ISO 20263:2024

Microbeam analysis - Analytical electron microscopy - Method for the determination of interface position in the cross-sectional image of the layered materials

This document specifies a procedure for the determination of the averaged interface position between two different layered materials recorded in the cross-sectional image of the multi-layered material. This document does not apply for determining the simulated interface of the multi-layered materials expected through the multi-slice simulation (MSS) method. This document is applicable to the cross-sectional images of multi-layered materials recorded using a transmission electron microscope (TEM) or a scanning transmission electron microscope (STEM) and cross-sectional elemental mapping images using an energy dispersive X-ray spectrometer (EDS) or an electron energy loss spectrometer (EELS). This document is also applicable to digitized images recorded on an image sensor built into a digital camera, a digital memory set in the PC or an imaging plate, where the digitalized image is obtained by converting an analogue image recorded on photographic film using an image scanner.

Analyse par microfaisceaux — Microscopie électronique analytique — Méthode de détermination de la position d'interface dans l'image de coupe transversale des matériaux en couches

General Information

• Status: Published
• Publication Date: 05-Nov-2024
• ICS: 37.020 - Optical equipment
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: ISO/TC 202/SC 3 - Analytical electron microscopy
• Drafting Committee: ISO/TC 202/SC 3 - Analytical electron microscopy
• Current Stage: 6060 - International Standard published
• Start Date: 06-Nov-2024
• Due Date: 28-Feb-2025
• Completion Date: 06-Nov-2024

Relations

• Revises: ISO 20263:2017 - Microbeam analysis - Analytical electron microscopy - Method for the determination of interface position in the cross-sectional image of the layered materials
• Effective Date: 02-Sep-2023

Overview - ISO 20263:2024 (Microbeam analysis, Analytical electron microscopy)

ISO 20263:2024 specifies a standardized procedure to determine the averaged interface position between two layered materials from cross‑sectional images. Applicable to images recorded by TEM or STEM and to cross‑sectional elemental maps from EDS or EELS, the method works with native digital images or images digitized from photographic film. It is intended for real experimental images (layer thicknesses from a few nanometres to a few micrometres) and explicitly does not apply to simulated interface positions generated by the multi‑slice simulation (MSS) method.

Key technical topics and requirements

• Scope and applicability

  • Cross‑sectional imaging of multi‑layered materials using TEM/STEM, and elemental mapping (EDS/EELS).
  • Digitized images from CCD/CMOS cameras, imaging plates, or scanned photographic film.

• Specimen preparation

  • Guidance on preparing cross‑sectional specimens (FIB thinning, ion‑milling, ultra‑microtomy are referenced concepts).
  • Requirements to ensure representative, artefact‑minimized images for interface analysis.

• Image analysis workflow

  • ROI (Region of Interest) selection and image classification to isolate the interface area.
  • Acquisition of an averaged intensity profile across the ROI.
  • Moving‑average processing to reduce noise and intensity fluctuations.
  • Differential processing (difference between adjacent profile points) to locate the interface transition.
  • Determination of the averaged interface position from processed profiles.

• Uncertainty and calibration

  • Procedures to estimate measurement uncertainty, including contributions from each processing step.
  • Pixel‑size calibration and scale unit considerations (see Annex C).

• Supporting material

  • Examples of processing real TEM/STEM images (Annex A) and principal applications of the method (Annex B).

Practical applications and users

This ISO standard is directly useful for:

• Materials scientists and electron microscopists performing layer thickness and interface characterization.
• Semiconductor manufacturers, coating and thin‑film developers, sensor and optical component producers for quality control, process verification and failure analysis.
• Laboratories producing cross‑sectional elemental maps (EDS/EELS) that require objective, repeatable interface position measurement.

Key practical benefits:

• Provides a reproducible, documented method to measure interface position and layer thickness from real cross‑sectional images.
• Improves comparability of results across labs and instruments by standardizing ROI selection, filtering and differential analysis.

Related standards

• ISO 29301 (image and detector terminology) - cited for camera and imaging plate definitions.
• ISO 15932 (TEM image processing terms) - cited for FFT/IFFT terminology.

