCE: ISO/TS 80004-1:2015, 2.5, modifiée — La Note 1 à l’article et la source ont été supprimées.]

3.1.3
particule
élément de matière isolé possédant des limites physiques définies

[SOURCE: ISO/TR 16197:2014, 3.10, modifiée — Les Notes 1, 2 et 3 à l’article et la source ont été

supprimées.]
3.1.4
particule primaire

particule source initiale (3.1.3) des agglomérats (3.1.5) ou des agrégats (3.1.6) ou de mélanges de ceux-

[SOURCE: ISO 26824:2013, 1.4, modifiée — Les Notes 1, 2 et 3 à l’article ont été supprimées.]

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: Le terme «agglomérat» découle du terme latin «agglomerare» qui signifie «former une boule».

Note 2 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.
2 © ISO 2021 – Tous droits réservés
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ISO 19749:2021(F)

Note 3 à l'article: Les agglomérats sont également appelés particules secondaires et les particules sources

initiales sont appelées particules primaires (3.1.4).
[SOURCE: ISO 26824:2013, 1.2, modifiée — La Note 1 à l’article a été ajoutée.]
3.1.6
agrégat

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

résultante est significativement plus petite que la somme des aires de surface 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, ou sinon d’anciennes particules

primaires combinées (3.1.4).

Note 2 à l'article: Le terme «agrégat» découle du terme latin «aggregat» qui signifie «rassemblé».

Note 3 à l'article: La Figure 1 montre des exemples de particules individuelles, d’agglomérats (3.1.5) de particules

et d’agrégats de particules.

NOTE Les images sont des vues en projection obtenues à partir de certains angles d’objets 3D. La taille

observable des particules peut varier sensiblement selon l’angle de vue.

Figure 1 — Images MEB de particules d’or (gauche), d’agrégats de noir de carbone (milieu) et

d’agglomérats de corindon (droite)

[SOURCE: ISO 26824:2013, 1.3, modifiée — Les Notes 2 et 3 à l’article ont été ajoutées.]

3.1.7
nanoparticule

nano-objet (3.1.2) dont toutes les dimensions externes sont à l’échelle nanométrique (3.1.1) 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 — La Note 1 à l’article a été supprimée.]

3.1.8
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 (3.1.3). Cependant, il est

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

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ISO 19749:2021(F)
3.1.9
distribution granulométrique

distribution de le quantité de particules (3.1.3) en fonction de leur taille (3.1.8)

[SOURCE: ISO/TS 80004-6:2021, 4.1.2, modifiée — Les Notes 1 et 2 à l’article ont été supprimée.]

3.1.10
forme d’une particule
forme géométrique externe d’une particule (3.1.3)

Note 1 à l'article: La description de la forme nécessite 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.]

3.1.11
échantillon pour analyse

prise de matériau, issue de l’échantillon d’origine ou d’un échantillon composite, au moyen d’une

méthode appropriée de traitement préalable des échantillons, et ayant la taille (volume/masse)

nécessaire pour les essais ou l’analyse souhaités

Note 1 à l'article: En chimie analytique l’échantillon est une prise de matériau réalisée dans un volume de

matériau plus important. Le terme doit être défini, par exemple échantillon global, échantillon représentatif,

échantillon primaire, échantillon massif, échantillon pour essai. Le terme «échantillon» implique l’existence

d’une erreur d’échantillonnage, c’est-à-dire que les résultats obtenus à partir des prises réalisées ne sont que des

estimations de la concentration d’un constituant ou de la quantité d’une propriété présente dans le matériau de

base. Si l’erreur d’échantillonnage est nulle ou négligeable, la prise effectuée est une prise d’essai, une aliquote

ou un spécimen. Le terme «spécimen» est utilisé pour indiquer une prise effectuée dans des conditions où la

variabilité d’échantillonnage ne peut pas être évaluée (généralement en raison de la variation de la population),

et où elle est considérée nulle par commodité. Il convient d’établir la façon dont l’échantillon est sélectionné dans

un plan d’échantillonnage.
[SOURCE: ISO 11074:2015, 4.1.3, modifiée — La Note 1 à l’article a été ajoutée.]
3.2 Termes «cœur»: analyse d’image
3.2.1
image binaire

image numérisée constituée d’une matrice de pixels (3.2.2), 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]
3.2.2
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.]

