ISO/TS 22292:2021
(Main)Nanotechnologies — 3D image reconstruction of rod-supported nano-objects using transmission electron microscopy
Nanotechnologies — 3D image reconstruction of rod-supported nano-objects using transmission electron microscopy
This document provides guidance for sample preparation, data acquisition by transmission electron microscopy, data processing, and three-dimensional image reconstruction to measure size and shape parameters of nano-objects on rod-shaped supports. The method is applicable to samples dispersed on or within an electron-transparent rod-shaped support.
Nanotechnologies — Reconstruction d'images 3D de nano-objets soutenus par des tiges à l'aide de la microscopie électronique à transmission
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TECHNICAL ISO/TS
SPECIFICATION 22292
First edition
2021-06
Nanotechnologies — 3D image
reconstruction of rod-supported nano-
objects using transmission electron
microscopy
Nanotechnologies — Reconstruction d'images 3D de nano-objets
soutenus par des tiges à l'aide de la microscopie électronique à
transmission
Reference number
ISO/TS 22292:2021(E)
©
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 22292:2021(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
---------------------- Page: 2 ----------------------
ISO/TS 22292:2021(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 Nanotechnology-related terms . 2
3.2 Instrument-related terms . 2
3.3 Measurement-related terms. 3
4 Sample considerations . 4
4.1 General . 4
4.2 Choice of sample rod diameter . 4
5 Instrument factors . 4
5.1 Microscope set up . 4
5.1.1 General. 4
5.1.2 Acceleration voltage . . 5
5.1.3 Convergence semi-angle . 5
5.1.4 Collection angle . 5
5.1.5 Microscope magnification . 5
5.1.6 Number of pixels of the detector . 5
5.1.7 Image acquisition time . 6
5.2 Microscope calibration . 6
6 Image capture (data acquisition) . 7
6.1 General . 7
6.2 Procedure . 7
7 Data alignment and volume reconstruction .10
7.1 General .10
7.2 Procedure .10
8 Reconstructed volume evaluation and data analysis .10
8.1 General .10
8.2 Identification of nanoparticles and 3D volume .10
8.3 Thresholding for measurand extraction .11
9 Expression of results .13
9.1 Extracting parameters for each well-separated nano-object.13
9.2 Measurement uncertainty .14
9.3 Sources of errors .14
9.3.1 Error arising from sample that is not representative of the object of interest .14
9.3.2 Acquisition of 2D projected images .14
9.3.3 Instrument calibration . .16
9.3.4 Alignment of the projected images .16
9.3.5 Reconstruction of the 3D volume .16
9.3.6 Discrete representation of the objects (nanoparticles) in 3D .16
9.3.7 Interpretation of the obtained measurands .17
9.3.8 Limited number of observed objects (nanoparticles) .17
10 Test report .19
Annex A (informative) Sample preparation .21
Annex B (informative) STEM set up .28
Annex C (normative) Tomography reconstruction and visualization software packages .30
Annex D (informative) Microscope data collection parameters .32
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ISO/TS 22292:2021(E)
Annex E (informative) Case study: Metal nanoparticle, ILC results .33
Annex F (informative) Case study — Organic nanoparticles — Sample preparation and use.38
Annex G (informative) Particle distortions arising from FBP and SIRT reconstruction methods .39
Annex H (informative) Uncertainty budget .41
Bibliography .43
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ISO/TS 22292:2021(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.
© ISO 2021 – All rights reserved v
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ISO/TS 22292:2021(E)
Introduction
Electron tomography, in transmission electron microscope (TEM), has impact on nanotechnology
and nanomaterial metrology like that of computer tomography in medicine. For example, industries
using nanotechnologies have requirements to verify materials, processes and products. Quantitative
measurement at the nanoscale, including three-dimensional (3D) image reconstruction of nano-objects
using TEM, responds to this need.