Keywords: ISO 20263:2024, microbeam analysis, analytical electron microscopy, TEM, STEM, interface position, cross‑sectional imaging, EDS, EELS, ROI, moving average, differential processing.

Standards Content (Sample)

International
Standard
ISO 20263
Second edition
Microbeam analysis — Analytical
2024-11
electron microscopy — Method
for the determination of interface
position in the cross-sectional
image of the layered materials
Analyse par microfaisceaux — Microscopie électronique
analytique — Méthode de détermination de la position
d'interface dans l'image de coupe transversale des matériaux en
couches
Reference number
© ISO 2024
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
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or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: [phone]
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and abbreviated terms . 1
3.1 Terms and definitions .1
3.2 Abbreviated terms .4
4 Specimen preparation for cross-sectional imaging . 4
4.1 General .4
4.2 Requirements for the cross-sectional specimen .5
5 Determination of an interface position . 6
5.1 General .6
5.2 Preliminary considerations .6
5.2.1 Ideal model of an interface .6
5.2.2 More realistic model of an interface .6
5.2.3 Dealing with intensity fluctuations in the image .7
6 Detailed procedure for determining the position of the interface . 8
6.1 General .8
6.2 Preparing cross-sectional TEM/STEM image .10
6.2.1 Preparing digitized Image .10
6.2.2 Displaying the digitized image .11
6.3 Setting the ROI .11
6.3.1 General .11
6.3.2 Classification of image.11
6.3.3 Procedure of setting the ROI . 12
6.4 Acquisition of the averaged intensity profile .16
6.5 Moving-averaged processing .19
6.6 Differential processing . 20
6.7 Determination of interface position .21
7 Uncertainty .21
7.1 Uncertainty accumulating from each step of the procedure.21
7.2 Uncertainty of measurement result on image analysis . 22
Annex A (informative) Examples of processing the real TEM/STEM images for three image
types .24
Annex B (informative) Two main applications of the method .38
Annex C (informative) Calibration of scale unit: Pixel size calibration .45
Bibliography . 47

iii
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
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
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This document was prepared by Technical Committee ISO/TC 202, Microbeam analysis, Subcommittee SC 3,
Analytical electron microscopy.
This second edition cancels and replaces the first edition (ISO 20263:2017), which has been technically
revised.
The main changes are as follows:
— introduction has been revised;
— terms and sources in Clause 3 have been revised;
— subclauses 5.2.2, 5.2.3, 6.1, 6.2.1, 6.7, 7.1, 7.2, A.4.1, B.2.3 and B.2.4 have been revised;
— figures have been revised.
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.

iv
Introduction
Multi-layered materials are widely used in the production of semiconductor devices, various kinds of sensors,
coating films for optical element, new functional materials, etc. One of the factors used to determine the
characteristics of multi-layered materials is the layer thickness, for evaluation of products and verification
of the production process. In practice, measuring the total thickness and/or the thickness of each layer
and checking the uniformity of thickness and/or flatness of the interface are often done using recorded
images of the materials. Evaluations can be made from the cross-sectional TEM/STEM images by accurately
determining the averaged interface position between two different layered materials.
In relation to the determination of the interface position in the HR atomic imaging, analysis by the multi-
slice simulation (MSS) method can be applied for the target measurement, if the atomic structural models
can be constructed. However, in real materials, there are a lot of cases when they cannot, such as:
— the interface between amorphous layers, or layers of amorphous substance and crystal;
— the interface recorded in low-resolution image in which the atomic columns cannot be identified:
— for very thick single-layered material;
— for thick multi-layered material.
This document gives the method to determine the averaged interface position using a differential processing
of the accumulated intensity profile obtained from the ROI set in the cross-sectional TEM/STEM image of
the multi-layered material. The thickness of the layer that can be applied ranges from a few nanometers to a
few micrometers. Thus, this document is not intended for the determination of the simulated position of the
layer interface analysed by the MSS method.

