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
ISO TS 22292:2021 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.
General Information
Standards Content (Sample)
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
ISO/TS 22292:2021(E)
© ISO 2021
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ii © ISO 2021 – All rights reserved
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
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
iv © ISO 2021 – All rights reserved
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
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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/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.
vi © ISO 2021 – All rights reserved
ISO/TS 22292:2021(E)
Figure 1 — Procedure steps
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/
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.
2 © ISO 2021 – All rights reserved
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]
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);
4 © ISO 2021 – All rights reserved
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
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 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 pixels (bin
the pixels). In such case, the necessary magnification should be estimated for the combined pixel size.
Combining (binning) the camera pixels results in a corresponding decrease of the field of view as
2 2
compared to binning 1 images. For example, a 1 000 nm × 1 000 nm field of view with 0,5 nm per pixel
obtainable with 1 000 pixel × 1 000 pixel camera would be reduced to 500 nm × 500 nm if the camera
is binned by 2 while maintaining sampling 0,5 nm per pixel.
5.1.7 Image acquisition time
Image acquisition time shall be chosen such that enough signal to noise ratio is obtained in the projected
images to allow for projected image alignment and for reconstruction. The acquisition time needs to be
chosen such that the sample drift is less than the pixel size at the sample plane. If the number of counts
per pixel is too low at the drift limited acquisition time, either the microscope beam current can be
increased or multiple images at each tilt can be acquired. Typically, about 100 electrons per pixel of the
[12]
detector are sufficient for alignment and reconstruction .
5.2 Microscope calibration
To ensure correct microscope calibration, the microscope shall be calibrated under the same imaging
conditions as used for the tomography data acquisition. The calibration shall be performed either
immediately before or immediately after the data acquisition. Identical conditions in this case refers
to using the same lens currents, the objective lens and the intermediate and projector lens system
currents, as used for the tomography data acquisition. The calibration of the TEM instrument shall be
performed as described in ISO 21363.
NOTE Calibrating before or after collection of projected images is equivalent as long as the lens settings are
not changed between calibration and collection of projected images.
For precise size measurement by using TEM, the same condition of TEM lens and specimen height
between a measurement specimen and a calibration specimen is important. An internal reference
length for TEM instruments must be calibrated using calibration standards. All size measurements
should be done with the same lens and specimen height conditions of calibration. Focusing condition
of the lens can affect the size measurement. A Scherzer defocus condition is generally used. The zero
defocus is defined using Fresnel fringe at first, then focusing goes to Scherzer defocus. Specimen height
should be at the eucentric position. It is important to obtain images at eucentric height and the same
defocus condition at magnifications as used explicitly for the instrument’s calibration.
For most microscopes the selection of identical conditions is achieved by selecting the same nominal
magnification and same focus value of the objective lens current as used for the tilt series acquisition.
The sample focusing shall be achieved by utilizing the mechanical Z height of the stage. Focusing by
changing the imaging lens in a TEM or probe forming lens in STEM should be limited to a range no more
than ±1 µm to prevent magnification change or image rotation. The lens currents must be same for
microscope calibration as for data collection. Calibration should be performed at the eucentric height
and at the limits of the range of lens current focusing. For calibration a suitable calibration sample with
[13]
known dimensions shall be used. An example of such sample is available from several suppliers .
The pixel size obtained using the above calibration procedure shall be used to calibrate the
reconstructed 3D volume.
See Annex D for recommended microscope and data collection parameters to record. See Annex H for
an example of uncertainty budget.
6 © ISO 2021 – All rights reserved
ISO/TS 22292:2021(E)
6 Image capture (data acquisition)
6.1 General
This clause covers image capture to obtain projected images in 2D.
NOTE ISO 21363 provides extensive information on collection of projected images, that is also applicable to
collection of projected images for electron tomography reconstruction.
6.2 Procedure
The procedure is as follows.
a) The data shall be collected in a transmission electron microscope (TEM) in conventional TEM
mode. Alternatively, a scanning TEM (STEM) mode can be used, see Annex B. The TEM shall be
operated at incident energy between 100 kV and 400 kV. The microscope can have thermal electron
source (LaB ), field assisted thermal electron source (Schottky electron source) or a field emission
electron source. The point resolution of the microscope shall be 0,3 nm or less. The information
limit of the microscope shall be 0,2 nm or less.
b) The microscope shall allow sample to be tilted over −90° to +90° around at least one axis
perpendicular to the incident beam. The microscope vacuum pressure near the sample chamber
−7
shall be 1 × 10 torr or less.
c) Data acquisition shall be bright field TEM. Other methods such as scanning TEM (STEM) bright
field (BF-STEM), annular dark field (ADF-STEM), and high angle annular dark field HAADF-STEM
are acceptable if they provide contrast that is predominantly monotonic with sample thickness
regardless of tilt angle. See Annexes B and C for examples.
d) The sample shall be mounted as depicted in Figure 2 with the sample long axis perpendicular to the
electron beam and parallel with the tilt axis of the sample stage.