3.2.3
résolution de pixels
nombre de pixels (3.2.2) d’imagerie par unité de distance d’un détecteur
Note 1 à l'article: L’unité type est souvent exprimée en points par pouce (ppp).
[SOURCE: ISO 29301:2017, 3.24, modifiée]
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ISO 19749:2021(F)
3.3 Termes «cœur»: symboles statistiques et définitions
3.3.1
moyenne arithmétique
somme des valeurs divisée par le nombre de valeurs

Note 1 à l'article: Voir l’ISO 9276-1:1998 pour les autres mesures de quantité et types de distributions.

3.3.2
écart-type

mesure de la dispersion d’une série de résultats autour de leur moyenne, égale à la racine carrée positive

de la variance et estimée par la racine carrée positive du carré moyen
[SOURCE: ISO 4259-1:2017, 3.21]
3.3.3
coefficient de variation
rapport de l’écart-type (3.3.2) à la moyenne arithmétique (3.3.1)
[SOURCE: ISO 27448:2009, 3.11]
3.3.4
erreur-type relative (RSE)
erreur-type (SE ) divisée par la moyenne (x ) et exprimée en pourcentage
3.3.5
analyse de variance
ANOVA

méthode consistant à séparer la variation totale d’une variable de réponse en composantes associées à

des sources spécifiques de variation
3.3.6
valeur p

probabilité d’obtenir la valeur de la statistique de test observée ou toute autre valeur défavorable à

l’hypothèse nulle

Note 1 à l'article: Si l’hypothèse nulle était vraie et si l’expérience a été répétée un grand nombre de fois, une

valeur p est la probabilité qu’une valeur au moins aussi extrême que la statistique d’essai calculée soit observée.

Note 2 à l'article: Lors d’un test d’hypothèse, une expression affirmant que le paramètre nul est le paramètre

vrai est appelée hypothèse nulle. L’objectif d’un test d’hypothèse est de déterminer si les données fournissent

des preuves contre l’hypothèse nulle. Lorsqu’une statistique obtenue est très différente du paramètre nul,

l’hypothèse nulle peut être rejetée. Une hypothèse alternative ou de recherche est une hypothèse indiquant que

le paramètre vrai n’est pas (ou est inférieur ou supérieur) le paramètre nul. Il s’agit de l’hypothèse correspondant

à la question de recherche. Le but d’un test d’hypothèse est de rejeter l’hypothèse nulle en faveur de l’hypothèse

de recherche.

[SOURCE: ISO/TR 14468:2010, 3.13, modifiée — La Note 1 à l’article a été modifié et la Note 2 à l’article

a été ajoutée.]
3.3.7
écart résiduel

différence entre la valeur observée de la variable de réponse et sa valeur estimée

3.3.8
écart-type résiduel
dispersion des valeurs d’information autour de la ligne de régression calculée

Note 1 à l'article: C’est un indice de performance qui décrit la fidélité (3.5.3) de l’étalonnage.

Note 2 à l'article: Dans le présent document, l’écart-type (3.3.2) de la méthode est l’écart-type de la performance

de l’étalonnage.
© ISO 2021 – Tous droits réservés 5
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ISO 19749:2021(F)

[SOURCE: ISO 8466-1:1990, 2.5, modifiée — Le symbole a été supprimé et une révision éditori

...