v
International Standard ISO 20263:2024(en)
Microbeam analysis — Analytical electron microscopy —
Method for the determination of interface position in the
cross-sectional image of the layered materials
1 Scope
This document specifies a procedure for the determination of the averaged interface position between
two different layered materials recorded in the cross-sectional image of the multi-layered material. This
document does not apply for determining the simulated interface of the multi-layered materials expected
through the multi-slice simulation (MSS) method.
This document is applicable to the cross-sectional images of multi-layered materials recorded using a
transmission electron microscope (TEM) or a scanning transmission electron microscope (STEM) and cross-
sectional elemental mapping images using an energy dispersive X-ray spectrometer (EDS) or an electron
energy loss spectrometer (EELS). This document is also applicable to digitized images recorded on an image
sensor built into a digital camera, a digital memory set in the PC or an imaging plate, where the digitalized
image is obtained by converting an analogue image recorded on photographic film using an image scanner.
2 Normative references
There are no normative references in this document.
3 Terms, definitions and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1.1
cross-sectional image
TEM/STEM image of the multi-layered materials along a plane perpendicular to the stacking direction
3.1.2
differential processing
calculation of the difference between the values of adjacent pixel data in the intensity profile
3.1.3
digital camera
device that detects the image using a chip-arrayed image sensor (3.1.12), such as a charge-coupled device (CCD)
or complementary metal-oxide semiconductor (CMOS), which converts a visual image to an electric signal
[SOURCE: ISO 29301:2023, 3.7]
3.1.4
dot pitch
distance between adjacent pixels in pixel-based devices

3.1.5
elemental mapping image
image produced by the selected signal which is attributed to a particular element, from the EDS/EELS
spectrum
3.1.6
FIB thinning
site-specific thinning technique by using focused ion beam to thin a particular region of the specimen
3.1.7
filtering mask
mask to define the cut–off frequency in the reciprocal space
3.1.8
fast Fourier transformation
FFT
efficient algorithm to compute the discrete Fourier transform
[SOURCE: ISO 15932:2013, 5.4.1.1]
3.1.9
inverse fast Fourier transformation
IFFT
efficient algorithm to compute the inverse of the discrete Fourier transform
[SOURCE: ISO 15932:2013, 5.4.1.2]
3.1.10
image file format
format for saving an image as a computer file according to a predetermined rule
3.1.11
image scanner
device that converts an analogue image into a digitized image with the desired resolution
EXAMPLE There are mainly two different types of scanners: flatbed type and drum type.
[SOURCE: ISO 29301:2023, 3.17, modified — example has been added.]
3.1.12
image sensor
device, such as a charge-coupled device (CCD) array or complementary metal-oxide semiconductor (CMOS)
sensor, which converts visual image information to an electric signal, built-in digital camera (3.1.3) or other
imaging devices
3.1.13
intensity profile
signal intensity distribution along a line specified in the image
3.1.14
interface
boundary surface at the junction of two different layers of materials recorded in the cross-sectional image
(3.1.1) of the multi-layered materials
3.1.15
ion-milling
thinning technique of sputtering the specimen with an inert gas
[SOURCE: ISO 15932:2013, 4.1]
3.1.16
imaging plate
IP
electron image detector consisting of a film with a thin active layer embedded with specifically designed
phosphors
[SOURCE: ISO 29301:2023, 3.16]
3.1.17
low pass filter
filter to pass signals of frequencies lower than the cut-off frequency
3.1.18
moving average
calculation for averaging the selected dataset which is picked out from equal number of dataset on either
side of a central data
3.1.19
multi-slice simulation
MSS
computer simulation method of high-resolution TEM images, which treats electrons as incoming waves and
treats the interactions with matter as occurring on multiple successive single slices of the specimen
[SOURCE: ISO 15932:2013, 6.4.1, modified — “algorithm for the simulation” has been replaced by “computer
simulation method”.]
3.1.20
multi-layered material
laminated material which is fabricated by alternating layers of at least two kinds of materials on the
substrate
3.1.21
photographic film
sheet or a roll of thin plastic coated by photographic emulsion for recording an image
[SOURCE: ISO 29301:2023, 3.23]
3.1.22
pixel
smallest unit element that makes up the digital image
3.1.23
region of interest
ROI
sub-dataset picked out from the entire dataset for a specific purpose
3.1.24
ultra-microtome
thin sectioning instrument to prepare the specimen thin enough for TEM observation by using glass or
diamond knives
3.1.25
zone axis
crystallographic direction, designated [u v w], defined by the intersection of a number of crystal planes
(h ,k ,l …….h ,k ,l ) such that all of the planes satisfy the so-called Weiss zone law; hu + kv + lw = 0
1 1 1 i i i
[SOURCE: ISO 29301:2023, 3.36]

