SIST EN ISO 19749:2023
(Main)Nanotechnologies - Measurements of particle size and shape distributions by scanning electron microscopy (ISO 19749:2021)
Nanotechnologies - Measurements of particle size and shape distributions by scanning electron microscopy (ISO 19749:2021)
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.
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.
This document applies also to SEM-based size and shape measurements of larger than nanoscale particles.
Nanotechnologien - Messung der Partikelgrößenverteilung und Partikelformverteilung mit Rasterelektronenmikroskopie (ISO 19749:2021)
Dieses Dokument legt Verfahren zur Bestimmung von Größen- und Formverteilungen von Nanopartikeln durch Erfassung und Auswertung von Bildern des Rasterelektronenmikroskops sowie durch die Ermittlung und Berichterstattung genauer Ergebnisse fest.
ANMERKUNG 1 Dieses Dokument ist anwendbar für Partikel mit einer unteren Größengrenze, die von der erforderlichen Unsicherheit und von der geeigneten Leistung des REM abhängt, die zunächst entsprechend den in diesem Dokument beschriebenen Anforderungen nachgewiesen werden muss.
ANMERKUNG 2 Dieses Dokument ist auch anwendbar für REM-basierte Größen- und Formmessungen von Partikeln, die größer als im Nanomaßstab sind.
Nanotechnologies - Détermination de la distribution de taille et de forme des particules par microscopie électronique à balayage (ISO 19749:2021)
Nanotehnologije - Meritve porazdelitve velikosti in oblike delcev s skenirno elektronsko mikroskopijo (ISO 19749:2021)
Ta dokument določa metode za določanje velikosti in oblike razporeditve nanodelcev z uporabo in ocenjevanjem slik vrstičnega elektronskega mikroskopa in pridobivanjem in poročanje točnih rezultatov.
Ta dokument se uporablja za delce z manjšo omejitvijo velikosti, ki je odvisna od zahtevane negotovosti in ustrezne zmogljivosti vrstičnega elektronskega mikroskopa, ki jo je treba najprej dokazati v skladu z zahtevami, opisanimi v tem dokumentu.
Ta dokument se uporablja tudi za merjenje velikosti in oblike delcev, ki so večji od nanodelcev, z vrstičnim elektronskim mikroskopom.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 19749:2023
01-maj-2023
Nanotehnologije - Meritve porazdelitve velikosti in oblike delcev s skenirno
elektronsko mikroskopijo (ISO 19749:2021)
Nanotechnologies - Measurements of particle size and shape distributions by scanning
electron microscopy (ISO 19749:2021)
Nanotechnologien - Messung der Partikelgrößenverteilung und Partikelformverteilung mit
Rasterelektronenmikroskopie (ISO 19749:2021)
Nanotechnologies - Détermination de la distribution de taille et de forme des particules
par microscopie électronique à balayage (ISO 19749:2021)
Ta slovenski standard je istoveten z: EN ISO 19749:2023
ICS:
07.120 Nanotehnologije Nanotechnologies
SIST EN ISO 19749:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 19749:2023
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SIST EN ISO 19749:2023
EN ISO 19749
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2023
EUROPÄISCHE NORM
ICS 07.120
English Version
Nanotechnologies - Measurements of particle size and
shape distributions by scanning electron microscopy (ISO
19749:2021)
Nanotechnologies - Détermination de la distribution de Nanotechnologien - Messung der
taille et de forme des particules par microscopie Partikelgrößenverteilung und Partikelformverteilung
électronique à balayage (ISO 19749:2021) mit Rasterelektronenmikroskopie (ISO 19749:2021)
This European Standard was approved by CEN on 10 March 2023.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 19749:2023 E
worldwide for CEN national Members.
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SIST EN ISO 19749:2023
EN ISO 19749:2023 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 19749:2023
EN ISO 19749:2023 (E)
European foreword
The text of ISO 19749:2021 has been prepared by Technical Committee ISO/TC 229 "Nanotechnologies”
of the International Organization for Standardization (ISO) and has been taken over as
EN ISO 19749:2023 by Technical Committee CEN/TC 352 “Nanotechnologies” the secretariat of which
is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by September 2023, and conflicting national standards
shall be withdrawn at the latest by September 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 19749:2021 has been approved by CEN as EN ISO 19749:2023 without any modification.
3
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SIST EN ISO 19749:2023
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SIST EN ISO 19749:2023
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
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SIST EN ISO 19749:2023
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 © ISO 2021 – All rights reserved
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SIST EN ISO 19749:2023
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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SIST EN ISO 19749:2023
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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SIST EN ISO 19749:2023
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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SIST EN ISO 19749:2023
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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SIST EN ISO 19749:2023
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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SIST EN ISO 19749:2023
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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SIST EN ISO 19749:2023
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
x
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.]
© ISO 2021 – All rights reserved 3
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SIST EN ISO 19749:2023
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 1
...
SLOVENSKI STANDARD
oSIST prEN ISO 19749:2023
01-januar-2023
Nanotehnologije - Meritve porazdelitve velikosti in oblike delcev s skenirno
elektronsko mikroskopijo (ISO 19749:2021)
Nanotechnologies - Measurements of particle size and shape distributions by scanning
electron microscopy (ISO 19749:2021)
Nanotechnologien - Messung der Partikelgrößenverteilung und Partikelformverteilung mit
Rasterelektronenmikroskopie (ISO 19749:2021)
Nanotechnologies - Détermination de la distribution de taille et de forme des particules
par microscopie électronique à balayage (ISO 19749:2021)
Ta slovenski standard je istoveten z: prEN ISO 19749
ICS:
07.120 Nanotehnologije Nanotechnologies
oSIST prEN ISO 19749:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 19749:2023
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oSIST prEN ISO 19749:2023
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
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oSIST prEN ISO 19749:2023
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 © ISO 2021 – All rights reserved
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oSIST prEN ISO 19749:2023
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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oSIST prEN ISO 19749:2023
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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oSIST prEN ISO 19749:2023
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
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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
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Attention is drawn to the possibility that some of the elements of this document may be the subject of
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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
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oSIST prEN ISO 19749:2023
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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oSIST prEN ISO 19749:2023
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
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oSIST prEN ISO 19749:2023
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.]
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oSIST prEN ISO 19749:2023
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
x
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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oSIST prEN ISO 19749:2023
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.
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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
x
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.]
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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 gr
...
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