ISO 29301:2010
(Main)Microbeam analysis — Analytical transmission electron microscopy — Methods for calibrating image magnification by using reference materials having periodic structures
Microbeam analysis — Analytical transmission electron microscopy — Methods for calibrating image magnification by using reference materials having periodic structures
ISO 29301:2010 specifies a calibration procedure applicable to images recorded over a wide magnification range in a transmission electron microscope (TEM). The reference materials used for calibration possess a periodic structure, such as a diffraction grating replica, a super-lattice structure of semiconductor or an analysing crystal for X-ray analysis, and a crystal lattice image of carbon, gold or silicon. ISO 29301:2010 is applicable to the magnification of the TEM image recorded on a photographic film, or an imaging plate, or detected by an image sensor built into a digital camera. ISO 29301:2010 also refers to the calibration of a scale bar. ISO 29301:2010 does not apply to the dedicated critical dimension measurement TEM (CD-TEM) and the scanning transmission electron microscope (STEM)
Analyse par microfaisceaux — Microscopie électronique en transmission analytique — Méthodes d'étalonnage du grandissement d'image au moyen de matériaux de référence de structures périodiques
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 29301
First edition
2010-06-01
Microbeam analysis — Analytical
transmission electron microscopy —
Methods for calibrating image
magnification by using reference
materials having periodic structures
Analyse par microfaisceaux — Microscopie électronique en
transmission analytique — Méthodes d'étalonnage du grandissement
d'image au moyen de matériaux de référence de structures périodiques
Reference number
ISO 29301:2010(E)
©
ISO 2010
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ISO 29301:2010(E)
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ISO 29301:2010(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms, definitions and abbreviated terms.1
4 Image magnification.5
4.1 Definition of the image magnification .5
4.2 Expressing magnification.6
5 Reference materials .7
5.1 General .7
5.2 Requirements for CRM/RM.7
5.3 Storage and handling.7
6 Calibration procedures .7
6.1 General .7
6.2 Mounting CRM/RM.8
6.3 Setting TEM operating conditions for calibration.8
6.4 Capturing digitized image.10
6.5 Digitizing the image recorded on photographic film .11
6.6 Measurement of the angle-corrected distance D , from the digitized image.12
t
6.7 Digitization of reference scale for pixel size calibration .15
6.8 Calibration of image magnification .16
6.9 Calibration of scale bar.18
6.10 Calibration procedure for length measurements using photographic film only .19
7 Accuracy of image magnification .19
8 Uncertainty of measurement result .20
9 Calibration report .21
9.1 General .21
9.2 Contents of calibration report.22
Annex A (informative) Parameters that influence the resultant magnification of a TEM .23
Annex B (normative) Flowchart of image-magnification calibration procedure .24
Annex C (normative) How to decide the number of lines for averaging.25
Annex D (informative) Reference materials for magnification calibration.28
Annex E (informative) Example of test report for calibration of TEM magnification .31
Bibliography.40
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ISO 29301:2010(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 29301 was prepared by Technical Committee ISO/TC 202, Microbeam analysis, Subcommittee SC 3,
Analytical electron microscopy.
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ISO 29301:2010(E)
Introduction
The transmission electron microscope is widely used to investigate the micro/nano-structure of a range of
important materials such as semiconductors, metals, nano-particles, polymers, ceramics, glass, food and
biological materials. This International Standard is relevant to the need for magnification calibration of the
images. It describes the requirements for calibration of the image magnification in the transmission electron
microscope using a certified reference material or a reference material having periodic structures.
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INTERNATIONAL STANDARD ISO 29301:2010(E)
Microbeam analysis — Analytical transmission electron
microscopy — Methods for calibrating image magnification by
using reference materials having periodic structures
1 Scope
This International Standard specifies a calibration procedure applicable to images recorded over a wide
magnification range in a transmission electron microscope (TEM). The reference materials used for calibration
possess a periodic structure, such as a diffraction grating replica, a super-lattice structure of semiconductor or
an analysing crystal for X-ray analysis, and a crystal lattice image of carbon, gold or silicon. This International
Standard is applicable to the magnification of the TEM image recorded on a photographic film, or an imaging
plate, or detected by an image sensor built into a digital camera. This International Standard also refers to the
calibration of a scale bar. This International Standard does not apply to the dedicated critical dimension
measurement TEM (CD-TEM) and the scanning transmission electron microscope (STEM).