Key
1 sample stage clamp 4 carbon rod with nanoparticles
2 tilt axis = sample holder axis A SEM image of sample tip with sample rod
3 sample tip with carbon rod
Figure 2 — Sample rod at the tip of a TEM holder
ISO/TS 22292:2021(E)
e) The electron beam convergence angle and collection angle, as determined by objective aperture
shall be selected such that the contribution of diffraction contrast is small. Typically, ~100 mrad
collection angle is sufficiently large at 300 kV to avoid artefacts arising from diffraction contrast.
Typically, the smallest effect of diffraction contrast can be achieved by using no objective aperture.
The image contrast of nanoparticles shall be maximized while keeping the diffraction contrast to a
minimum.
f) Eucentric height shall be adjusted by minimizing the sample lateral movement when the sample
is tilted over the entire −90° to +90° tilt. Sample focusing using objective lens excitation shall be
limited to ±1 µm range to prevent change in magnification and sample rotation. Sample focusing
exceeding the ±1 µm range shall be achieved by adjusting mechanical Z height of the sample stage.
g) The lateral (X, Y) positioning of the sample on the camera within ±1 µm range shall be done using
deflector coils. Outside the ±1 µm range mechanical movement of the sample stage shall be used.
h) The microscope magnification shall be set up to acquire about 5 pixels per desired resolution in the
reconstructed volume. For example (0,2 × 0,2) nm image pixel size shall be used for 1 nm intended
resolution of the 3D reconstructed volume.
i) The data can be collected manually, or an automated data acquisition package can be used.
j) The bit depth of the saved images shall be 16 bit or more. The bit depth should be chosen such that
the digitization of the data does not lead to loss of dynamic range or insufficient sampling of image
greyscale levels. The camera output can provide more than 16-bit dynamic range. The data should
be saved with at least 16-bit dynamic range.
k) The field of view should include sufficient number of nanoparticles, as shown in Figure 3 to ensure
a maximum number of well-separated particles. The number achievable will vary from sample to
sample and also will vary for deposited vs embedded particles. To obtain sufficient total number of
nanoparticles, multiple fields of view along the rod axis can be collected. For example, two fields of
view, about 500 nm each, can be obtained with a 1000 nm long sample rod.
NOTE 2 ISO 21363 provides guidance on the number of nanoparticles needed for statistical analysis.
8 © ISO 2021 – All rights reserved
ISO/TS 22292:2021(E)
Key
1 carbon rod
2 vacuum
3 clustered nanoparticles not suitable for analysis
4 well separated nanoparticles suitable for analysis
Figure 3 — Experimental projected TEM image showing 20 nanoparticles visible within the
field of view
l) Projected images shall be acquired in 3° or less increments from −90° to +90° stage tilt. Here 0°
corresponds to tilt value where the sample rod is not tilted and corresponds to sample exchange
position in most microscopes.
Suggestion for file naming: file names should indicate the nominal tilt at which they were acquired.
For example, the positive tilt angles can be marked by underscore “XXXXXXXX_zzz.tiff” while the
negative tilt angles can be marked by a hyphen “-” such as “XXXXXXXX_-zzz.tiff”. Here “XXXXXXXX”
is arbitrary name up to 8 characters long, “zzz” is image number. The projected data can be saved
as uncompressed tiff or in Gatan Digital Micrograph format. When Gatan Digital Micrograph
file format is used, the file name extension can reflect that by using a “.dm3” or “.dm4” filename
extension.
NOTE 3 Stack data formats, such as MRC and various binary formats can be used.
Use of data formats with publicly available documentation of the format are recommended.
m) The images acquisition time shall be selected such that about 100 electrons per pixel are collected
in each image of the tilt series.
n) The procedure for individual image acquisition described in ISO 21363 shall be used.