TEM, a two-dimensional (2D) imaging instrument, can provide 2D projection images of materials at
the nanoscale, in the length range from below 1 nm to above 100 nm. From multiple 2D TEM images
collected at suitable tilt increments, the 3D shape, size and volume parameters can be determined. This
document describes sample preparation, instrumentation setup, data acquisition and processing for
3D image reconstruction of nano-objects using TEM, from which dimensional parameter values can
be determined and interpreted. Variation in methodology for use with scanning transmission electron
microscopy (STEM) is included in an informative annex.
The method described herein is limited to samples dispersed on or within an electron-transparent
rod-shaped support. This method is particularly useful when the detailed shape of a limited number
of objects, such as nanoparticles, is sought. For example, when 2D measurements yield a non-uniform
distribution of objects, 3D image reconstruction can be used applied to study a small number of the
objects in more detail. A variant of sample preparation is described that allows 3D reconstruction to
be used in conjunction with 2D TEM analysis of a sample area of interest, such as an area containing
outliers.
Potential applications for 3D image reconstruction of nano-objects using TEM are broad and might
include validation of metrological artefacts, such as polystyrene latex nanoparticles, and site-
specific analysis of interfaces buried within devices, and measurement of individual objects such as
nanoparticles. The method might also be utilized to obtain detailed shape of non-symmetric nano-
objects such as nanorods and nanocrystals.
Other applications include calibration for a variety of nanoscale characterization tools, particularly
nanoscale characterization instruments and artefacts, to ensure that they are applied in a consistent
way.
Case studies are provided in informative annexes, including variations of sample preparation, data
acquisition, alignment and reconstruction methods. It is noted that placing of alternative data
acquisition, alignment and reconstruction methods in annexes does not imply that a method is inferior
to the one described in the main body of the document. Conversely, such might be the subject of future
revisions of this document. However, the process, from sample preparation on a rod-shaped support to
extraction of measurands, has been tested in accordance with the steps described in this document and
tested on samples described in the annexes.
Figure 1 summarizes the procedure steps in this document. Normative aspects are highlighted in red.
Informative aspects are highlighted in blue and appear in annexes. Additional annexes not listed in this
figure are also included.
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ISO/TS 22292:2021(E)
Figure 1 — Procedure steps
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TECHNICAL SPECIFICATION ISO/TS 22292:2021(E)
Nanotechnologies — 3D image reconstruction of rod-
supported nano-objects using transmission electron
microscopy
1 Scope
This document provides guidance for sample preparation, data acquisition by transmission electron
microscopy, data processing, and three-dimensional image reconstruction to measure size and shape
parameters of nano-objects on rod-shaped supports. The method is applicable to samples dispersed on
or within an electron-transparent rod-shaped support.
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 99:2007, International vocabulary of metrology — Basic and general concepts and
associated terms (VIM)
ISO/TR 945-2:2011, Microstructure of cast irons — Part 2: Graphite classification by image analysis
ISO/TS 10797:2012, Nanotechnologies — Characterization of single-wall carbon nanotubes using
transmission electron microscopy
ISO 21363, Nanotechnologies — Measurements of particle size and shape distributions by transmission
electron microscopy
ISO/TS 24597:2011, Microbeam analysis — Scanning electron microscopy — Methods of evaluating image
sharpness
ISO 26824:2013, Particle characterization of particulate systems — Vocabulary
ISO/TS 80004-1:2015, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2:2015, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-6:2021, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
ISO/TS 80004-8:2020, Nanotechnologies — Vocabulary — Part 8: Nanomanufacturing processes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/IEC Guide 99:2007,
ISO/TR 945-2:2011, ISO/TS 10797:2012, ISO/TS 24597:2011, ISO 26824:2013, ISO/TS 80004-1:2015,
ISO/TS 80004-2:2015, ISO/TS 80004-6:2021, ISO/TS 80004-8:2020 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/
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ISO/TS 22292:2021(E)
3.1 Nanotechnology-related terms
3.1.1
nanoscale
length range approximately from 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
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.1.3
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each
other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3.1.4
nanoparticle
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as
nanofibre or nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — the abbreviation "NP" has been deleted".]