2 Normative references
The following referenced documents are indispensable for the application 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 Guide 30:1992, Terms and definitions used in connection with reference materials
ISO Guide 34:2000, General requirements for the competence of reference material producers
ISO Guide 35:2006, Reference materials — General and statistical principles for certification
ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories
ISO/IEC Guide 98-3:2008, Uncertainty of measurement — Part 3: Guide to the expression of uncertainty in
measurement (GUM:1995)
3 Terms, definitions and abbreviated terms
For the purposes of this document, the terms and definitions in ISO Guide 30 and the following apply.
3.1
alignment
series of operations to align the incident direction of the electron beam to the optical axis using deflectors
and/or mechanical knobs
3.2
beam damage
specimen damage generated by irradiation with the electron beam
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ISO 29301:2010(E)
3.3
certified reference material
CRM
reference material, accompanied by a certificate, one or more of whose property values are certified by a
procedure which establishes its traceability to an accurate realization of the unit in which the property values
are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of
confidence
NOTE For the purposes of this International Standard, a CRM possesses periodic structure(s), with the desired range
of periodic interval and accuracy, to be used for the calibration of the image magnification.
3.4
contamination
formation of a deposited layer of any material due to the interaction of the electron beam with the sample
and/or its immediate environment
3.5
crystal orientation
direction of crystal which is represented by crystal index
NOTE During TEM imaging, it is often useful to have a crystalline specimen aligned such that a specific (low index)
zone axis is parallel, or nearly parallel, to the beam direction (optical axis).
3.6
defocus
focusing condition in which the vertical positioning of the specimen is not coincident with the object plane of
the objective lens
NOTE Over-focus condition is that the specimen height is nearer the lens than the object plane, under-focus
condition is that the specimen height is further from the lens than the object plane.
3.7
diffraction grating replica
shadow-casting carbon replica film constituting a grating which contains 500 to 2 000 parallel grooves per
millimetre, or cross-line grating with a similar line spacing
NOTE A diffraction grating replica can be used as a reference material for calibration of the image magnification in
the low to medium-low magnification range.
3.8
digital camera
device that detects the image using a chip-arrayed image sensor, such as a charge-coupled device (CCD) or
complementary metal-oxide semiconductor (CMOS), that converts a visual image to an electric signal
3.9
dynamic range
range of detectable electron doses illuminated on the detector, in which the image signal can be detected
properly
3.10
excitation current
electric current applied to the coil of the magnetic lens
3.11
glass scale
ruler on which a fine scale is drawn and utilized as the reference scale to measure the distance in the digitized
image after digitizing it with an image scanner
NOTE The transparency and thermal stability of the glass scale are convenient to get the digitized reference image
with a transmitted image scanner and to make the contact image on the imaging plate.
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ISO 29301:2010(E)
3.12
goniometer stage
device to move the specimen laterally and vertically, and to tilt the specimen by tilting the specimen holder
around the longitudinal holder axis
3.13
horizontal field width
HFW
original length corresponding to full width in the horizontal direction on a magnified image
3.14
image
two-dimensional projection of the specimen structure generated by TEM
NOTE A photographic film, an imaging plate, and an image sensor built into a digital camera are examples of devices
for detecting the image.
3.15
image file format
processing method to encode the image information for storage in a computer file
3.16
image magnification
ratio of the linear dimension of the specific structure/scaling on the image detector, such as a photographic
film, an imaging plate, or an image sensor built into a digital camera, to the corresponding linear dimension of
the structure/scaling on the specimen
3.17
imaging plate
IP
electron image detector consisting of a film with a thin active layer embedded with specifically designed
phosphors
3.18
image scanner
device that converts an analogue image into a digitized image with the desired resolution
NOTE There are mainly two different types of scanners: flatbed type and drum type.
3.19
image sensor
device, such as a charge-coupled device (CCD) array or complementary metal-oxide semiconductor (CMOS)
sensor, that converts visual image information to an electric signal, built-in digital camera or other imaging
devices
3.20
image wobbler
deflection coil to change direction of incident electron beam onto the specimen
NOTE This coil is activated in a periodic manner with the aim of identifying easily the place of focus.
3.21
just focus
focusing condition in which the specimen height coincides with the object plane of the objective lens
3.22
lattice image
image consisting of interference fringes formed by the interaction between the transmitted electron beam and
diffracted electron beam from a specific crystal plane
NOTE Lattice fringes can be used to calibrate image magnification at the high end of the magnification range.