3.2 Abbreviated terms
AEM Analytical electron microscope/microscopy
CCD Charge coupled device
CMOS Complementary metal oxide semiconductor
CRT Cathode ray tube
EDS/EDX Energy dispersive X-ray spectrometer/spectroscopy
Although "EDX" and "EDS" are interchangeable, this document uses "EDS".
EELS Electron energy loss spectrometer/spectroscopy
FFT Fast Fourier transformation
FIB Focused ion beam
HREM High-resolution transmission electron microscope/microscopy
IFFT Inverse fast Fourier transformation
MSS Multi-slice simulation
ROI Region of interest
STEM Scanning transmission electron microscope/microscopy
TEM Transmission electron microscope/microscopy
4 Specimen preparation for cross-sectional imaging
4.1 General
To determine the interface potion of the multi-layered materials stacked on a substrate, the specimen
observed by TEM/STEM shall be cut into a cross-sectional thin slice perpendicular to the stacking direction
of the multi-layered thin film, using the techniques of ultra-microtome, ion-milling, FIB thinning, chemical
etching and so on. In order to keep the thickness information of the layered materials with an accuracy of
1 %, the cut out angle α [shown in Figure 1, a)] shall be 90° ± 6°.

a) Multi-layered specimen b) TEM/STEM observation
c) Cross-sectional imaging
Key
1 substrate
2 multi-layered materials
3 direction of electron beam
4 thin slice of the specimen
5 cross-sectional TEM/STEM image
6 arrows indicate interface positions
Figure 1 — Specimen preparation for cross-sectional imaging
4.2 Requirements for the cross-sectional specimen
Ensure that the specimen:
— provides a good and sharp contrast and clear and straight interface for the multi-layered materials in the
TEM/STEM/elemental mapping image,
— can be cleaned to remove contamination without causing mechanical/electrical damage or distortion,
— has a smooth surface on both sides and identical thickness, at least within the area used for the
determination process of interface position,
— is aligned to a low-index zone axis along the electron optical axis, if the specimen region is a single
crystal.
5 Determination of an interface position
5.1 General
It is important to determine the position of an interface in a layered material from its cross-sectional
TEM/STEM/elemental mapping image, objectively and uniquely. In Clause 6, the main scheme for the
determination of the interface position, as prescribed by this document, is explained.
5.2 Preliminary considerations
5.2.1 Ideal model of an interface
Ideally, the interface between two kinds of materials, M and M , show straight edge [see Figure 2 a)]. In this
1 2
case, it is easy to find the interface positions (S and S ) uniquely from the intensity profile [see Figure 2 b)]
1 2
along a line (L) perpendicular to the interface.
a) Ideal interface (S and S) model between two kinds of layers, M and M
1 2 1 2
b) Intensity profile along an arrow line, L, in a), perpendicular to the interfaces (S and S )
1 2
Figure 2 — Ideal interface model
5.2.2 More realistic model of an interface
In general, the interface will not be in a straight line. It typically appears as a region with a gradated intensity
distribution between layers M and M [see Figure 3 a)]. In this case, it is not easy to find the accurate
1 2
interface position from its intensity profile which is normally s-shaped [see Figure 3 b)]. This document
defines the interface position at the steepest tilt angle in the slope. Differential processing of the intensity
profile is the most suitable method to determine the interface position as defined above. Figure 3 c) shows
the differential curve of the intensity profile on in Figure 3 b). Pixel positions on the x-axis corresponding to