3.2 Instrument-related terms
3.2.1
scanning electron microscopy
SEM
method that examines and analyses the physical information (such as secondary electron, backscattered
electron, absorbed electron and X-ray radiation) obtained by generating electron beams and scanning
the surface of the sample in order to determine the structure, composition and topography of the
sample
[SOURCE: ISO/TS 80004-6:2021, 4.5.5]
3.2.2
scanning transmission electron microscopy
STEM
method that produces magnified images or diffraction patterns of the sample by a finely focused
electron beam, scanned over the surface and which passes through the sample and interacts with it
Note 1 to entry: Typically uses an electron beam with a diameter of less than 1 nm.
Note 2 to entry: Provides high-resolution imaging of the inner microstructure and the surface of a thin sample
(or small particles), as well as the possibility of chemical and structural characterization of micrometre and sub-
micrometre domains through evaluation of the X-ray spectra and the electron diffraction pattern.
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ISO/TS 22292:2021(E)
[SOURCE: ISO/TS 80004-6:2013, 4.5.7]
3.2.3
transmission electron microscope
TEM
method that produces magnified images or diffraction patterns of the sample by an electron beam
which passes through the sample and interacts with it
[SOURCE: ISO/TS 80004-6:2021, 4.5.6]
3.2.4
focused ion beam instrument
FIBI
instrument and method that allows to fabricate objects at nanoscale using a focused ion beam (FIB),
typically Gallium, and observe the fabricated area using an SEM column located in the same instrument
chamber
Note 1 to entry: For FIB lithography, refer to ISO/TS 80004-8:2020, 7.1.9.
Note 2 to entry: For FIB focused ion-beam deposition refer to ISO/TS 80004-8:2020, 7.2.12.
3.2.5
dual beam instrument
DBI
instrument combining the instruments used in the SEM (3.2.1) and FIB (3.2.4) methods
3.3 Measurement-related terms
3.3.1
Feret diameter
distance between two parallel tangents on opposite sides of the image of a particle
[SOURCE: ISO 26824:2013, 8.6]
3.3.2
maximum Feret diameter
maximum value of Feret diameter of an object, whatever its orientation
[SOURCE: ISO/TR 945-2:2011, 2.1]
3.3.3
minimum Feret diameter
minimum value of Feret diameter of an object whatever its orientation
[SOURCE: ISO 21363:2020, 3.4.5]
3.3.4
pixel
smallest non-divisible image-forming unit on a digitized TEM image
[SOURCE: ISO/TS 24597:2011, 3.1, modified — the abbreviation "TEM" has been changed to "SEM".]
3.3.5
measurand
quantity intended to be measured
[SOURCE: ISO/IEC Guide 99: 2007, 2.3]
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ISO/TS 22292:2021(E)
4 Sample considerations
4.1 General
Clause 4 discusses physical properties of the sample rod. For methods that can be applied to prepare
the sample rod, see Annexes A, E and F.
4.2 Choice of sample rod diameter
Sample rod diameter considerations apply to both TEM and scanning TEM (see Annex B) as follows:
a) The sample shall be rod-shaped with cross section shape no more that 50 % different from circular
cross section (1:1,5 ratio of axis length for elliptical sample rod cross section);
NOTE 1 Rectangular cross-section that does not exceed the 1:1,5 aspect ratio is acceptable.
b) The sample rod shall be made of low atomic number material such as carbon;
c) The sample rod diameter shall be less than one inelastic mean free path for the incident electron
energy in the TEM chosen. For example, at 300 keV incident electron energy a carbon rod with less
than 250 nm diameter shall be utilized;
d) Sample rod diameter that exceeds twice the inelastic mean free path shall be avoided to reduce the
[5]
effect of plural electron scattering in the sample rod and the associated loss of spatial resolution
[6][7]
;
e) The effect of geometrical broadening of the electron beam shall be kept at a small fraction of desired
resolution of the final 3D reconstructed volume. The geometrical broadening can be estimated
[1]
from instrument convergence semi-angle and collection semi-angle ;
f) To ensure adequate image resolution, the sample rod diameter shall not exceed two times the depth
[15]
of focus .