INTERNATIONAL ISO
STANDARD 19749
First edition
2021-05
Nanotechnologies — Measurements of
particle size and shape distributions
by scanning electron microscopy
Nanotechnologies — Détermination de la distribution de taille et de
forme des particules par microscopie électronique à balayage
PROOF/ÉPREUVE
Reference number
ISO 19749:2021(E)
ISO 2021
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ISO 19749:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
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ISO 19749:2021(E)
Contents Page

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

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

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

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

3 Terms and definitions ..................................................................................................................................................................................... 2

3.1 General terms ........................................................................................................................................................................................... 2

3.2 Core terms: image analysis ........................................................................................................................................................... 4

3.3 Core terms: statistical symbols and definitions ......................................................................................................... 4

3.4 Core terms: measurands and descriptors ........................................................................................................................ 6

3.5 Core terms: metrology ...................................................................................................................................................................... 8

3.6 Core terms: scanning electron microscopy .................................................................................................................10

4 General principles ............................................................................................................................................................................................11

4.1 SEM imaging ...........................................................................................................................................................................................11

4.2 SEM image-based particle size measurements ........................................................................................................12

4.3 SEM image-based particle shape measurements ...................................................................................................13

5 Sample preparation ........................................................................................................................................................................................13

5.1 Sample preparation fundamental information ........................................................................................................13

5.2 General recommendations.........................................................................................................................................................14

5.3 Ensuring good sampling of powder or dispersion-in-liquid raw materials ....................................14

5.3.1 Powders ................................................................................................................................................................................14

5.3.2 Nanoparticle dispersions in liquids ............................................................................................................15

5.4 Ensuring representative dispersion ..................................................................................................................................15

5.5 Nanoparticle deposition on a substrate .........................................................................................................................15

5.5.1 General...................................................................................................................................................................................15

5.5.2 Nanoparticle deposition on wafers and chips of silicon or other materials .............16

5.5.3 Nanoparticle deposition on TEM grids ......................................................................................................17

5.6 Number of samples to be prepared ....................................................................................................................................18

5.7 Number of particles to be measured for particle size determination ..................................................18

5.8 Number of particles to be measured for particle shape determination .............................................19

6 Qualification of the SEM for nanoparticle measurements .....................................................................................19

7 Image acquisition .............................................................................................................................................................................................19

7.1 General ........................................................................................................................................................................................................19

7.2 Setting suitable image magnification and pixel resolution ............................................................................23

8 Particle analysis .................................................................................................................................................................................................24

8.1 Particle analysis fundamental information .................................................................................................................24

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

8.3 Automated particle analysis .....................................................................................................................................................25

8.4 Automated particle analysis procedure example ...................................................................................................26

9 Data analysis ..........................................................................................................................................................................................................26

9.1 General ........................................................................................................................................................................................................26

9.2 Raw data screening: detecting touching particles, artefacts and contaminants .........................27

9.3 Fitting models to data ....................................................................................................................................................................27

9.4 Assessment of measur ement uncertainty ....................................................................................................................27

9.4.1 General...................................................................................................................................................................................27

9.4.2 Example: measurement uncertainty for particle size measurements ............................28

9.4.3 Bivariate analysis ..........................................................................................................................................................28

10 Reporting the results ....................................................................................................................................................................................29

Annex A (normative) Qualification of the SEM for nanoparticle measurements ................................................30

Annex B (informative) Cross-sectional titanium dioxide samples preparation ...................................................35

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
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ISO 19749:2021(E)

Annex C (informative) Case study on well-dispersed 60 nm size silicon dioxide nanoparticles ........37

Annex D (informative) Case study on 40 nm size titanium dioxide nanoparticles ...........................................45

Annex E (informative) Example for extracting particle size results of SEM-based

nanoparticle measurements using ImageJ .............................................................................................................................54

Annex F (informative) Effects of some image acquisition parameters and thresholding

methods on SEM particle size measurements ....................................................................................................................56

Annex G (informative) Example for reporting results of SEM-based nanoparticle measurements 60

Bibliography .............................................................................................................................................................................................................................70

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ISO 19749:2021(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 jointly by Technical Committee ISO/ TC 229, Nanotechnologies, and

Technical Committee IEC/TC 113, Nanotechnology for electrotechnical products and systems.