the minimal value and maximal value in the curve show the interface positions (S and S ) on both sides of
1 2
the layer M .
a) Realistic interface (S and S) model between two kinds of layers, M and M
1 2 1 2
b) Intensity profile along an arrow line, L, in a), perpendicular to the interfaces (S and S )
1 2
c) Differential curve of intensity profile
NOTE Positions of minimal and maximal value correspond to the interfaces (S and S ) defined in this document.
1 2
Figure 3 — Realistic interface model
5.2.3 Dealing with intensity fluctuations in the image
Unlike models described in 5.2.1 and 5.2.2, the actual cross-sectional TEM/STEM/elemental mapping image
has a domain-like intensity fluctuation, background noise and sometimes (in the high-resolution images)

periodic modulation of the intensity due to atomic column structures. Because of this non-uniformity in
the intensity in the image, follow the steps a) to g) sequentially for obtaining the desired smooth intensity
profile with a plateau and well-defined slope.
NOTE 1 Details of the procedure are described in Clause 6.
a) Prepare the cross-sectional TEM/STEM/elemental mapping digital image.
b) Set the direction of the interface parallel to the y-axis of the monitor screen.
c) Set the ROI area in the image.
d) Obtain the averaged intensity profile by adding up and averaging the line profiles in the direction
perpendicular to the interface (parallel to the x-axis of the monitor screen) over the entire interface
direction (parallel to the y-axis of the monitor screen) in the ROI.
e) Apply “moving-average” processing to the averaged intensity profile produced by the previous step, d).
This will remove small noise from the boundary region contributing to the slope of the interface.
f) Apply differential processing to the moving-averaged intensity profile obtained in e).
g) Determine the interface position as the pixel coordinate on the x-axis which either corresponds to the
maximal or minimal value in the differential curve.
6 Detailed procedure for determining the position of the interface
6.1 General
As described in the previous clause, the interface position can be determined through the differential
processing of the intensity profile in the ROI.
In the differential processing, a noise component existing in the intensity profile becomes an obstacle to find
the correct interface position. Therefore, it is necessary to remove the noise components in advance through
the average processing and the moving-averaged processing.
Also, in an atomic resolution image with an atomic column arrangement along the interface, the oscillated
component depends on the atomic column which cannot be eliminated even in the averaged intensity profile.
This is an obstacle to the extraction of the correct interface position by differential processing. Therefore, for
such an image, pre-processing for obscuring the atomic column structure is essential through the processing
of FFT/low pass filtering/IFFT.
Figure 4 shows a flow chart of the interface position determination procedure described in 5.2.3. In Clause 6
details of each procedure will be described.