NOTE 2 Typical imaging conditions in conventional TEM mode at 300 keV electron energy allow for about
250 nm sample rod diameter.
NOTE 3 The choice of imaging parameters for TEM and STEM tomography, depth focus, and rod diameter is
described in detail in Reference [15].
5 Instrument factors
5.1 Microscope set up
5.1.1 General
This clause provides guidance on conventional parallel beam transmission electron microscope (TEM)
instrumentation set up for data acquisition. For scanning TEM (STEM) instrumentation set up, see
Annex B.
The critical set up parameters for TEM data acquisition are:
a) acceleration voltage (see 5.1.2);
b) convergence semi-angle (see 5.1.3);
c) collection angle (see 5.1.4);
d) microscope magnification (see 5.1.5);
e) number of pixels of the detector (see 5.1.6);
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ISO/TS 22292:2021(E)
f) image acquisition time (see 5.1.7).
5.1.2 Acceleration voltage
The acceleration voltage shall be selected as described in A.2.4 d) on sample thickness. Typically, 300 kV
or 200 kV should be used.
Using the maximum voltage available on a particular TEM is preferred. Using the maximum available
voltage means maximum allowable sample rod diameter and maximum depth of focus. For example,
using 300 kV rather than 200 kV allows for 250 nm diameter carbon rod rather than 200 nm diameter
rod at 200 kV.
5.1.3 Convergence semi-angle
The convergence semi-angle shall be selected so that the illumination at the sample plane is uniform
over the imaged area of the sample. Furthermore, the illumination uniformity has to be such that the
apparent sample focus does not visibly change over the observed area.
NOTE The illumination uniformity can be verified by performing an intensity profile across the image (e.g.
diagonally from corner to corner of the image). The uniformity of the defocus value is typically not a concern
and it can be verified by performing a fast Fourier transform (FFT) of sub areas of the image. For example, a
2048 × 2048 image can be divided into 256 × 256 pixels regions of interest. The image composed of absolute
value of FFT can be compared among the various regions of interest to ensure that the FFT does not vary too
much from region to region. An FFT that has same numbers of circular rings over an arbitrary sub-field of view is
an indication that illumination is sufficiently uniform.
5.1.4 Collection angle
In conventional TEM mode the collection angle is selected by the objective aperture size. The collection
angle is determined as the square root of the sum of squares of the convergence angle and acceptance
angle of the objective aperture. In practise the convergence angle in TEM mode is much smaller than
the acceptance angle of the objective aperture. Therefore, the collection angle is determined by the
objective aperture acceptance angle alone.
The main criteria for collection angle is the avoidance of diffraction contrast while maximizing the
[9][10]
contrast in the image. The contrast increases with decreasing collection angle, but at the same
time, the diffraction contrast increases with decreasing collection angle. The presence of diffraction
[9][10]
contrast could make the data unsuitable for 3D reconstruction. The number of counts collected by
the detector decreases with decreasing collection angle leading to increase in acquisition time.
5.1.5 Microscope magnification
Microscope magnification and detector pixel size are critical parameters to ensure correct sampling of
the object for 3D reconstruction. The higher the desired number sampling, the lower the effect of the
detector point spread function. At the same time high sampling, i.e. high microscope magnification and
small pixel size, decreases the field of view that can be acquired without region stitching by subsequent
offset acquisitions. High magnification also decreases the number of counts per pixel at a given beam
current density and acquisition time. Typically, the magnification is chosen so that the desired projected
[11]
image resolution is sampled by 5 pixels or more .
EXAMPLE If a desired projected image resolution is 1 nm, the pixel size is chosen to be about 0,2 nm. A 2 048
pixel × 2 048 pixel camera can then cover a 410 nm × 410 nm field of view that is adequate for most practical
purposes. For example, a camera with physical 5 µm × 5 µm pixel size and 0,2 nm pixel size at the sample plane
requires microscope magnification 25,000×. Typically, a somewhat higher magnification, for example, 30,000×,
can be chosen to slightly oversample the object.