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.
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ISO 19749:2021(E)
Introduction

This document provides guidance for measuring and reporting the size and shape distributions of

nanometer-scale particles using images acquired by the scanning electron microscope (SEM). This

document applies to the SEM-based measurement of larger particles also. Nanoparticles are three-

dimensional (3D) objects, but the SEM image is only a two-dimensional (2D) representation of the 3D

shape from a certain viewing angle. The SEM image carries valuable information about the size and

shape of particles. While the SEM image does contain a certain amount of 3D information, for sake of

simplicity, this document does not deal with reconstructing 3D information. Rigorous three-dimensional

characterization of nanoparticles would include size, shape, surface structure (e.g. texture), surface

and internal material composition, and their locations in the investigated 3D volume. This document

deals with two attributes of morphology, size and shape, for discrete and aggregated nano-objects

(materials with at least one dimension in the nanometer-scale, i.e. within 1 nm to 100 nm). Suitable

sample preparation is essential to obtaining high-quality electron microscope images and 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. Typical guidance suggests that

a large number, several hundreds or thousands of particles need to be measured for statistically sound

size and shape distribution results. The actual number of nano-objects needed to be measured depends

on the sample, the required uncertainty and on the performance of the SEM. Statistical evaluation of

the data and the evaluation of uncertainty of the measurands are included as part of the measurement

and reporting procedures.

This document contains measurement procedures, particle and data analysis and reporting clauses. In

the Annexes, there are specific examples for measurements and guidance for the qualification of the

SEM for reliable quantitative measurements. Automation of the image acquisition and data analysis can

reduce cost and improve the quality of the results. Measurements of samples of discrete nanoparticles

are generally easier to carry out with automated image acquisition and particle analysis systems.

Measurements of complex discrete nanoparticles, and aggregates or agglomerates of nanoparticles

may require operator-assisted image acquisition and analysis. Evaluation of particle shape is facilitated

by many pertinent analysis software solutions that allow for automatic selection of various shape

attributes as well.
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INTERNATIONAL STANDARD ISO 19749:2021(E)
Nanotechnologies — Measurements of particle size and
shape distributions by scanning electron microscopy
1 Scope

This document specifies methods of determining nanoparticle size and shape distributions by acquiring

and evaluating scanning electron microscope images and by obtaining and reporting accurate results.

NOTE 1 This document applies to particles with a lower size limit that depends on the required uncertainty

and on the suitable performance of the SEM, which is to be proven first -according to the requirements described

in this document.

NOTE 2 This document applies also to SEM-based size and shape measurements of larger than nanoscale

particles.
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/IEC Guide 99, International vocabulary of metrology — Basic and general concepts and associated

terms (VIM)

ISO 9276-1, Representation of results of particle size analysis — Part 1: Graphical representation

ISO 9276-2, Representation of results of particle size analysis — Part 2: Calculation of average particle

sizes/diameters and moments from particle size distributions

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

curve to a reference model

ISO 9276-5, Representation of results of particle size analysis — Part 5: Methods of calculation relating to

particle size analyses using logarithmic normal probability distribution

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

representation of particle shape and morphology. This document provides a detailed list of other shape

parameters for size, macroshape descriptors, geometrical descriptors, proportion descriptors, and

mesoshape descriptors

ISO 13322-1, Particle size analysis — Image analysis methods — Part 1: Static image analysis methods

ISO 26824, Particle characterization of particulate systems — Vocabulary

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

magnification by using reference materials with periodic structures
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-3, Nanotechnologies – Vocabulary – Part 3: Carbon nano-objects
ISO/TS 80004-4, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials

ISO/TS 80004-6, Nanotechnologies – Vocabulary – Part 6: Nano-object characterization

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ISO 19749:2021(E)
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 9276-6, ISO 26824,

ISO/TS 80004-1, ISO/TS 80004-2, ISO/TS 80004-3, ISO/TS 80004-4, ISO/TS 80004-6, ISO/IEC Guide 99,

and the following 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 General terms
3.1.1
nanoscale
length range from approximately 1 nm to 100 nm

Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this

length range.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.1.2
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (3.1.1)

[SOURCE: ISO/TS 80004-3:2015, 3.1.3, modified — Note 1 to entry and the source have been deleted.]