ISO/TS 22292:2021(E)
7 Data alignment and volume reconstruction
7.1 General
This clause covers steps to obtain a 3D reconstruction from collected 2D projected images. One needs
to use a 3D reconstruction algorithm implemented in software. Filtered back projection (FPB) is
described here.
NOTE Alternative reconstruction algorithms, such as simultaneous iterative reconstruction technique
(SIRT), are described in Annex G.
7.2 Procedure
The procedure is as follows:
a) A suitable tomography reconstruction software package shall be utilized.
NOTE 1 See Annex C.
b) The images within the tilt series shall be aligned to eliminate lateral RMS displacement among
images in the tilt series to less than 1 pixel over the entire tilt range −90° to +90°. The alignment
can be performed manually or using an automated procedure. The nanoparticles themselves can
be utilized as fiducial markers for image alignment.
c) Using the aligned image tilt series, the 3D volume shall be reconstructed using standard filtered
back projection (FBP) algorithm. A linear ramp filter shall be used for the back-projection step. The
reconstruction shall be performed for example using a suitable package as listed in Annex C.
d) The reconstructed volume shall be saved as a cube of raw data with X, Y, Z axis and reconstructed
intensity in all three dimensions. For example, a reconstructed volume shall be saved as a volume
of 500 volume pixels × 400 volume pixels × 300 volume pixels, 16 bit per pixel.
NOTE 2 16 bit = 2 byte / pixel results in 0,25 GB uncompressed file for a 500 volume × 500 volume × 500
volume.
NOTE 3 The use of bit depth higher than 16 bit is acceptable.
8 Reconstructed volume evaluation and data analysis
8.1 General
Clause 8 describes how to extract data from the reconstructed 3D data cube.
The particles selected for analysis shall not be clustered and they shall be visibly separated from
adjacent particles.
Reliable separation of particles implies particle centre separation of about 2,5× their radius in case of
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spherical particles . When the particles are non-spherical, the distance between particles larger than
the 2,5× radius of the sphere fully enclosing the particles is recommended.
8.2 Identification of nanoparticles and 3D volume
The steps for identification of nanoparticles in the reconstructed 3D volume are as follows.
a) The first step toward extracting particle characteristics is the identification of the particles and
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their boundaries. This is achieved by suitable software. All processing shall be performed on the
3D volume. This document does not make use of 2D projections of the 3D volume.
NOTE 1 Figure 4 shows the workflow diagram for the steps outlined in 8.2. An open source TomoMi
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software can be used to perform the nanoparticle extraction .
10 © ISO 2021 – All rights reserved
ISO/TS 22292:2021(E)
b) The reconstructed 3D data volume shall be loaded in a suitable software. A 3D median filter with
3 pixel × 3 pixel × 3 pixel is applied to the reconstructed volume.
c) A small cube-shaped box of the volume is selected around each nanoparticle selected for analysis.
The nanoparticle selection is performed manually. The box around the nanoparticle should be about
40 % larger than the nanoparticle itself. Multiple nanoparticles are selected for a reconstructed 3D
data set. Each box with nanoparticle is assigned an identifier number (ID#).
A cube shaped volume about 50 pixel × 50 pixel × 50 pixel should be selected around a spherical
nanoparticle with a 30-pixel diameter.
d) The software shall perform local thresholding limited to the box with each selected nanoparticle.
The thresholding is defined in 8.3. The nanoparticle boundary is thus identified by its boundary.
e) The software shall identify the minimum and maximum Feret diameter. The minimum Feret
diameter F is obtained by finding the minimum distance connecting the nanoparticle internal
min
boundary in 3D. The maximum Feret diameter F is obtained by finding the maximum internal
max
distance connecting the nanoparticle boundary in 3D.
NOTE 2 The F and F obtained in 3D in general do not agree with F and F obtained from 2D
min max min max
projections, as described in ISO 21363.
f) The software shall calculate the Feret diameter ratio as F = F / F ;
rat max min
g) The software shall extract the nanoparticle volume V by counting the voxels enclosed inside
nanoparticle internal boundary;
h) The software shall export a CSV file with the following columns:
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ID# F F F Volume .
min max rat
8.3 Thresholding for measurand extraction
The method for local thresholding for detection of nanoparticle boundaries is as follows:
a) A volume containing an individual nanoparticle shall be selected, see 8.2 c).
b) A three-dimensional medial filter with 3 pixel diameter shall be applied to the volume containing
an individual nanoparticle. All voxels, including those of t
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