5.1.6 Number of pixels of the detector
The highest number of pixels on the camera should be used to achieve optimum image resolution and
field of view. For example, it is advisable to use binning 1 for a 2 048 pixel × 2 048 pixel camera so that
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ISO/TS 22292:2021(E)
there are 2 048 pixel × 2 048 pixel in the images. Number of pixels, their size, the field of view
...
TECHNICAL ISO/TS
SPECIFICATION 22292
First edition
Nanotechnologies — 3D image
reconstruction of rod-supported nano-
objects using transmission electron
microscopy
Nanotechnologies — Reconstruction d'images 3D de nano-objets
soutenus par des tiges à l'aide de la microscopie électronique à
transmission
PROOF/ÉPREUVE
Reference number
ISO/TS 22292:2021(E)
©
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 22292:2021(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
---------------------- Page: 2 ----------------------
ISO/TS 22292:2021(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 Nanotechnology-related terms . 2
3.2 Instrument-related terms . 2
3.3 Measurement-related terms. 3
4 Sample considerations . 3
4.1 General . 3
4.2 Choice of sample rod diameter . 4
5 Instrument factors . 4
5.1 Microscope set up . 4
5.1.1 General. 4
5.1.2 Acceleration voltage . . 4
5.1.3 Convergence semiangle . 5
5.1.4 Collection angle . 5
5.1.5 Microscope magnification . 5
5.1.6 Number of pixels of the detector . 5
5.1.7 Image acquisition time . 6
5.2 Microscope calibration . 6
6 Image capture (data acquisition) . 6
6.1 General . 6
6.2 Procedure . 7
7 Data alignment and volume reconstruction .10
7.1 General .10
7.2 Procedure .10
8 Reconstructed volume evaluation and data analysis .10
8.1 General .10
8.2 Identification of nanoparticles and 3D volume .10
8.3 Thresholding for measurand extraction .11
9 Expression of results .13
9.1 Extracting parameters for each well-separated nano-object.13
9.2 Measurement uncertainty .14
9.3 Sources of errors .14
9.3.1 Error arising from sample that is not representative of the object of interest .14
9.3.2 Acquisition of 2D projected images .14
9.3.3 Instrument calibration . .15
9.3.4 Alignment of the projected images .16
9.3.5 Reconstruction of the 3D volume .16
9.3.6 Discrete representation of the objects (nanoparticles) in 3D .16
9.3.7 Interpretation of the obtained measurands .17
9.3.8 Limited number of observed objects (nanoparticles) .17
10 Test report .19
Annex A (informative) Sample preparation .21
Annex B (informative) STEM set up .28
Annex C (normative) Tomography reconstruction and visualization software packages .30
Annex D (informative) Microscope data collection parameters .32
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ISO/TS 22292:2021(E)
Annex E (informative) Case study: Metal nanoparticle, ILC results .33
Annex F (informative) Case study — Organic nanoparticles — Sample preparation and use.38
Annex G (informative) Particle distortions arising from FBP and SIRT reconstruction methods .39
Annex H (informative) Uncertainty budget .41
Bibliography .43
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ISO/TS 22292:2021(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 PROOF/ÉPREUVE v
---------------------- Page: 5 ----------------------
ISO/TS 22292:2021(E)
Introduction
Electron tomography, in transmission electron microscope (TEM), has impact on nanotechnology
and nanomaterial metrology like that of computer tomography in medicine. For example, industries
using nanotechnologies have requirements to verify materials, processes and products. Quantitative
measurement at the nanoscale, including three-dimensional (3D) image reconstruction of nano-objects
using TEM, responds to this need.
TEM, a two-dimensional (2D) imaging instrument, can provide 2D projection images of materials at
the nanoscale, in the length range from below 1 nm to above 100 nm. From multiple 2D TEM images
collected at suitable tilt increments, the 3D shape, size and volume parameters can be determined. This
document describes sample preparation, instrumentation setup, data acquisition and processing for
3D image reconstruction of nano-objects using TEM, from which dimensional parameter values can
be determined and interpreted. Variation in methodology for use with scanning transmission electron
microscopy (STEM) is included in an informative annex.