Figure 4 — Flow chart of interface position determination procedure

6.2 Preparing cross-sectional TEM/STEM image
6.2.1 Preparing digitized Image
It is necessary to prepare a digitized cross-sectional image to determine the interface position. The bit depth
of digitization of the image shall be 8 bits or more. There are four ways of digitizing the image corresponding
to each image detection system shown in Table 1.
Table 1 — Comparison table for image detection
Image detection
Apparatus for digitization Pixel size
system
Photographic film Flatbed image scanner Determined by dot pitch applied to image scanner
Imaging plate Dedicated scanner Determined by LASER beam diameter for readout
Image sensor
Built in the digital camera Same size as that of the image sensor
(Digital camera)
Built in the PC connected to
Digital memory Determined by scanning condition of electron beam
the scanning device
a) Photographic film: Analogue image recorded on the photographic negative film shall be converted to a
digitized image using an image scanner with dot pitch less than 20 μm.
NOTE 1 Use a flatbed image scanner because it is easy to set the transparent scale in it for pixel size calibration
(Refer to ISO 29301).
b) Imaging plate (IP): The recorded image shall be read out with a dedicated image digitizer (IP reader)
connected to a PC.
c) Image sensor: The digitized image, captured by a digital camera, shall be saved on the memory in the PC
system as an image file with a reversible format.
NOTE 2 Ensure that the procedure for normalization of gain is performed to have a uniform background of
the image.
d) Digital memory: STEM and elemental mapping image, captured by the digital memory built into a PC,
shall be saved as an image file with a reversible format.
Before and during the execution of the digitization procedure, ensure the following.
— The correct sensitivity setting is used for the photographic film used, to generate the negative image
with proper density and contrast.
— Keep the exposure time to minimize the drift and reduce blurring in the recorded image.
— Do not use “binning” in the readout process of the magnified digital image from the digital camera.
— When using an image montage function, ensure that the seams of the image do not overlap with any of
the interface of the specimen.
— For saving the digitized images, an uncompressed image file format (ESP, PICT, TIFF or Windows bitmap),
or reversible (lossless) compressed image file format (GIF or PING) shall be used.
— Ethical digital imaging requires that the original uncompressed image file be stored on archival media,
e.g. CD-R, without any image manipulation or processing operation. All parameters of the production
and acquisition of this file and any subsequent processing steps shall be documented and reported to
ensure reproducibility.
Generally, acceptable imaging operations include gamma correction, histogram stretching and brightness
and contrast adjustments need not be reported. All other operations (such as Unsharp-Masking, Gaussian
Blur, etc.) shall be directly identified by the author as part of the experimental methodology. However, for

diffraction data or any other image data that is used for subsequent quantification, all imaging operations
shall be reported.
6.2.2 Displaying the digitized image
The digitized image used for further processing shall be orientated so that the interface is, as far as possible,
parallel to the y-axis of the monitor screen. If the interface is tilted with an angle, α, to the y-axis of the
monitor screen, it is necessary to measure and to record the tilting angle, α (see Figure 5).
Key
1 interface
2 y-axis of monitor screen
Figure 5 — Example of a tilted target image
6.3 Setting the ROI
6.3.1 General
This depends on the type of the image classified in 6.3.2.
6.3.2 Classification of image
Firstly, it is necessary to obtain an intensity profile in a direction across the interface. Ensure that the
intensity profile is as smooth as possible. To do this, set the ROI in the target image in advance. Then,
integrate the line profile measured across the interface, pixel by pixel, to the appropriate range along the
interface. In practice, the setting procedure depends on the image type classified by image resolution and
the atomic column arrangement recorded in high resolution image, as follows.
— Type 1: Low resolution images in which atomic column arrangements cannot be recognized. Figure 6 a)
shows an example.
— Type 2: High resolution images in which atomic column arranged at an angle θ to the interface [see
Figure 6 b)].
— Type 3: High resolution images in which atomic column is parallel to the interface [see Figure 6 c)].

a) Type 1 image b) Type 2 image
c) Type 3 image
Key
1 interface position
2 atomic column arrangement direction
Figure 6 — Examples of classified target image
6.3.3 Procedure of setting the ROI
6.3.3.1 ROI for type 1 image
The ROI shall cover the widest possible area in the image, including the interface.
Figure 7 shows examples of ROI settings for the type 1 image model. In a very low magnification image,
image distortion is sometimes observed at the edges. In such case, exclude the area containing distortions
from the ROI setting. Figure 7 a) and b) show examples of setting the ROI when the interface is displayed
in parallel to the y-axis of the monitor screen. In these cases, each side of the ROI area is parallel to the
x-axis or y-axis. Figure 7 c) shows an example of setting the ROI when the interface is inclined to the y-axis
of the monitor screen. In this case, the ROI shall be set; one side of the ROI is parallel to the direction of the
interface.
a) ROI contains one interface parallel b) ROI contains both interfaces parallel
to the y-axis to the y-axis
c) ROI contains both interfaces inclined to the y-axis
Key
1 region of interest (ROI)
Y y-axis of monitor screen
Figure 7 — Examples of ROI setting for type 1 image model
6.3.3.2 ROI for type 2 image
A schematic model of the type 2 image is shown in Figur
...