3.1.3
particle
minute piece of matter with defined physical boundaries

[SOURCE: ISO/TR 16197:2014, 3.10, modified — Notes 1, 2 and 3 to entry and the source have been

deleted.]
3.1.4
primary particle

original source particle (3.1.3) of agglomerates (3.1.5) or aggregates (3.1.6) or mixtures of the two

[SOURCE: ISO 26824:2013, 1.4, modified — Notes 1, 2 and 3 to entry have 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: Agglomerate originates from the Latin “agglomerare” meaning “to form into a ball”.

Note 2 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or

simple physical entanglement.

Note 3 to entry: Agglomerates are also termed secondary particles and the original source particles are termed

primary particles (3.1.4).
[SOURCE: ISO 26824:2013, 1.2, modified — Note 1 to entry has been added.]
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ISO 19749:2021(E)
3.1.6
aggregate

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

is significantly smaller than the sum of 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, or otherwise combined former primary particles

(3.1.4).

Note 2 to entry: Aggregate comes from the Latin “aggregat” meaning “herded together”.

Note 3 to entry: Figure 1 shows examples of individual, aggregate and agglomerate (3.1.5) particles.

NOTE The images are projected views from certain angles of the 3D objects. Depending on the viewing

angle, the observable size of particles can vary substantially.

Figure 1 — SEM images of individual gold (left) and carbon black aggregate (middle) and

corundum agglomerate (right) particles

[SOURCE: ISO 26824:2013, 1.3, modified — Notes 2 and 3 to entry have been added.]

3.1.7
nanoparticle

nano-object (3.1.2) with all external dimensions in the nanoscale (3.1.1) 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 — Note 1 to entry has been deleted.]
3.1.8
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 (3.1.3) size. However, it is recognized that the symbol

d is also widely used. Therefore, the symbol x may be replaced by d.
3.1.9
particle size distribution
distribution of particles (3.1.3) as a function of particle size (3.1.8)

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

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ISO 19749:2021(E)
3.1.10
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 breadth.

[SOURCE: ISO/TS 80004-6:2013, 3.1.3, modified — Note 1 to entry has been added.]
3.1.11
analytical sample

portion of material, resulting from the original sample or composite sample by means of an appropriate

method of sample pretreatment and having the size (volume/mass) necessary for the desired testing or

analysis

Note 1 to entry: the sample in analytical chemistry is a portion of material selected from a larger quantity of

material. The term needs to be qualified, for example, bulk sample, representative sample, primary sample,

bulked sample, test sample. The term 'sample' implies the existence of a sampling error, i.e. the results obtained

on the portions taken are only estimates of the concentration of a constituent or the quantity of a property present

in the parent material. If there is no or negligible sampling error, the portion removed is a test portion, aliquot

or specimen. The term 'specimen' is used to denote a portion taken under conditions such that the sampling

variability cannot be assessed (usually because the population is changing), and is assumed, for convenience, to

be zero. The manner of selection of the sample should be prescribed in a sampling plan.

[SOURCE: ISO 11074:2015, 4.1.3, modified — Note 1 to entry has been added.]
3.2 Core terms: image analysis
3.2.1
binary image

digitized image consisting of an array of pixels (3.2.2), 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 colors

[SOURCE: ISO 13322-1:2014, 3.1.2]
3.2.2
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.]
3.2.3
pixel resolution
number of imaging pixels (3.2.2) per unit distance of the detector
Note 1 to entry: The typical unit is sometimes expressed as dots per inch (dpi).