The method described herein is limited to samples dispersed on or within an electron-transparent
rod-shaped support. This method is particularly useful when the detailed shape of a limited number
of objects, such as nanoparticles, is sought. For example, when 2D measurements yield a non-uniform
distribution of objects, 3D image reconstruction can be used applied to study a small number of the
objects in more detail. A variant of sample preparation is described that allows 3D reconstruction to
be used in conjunction with 2D TEM analysis of a sample area of interest, such as an area containing
outliers.
Potential applications for 3D image reconstruction of nano-objects using TEM are broad and might
include validation of metrological artefacts, such as polystyrene latex nanoparticles, and site-
specific analysis of interfaces buried within devices, and measurement of individual objects such as
nanoparticles. The method might also be utilized to obtain detailed shape of non-symmetric nano-
objects such as nanorods and nanocrystals.
Other applications include calibration for a variety of nanoscale characterization tools, particularly
nanoscale characterization instruments and artefacts, to ensure that they are applied in a consistent way.
Case studies are provided in informative annexes, including variations of sample preparation, data
acquisition, alignment and reconstruction methods. It is noted that placing of alternative data
acquisition, alignment and reconstruction methods in annexes does not imply that a method is inferior
to the one described in the main body of the document. Conversely, such might be the subject of future
revisions of this document. However, the process, from sample preparation on a rod-shaped support to
extraction of measurands, has been tested in accordance with the steps described in this document and
tested on samples described in the annexes.
Figure 1 summarizes the procedure steps in this document. Normative aspects are highlighted in red.
Informative aspects are highlighted in blue and appear in annexes. Additional annexes not listed in this
figure are also included.
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ISO/TS 22292:2021(E)
Figure 1 — Procedure steps
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TECHNICAL SPECIFICATION ISO/TS 22292:2021(E)
Nanotechnologies — 3D image reconstruction of rod-
supported nano-objects using transmission electron
microscopy
1 Scope
This document provides guidance for sample preparation, data acquisition by transmission electron
microscopy, data processing, and three-dimensional image reconstruction to measure size and shape
parameters of nano-objects on rod-shaped supports. The method is applicable to samples dispersed on
or within an electron-transparent rod-shaped support.
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 21363, Nanotechnologies — Measurements of particle size and shape distributions by transmission
electron microscopy
ISO/IEC Guide 99:2007, International vocabulary of metrology — Basic and general concepts and
associated terms (VIM)
ISO/TR 945-2:2011, Microstructure of cast irons — Part 2: Graphite classification by image analysis
ISO/TS 10797:2012, Nanotechnologies — Characterization of single-wall carbon nanotubes using
transmission electron microscopy
ISO/TS 24597:2011, Microbeam analysis — Scanning electron microscopy — Methods of evaluating image
sharpness
ISO 26824:2013, Particle characterization of particulate systems — Vocabulary
ISO/TS 80004-1:2015, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2:2015, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-6:2021, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
ISO/TS 80004-8:2020, Nanotechnologies — Vocabulary — Part 8: Nanomanufacturing processes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/IEC Guide 99:2007,
ISO/TR 945-2:2011, ISO/TS 10797:2012, ISO/TS 24597:2011, ISO 26824:2013, ISO/TS 80004-1:2015,
ISO/TS 80004-2:2015, ISO/TS 80004-6:2021, ISO/TS 80004-8:2020 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/
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ISO/TS 22292:2021(E)
3.1 Nanotechnology-related terms
3.1.1
nanoscale
length range approximately from 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
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.1.3
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3.1.4
nanoparticle
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as
nanofibre or nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — the abbreviation "NP" has been deleted".]
3.2 Instrument-related terms
3.2.1
scanning electron microscopy
SEM
method that examines and analyses the physical information (such as secondary electron, backscattered
electron, absorbed electron and X-ray radiation) obtained by generating electron beams and scanning
the surface of the sample in order to determine the structure, composition and topography of the sample
[SOURCE: ISO/TS 80004-6:2021, 4.5.5]
3.2.2
scanning transmission electron microscopy
STEM
method that produces magnified images or diffraction patterns of the sample by a finely focused
electron beam, scanned over the surface and which passes through the sample and interacts with it
Note 1 to entry: Typically uses an electron beam with a diameter of less than 1 nm.
Note 2 to entry: Provides high-resolution imaging of the inner microstructure and the surface of a thin sample
(or small particles), as well as the possibility of chemical and structural characterization of micrometre and sub-
micrometre domains through evaluation of the X-ray spectra and the electron diffraction pattern.
[SOURCE: ISO/TS 80004-6:2013, 4.5.7]
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ISO/TS 22292:2021(E)
3.2.3
transmission electron microscope
TEM
method that produces magnified images or diffraction patterns of the sample by an electron beam
which passes through the sample and interacts with it
[SOURCE: ISO/TS 80004-6:2021, 4.5.6]
3.2.4
dual beam instrument and focused ion beam instrument
FIB
instrument and method that allows to fabricate objects at nanoscale using a focused ion beam
(FIB), typically Gallium, and observe the fabricated area using an SEM column located in the same
instrument chamber
Note 1 to entry: For FIB lithography, refer to ISO/TS 80004-8:2020, 7.1.9.
Note 2 to entry: For FIB focused ion-beam deposition refer to ISO/TS 80004-8:2020, 7.2.12.
3.3 Measurement-related terms
3.3.1
Feret diameter
distance between two parallel tangents on opposite sides of the image of a particle
[SOURCE: ISO 26824:2013, 8.6]
3.3.2
maximum Feret diameter
maximum value of Feret diameter of an object, whatever its orientation
[SOURCE: ISO/TR 945-2:2011, 2.1]
3.3.3
minimum Feret diameter
minimum value of Feret diameter of an object whatever its orientation
[SOURCE: ISO 21363:2020, 3.4.5]
3.3.4
pixel
smallest non-divisible image-forming unit on a digitized TEM image
[SOURCE: ISO/TS 24597:2011, 3.1, modified — the abbreviation "TEM" has been changed to "SEM".]
3.3.5
measurand
quantity intended to be measured
[SOURCE: ISO/IEC Guide 99: 2007, 2.3]
4 Sample considerations
4.1 General
Clause 4 discusses physical properties of the sample rod. For methods that can be applied to prepare
the sample rod, see Annexes A, E and F.
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ISO/TS 22292:2021(E)
4.2 Choice of sample rod diameter
Sample rod diameter considerations apply to both TEM and scanning TEM (see Annex B) as follows:
a) The sample shall be rod-shaped with cross section shape no more that 50 % different from circular
cross section (1:1.5 ratio of axis length for elliptical sample rod cross section);
NOTE 1 Rectangular cross-section that does not exceed the 1:1,5 aspect ratio is acceptable.
b) The sample rod shall be made of low atomic number material such as carbon;
c) The sample rod diameter shall be less than one inelastic mean free path for the incident electron
energy in the TEM chosen. For example, at 300 keV incident electron energy a carbon rod with less
than 250 nm diameter shall be utilized;
d) Sample rod diameter that exceeds twice the inelastic mean free path shall be avoided to reduce
the effect of plural electron scattering in the sample rod and the associated loss of spatial
[3][4][5]
resolution ;
e) The effect of geometrical broadening of the electron beam shall be kept at a small fraction of desired
resolution of the final 3D reconstructed volume. The geometrical broadening can be estimated
[6]
from instrument convergence semiangle and collection semiangle ;
f) To ensure adequate image resolution, the sample rod diameter shall not exceed two times the depth
[13]
of focus .
NOTE 2 Typical imaging conditions in conventional TEM mode at 300 keV electron energy allow for about
250 nm sample rod diameter.
NOTE 3 The choice of imaging parameters for TEM and STEM tomography, depth focus, and rod diameter is
described in detail in Reference [13].
5 Instrument factors
5.1 Microscope set up
5.1.1 General
This clause provides guidance on conventional parallel beam transmission electron microscope (TEM)
instrumentation set up for data acquisition. For scanning TEM (STEM) instrumentation set up, see
Annex B.