[SOURCE: ISO 29301:2017, 3.24, modified — the hyphen has been deleted in this term.]

3.3 Core terms: statistical symbols and definitions
3.3.1
arithmetic mean
sum of values divided by the number of values

Note 1 to entry: See ISO 9276-1:1998 for other quantity measures and types of distributions.

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ISO 19749:2021(E)
3.3.2
standard deviation

measure of the dispersion of a series of results around their mean, equal to the positive square root of

the variance and estimated by the positive square root of the mean square
[SOURCE: ISO 4259-1:2017, 3.21]
3.3.3
coefficient of variation
ratio of the standard deviation (3.3.2) to the arithmetic mean (3.3.1)
[SOURCE: ISO 27448:2009, 3.11]
3.3.4
relative standard error
standard error (SE ) divided by the mean (x ) and expressed as a percentage
3.3.5
analysis of variance
ANOVA

technique which subdivides the total variation of a response variable (2.2) into components associated

with defined sources of variation
3.3.6
p-value

probability of observing the observed test statistic value or any other value at least as unfavorable to

the null hypothesis

Note 1 to entry: If the null hypothesis were true and if the experiment were repeated many times, a p-value is the

probability that a value at least as extreme as the computed test statistic would be observed.

Note 2 to entry: In hypothesis testing, a statement claiming that the null parameter is the true parameter is

called the null hypothesis. The purpose of a hypothesis test is to determine whether the data provide evidence

against the null hypothesis. When a statistic is obtained that is very different from the null parameter, the null

hypothesis can be rejected. An alternative, or research hypothesis, is a hypothesis that states that the true

parameter is not (or is less than or is greater than) the null parameter; it is the hypothesis that corresponds

to the research question. The goal of a hypothesis test is to reject the null hypothesis in favour of the research

hypothesis.

[SOURCE: ISO/TR 14468:2010, 3.13, modified — Notes 1 and 2 to entry have been added.]

3.3.7
residual deviation

difference between the observed value of the response variable and the estimated value of the response

variable
3.3.8
residual standard deviation

scatter of the information values about the calculated regression line. It is a figure of merit, describing

the precision (3.5.3) of the calibrationNote 1 to entry: For this document, the standard deviation (3.3.2)

of the method means the standard of deviation of the calibration procedure.

[SOURCE: ISO 8466-1:1990, 2.5, modified — the symbol has been deleted and the entire entry has been

editorially revised.]
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ISO 19749:2021(E)
3.3.9
quantile plot

graphical method of comparing two distributions where the quantiles of the empirical (data)

distribution are plotted on the y-axis while the quantiles of the theoretical (reference) distribution with

the same mean and variance as the empirical distribution are plotted on the x-axis

Note 1 to entry: The quantile-quantile (q-q) plot is a probability plot, a graphical technique for determining if two

data sets come from populations with a common distribution. A q-q plot is a plot of the quantiles of the first data

set against the quantiles of the second data set. See Reference [21].
3.4 Core terms: measurands and descriptors
3.4.1
measurand
quantity intended to be measured
[SOURCE: ISO/IEC Guide 98-4:2012, 3.2.4]
3.4.2
Feret diameter

distance between two parallel lines which are tangent to the perimeter (3.4.5) of a particle (3.1.3)

[SOURCE: ISO 10788:2014, 2.1.4, modified — Note 1 to entry has been deleted.]
3.4.3
maximum Feret diameter
maximum length of an object whatever its orientation

[SOURCE: ISO/TR 945-2:2011, 2.1, modified — the word "Féret" in the term has been changed to "Feret".]

3.4.4
minimum Feret diameter
minimum length of an object whatever its orientation

Note 1 to entry: The Feret diameter (3.4.2) or Feret's diameter is a measure of an object size along a specified

direction; it is applied to projections of a three-dimensional object on a two-dimensional plane, see Figure 2. It is

also called
...

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