The critical set up parameters for TEM data acquisition are:
a) acceleration voltage (see 5.1.2);
b) convergence semi-angle (see 5.1.3);
c) collection angle (see 5.1.4);
d) microscope magnification (see 5.1.5);
e) number of pixels of the detector (see 5.1.6);
f) image acquisition time (see 5.1.7).
5.1.2 Acceleration voltage
The acceleration voltage shall be selected as described in A.2.4 d) on sample thickness. Typically, 300 kV
or 200 kV should be used.
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ISO/TS 22292:2021(E)
Using the maximum voltage available on a particular TEM is preferred. Using the maximum available
voltage means maximum allowable sample rod diameter and maximum depth of focus. For example,
using 300 kV rather than 200 kV allows for 250 nm diameter carbon rod rather than 200 nm diameter
rod at 200 kV.
5.1.3 Convergence semiangle
The convergence semi-angle shall be selected so that the illumination at the sample plane is uniform
over the imaged area of the sample. Furthermore, the illumination uniformity has to be such that the
apparent sample focus does not visibly change over the observed area.
NOTE The illumination uniformity can be verified by performing an intensity profile across the image (e.g.
diagonally from corner to corner of the image). The uniformity of the defocus value is typically not a concern
and it can be verified by performing a fast Fourier transform (FFT) of sub areas of the image. For example, a
2048 × 2048 image can be divided into 256 × 256 pixels regions of interest. The image composed of absolute
value of FFT can be compared among the various regions of interest to ensure that the FFT does not vary too
much from region to region. An FFT that has same numbers of circular rings over an arbitrary sub-field of view is
an indication that illumination is sufficiently uniform.
5.1.4 Collection angle
In conventional TEM mode the collection angle is selected by the objective aperture size. The collection
angle is determined as the square root of the sum of squares of the convergence angle and acceptance
angle of the objective aperture. In practise the convergence angle in TEM mode is much smaller than
the acceptance angle of the objective aperture. Therefore, the collection angle is determined by the
objective aperture acceptance angle alone.
The main criteria for collection angle is the avoidance of diffraction contrast while maximizing the
[7][8]
contrast in the image. The contrast increases with decreasing collection angle, but at the same
time, the diffraction contrast increases with decreasing collection angle. The presence of diffraction
[7][8]
contrast could make the data unsuitable for 3D reconstruction. The number of counts collected by
the detector decreases with decreasing collection angle leading to increase in acquisition time.
5.1.5 Microscope magnification
Microscope magnification and detector pixel size are critical parameters to ensure correct sampling of
the object for 3D reconstruction. The higher the desired number sampling, the lower the effect of the
detector point spread function. At the same time high sampling, i.e. high microscope magnification and
small pixel size, decreases the field of view that can be acquired without region stitching by subsequent
offset acquisitions. High magnification also decreases the number of counts per pixel at a given beam
current density and acquisition time. Typically, the magnification is chosen so that the desired projected
[9]
image resolution is sampled by 5 pixels or more .
EXAMPLE If a desired projected image resolution is 1 nm, the pixel size is chosen to be about 0,2 nm. A 2 048
pixel × 2 048 pixel camera can then cover a 410 nm × 410 nm field of view that is adequate for most practical
purposes. For example, a camera with physical 5 µm × 5 µm pixel size and 0,2 nm pixel size at the sample plane
requires microscope magnification 25,000×. Typically, a somewhat higher magnification, for example, 30,000×,
can be chosen to slightly oversample the object.
5.1.6 Number of pixels of the detector
The highest number of pixels on the camera should be used to achieve optimum image resolution and
field of view. For example, it is advisable to use binning 1 for a 2 048 pixel × 2 014 pixel camera so that
there are 2 048 pixel × 2 048 pixel in the images. Number of pixels, their size, the field of view and the
image resolution are related. See 5.1.5 for an example of magnification estimate.
If the point spread function of the camera is poor, it can be necessary to combine camera pixel
...
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