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ISO 29301:2010(E)
3.23
lattice spacing
crystallographic distance between two adjacent parallel planes with the same Miller indices, which can be
calculated from the value of the basic cell vector
3.24
magnetic hysteresis
physical phenomenon related to the magnetizing loop in which the magnetic field strength depends on the
direction of the adjustment of the exciting current for the magnetic lens
3.25
optical axis
straight line passing through the symmetrical centre of the magnetic field of the electron lens
NOTE The path of an electron beam along this axis goes through the lens without changing the direction.
3.26
photographic film
negative film
sheet or a roll of thin plastic coated by photographic emulsion for recording an image
3.27
pixel-resolution
number of imaging pixels per unit distance of the detector
NOTE Typical unit is sometimes expressed as dots per inch (dpi).
3.28
reference material
RM
material or substance, one or more of whose property values are sufficiently homogeneous and well
established to be used for the calibration of an apparatus, the assessment of a measurement method, or for
assigning values to materials
NOTE For the purpose of this International Standard, an RM possesses periodic pattern(s) with the desired range of
periodic interval and accuracy, to be used for the calibration of the image magnification.
3.29
region of Interest
ROI
a part region extracted from the whole area in the graph
3.30
specimen
small portion of a sample for observation
NOTE For TEM, a specimen has to be thin enough to transmit the electron beam.
3.31
specimen cartridge
part of specimen holder which supports a specimen and is attached to the tip of the specimen holder for use
3.32
specimen drift
unintentional movement of the specimen due to any source (thermal, mechanical, electric, charging)
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ISO 29301:2010(E)
3.33
specimen height
specimen position along the optical axis of the objective lens
NOTE 1 “Specimen height = 0” corresponds to the specimen position in correct focus under the standard excitation
condition of the objective lens.
NOTE 2 See Reference [2] in the Bibliography.
3.34
specimen holder
device that supports a specimen in the right position in the pole-piece gap of the objective lens
3.35
standard excitation condition
optimal condition for excitation current of the objective lens to focus the image
NOTE 1 This condition is provided by the TEM manufacturer for each instrument.
NOTE 2 Image magnification is generally measured under this condition; however, as long as reproducible conditions
are established, the magnification can be calibrated at any of the instrument settings.
3.36
super-lattice
stable periodic structure which is fabricated by alternating layers of at least two different kinds of materials
NOTE The super-lattice can be used as a reference material for calibration of image magnification from a medium-
high to high magnification range.
3.37
transmission electron microscope
TEM
instrument that produces magnified images or diffraction patterns of the specimen by an electron beam which
passes through the specimen and interacts with it
3.38
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
4 Image magnification
4.1 Definition of the image magnification
The image magnification (or scaling factor) of the TEM is defined by the ratio of the linear dimension of the
specific structure on the detected image to the corresponding linear dimension of the specific structure in the
specimen. There are three main kinds of image detectors: photographic film, imaging plate, and image sensor,
such as CCD array or CMOS sensor built in the digital camera.
In general, the value of image magnification detected on an image sensor is different from the value of image
magnification detected on the photographic film or imaging plate under the same electron optical conditions
for TEM imaging, because the image-detecting positions are different from each other (see Figure 1).
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ISO 29301:2010(E)
1
2
3
4
5
6
7
8
9
12
10
11
13
14
9
15
10
The digital camera (image sensor) position is different from the photographic film/imaging plate position.
Key
1 electron gun 9 monitor
2 condenser lens 10 computer
3 specimen 11 digital camera (image sensor) magnification; M < M
s g
4 objective lens 12 screen/mirror
5 1st magnified image 13 viewing screen
6 intermediate lens 14 photographic film/imaging plate magnification; M
f
7 2nd magnified image 15 digital camera (image sensor) magnification; M > M
is f
8 projector lens
Figure 1 — Detector position in TEM system
4.2 Expressing magnification
The magnification of an image recorded on the photographic film or the imaging plate, or detected by the
image sensor, is given by a number representing the number of times, and the number is accompanied by the
symbol “×” (e.g. 10 000×, 10k×, 1 000 000×, 1M× or ×10 000, ×10k, ×1 000 000, ×1M, where 10 000, 10k,
1 000 000 and 1M are magnitude numbers). Alternatively, introducing a scale bar having a length
corresponding to unit length on the specimen can be used to represent the magnification. The digitized image
should also indicate a magnification by detailing the number of pixels per unit distance of the raw data file.
NOTE The horizontal field width (HFW) is another way to define the scaling on a magnified image.
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ISO 29301:2010(E)
5 Reference materials
5.1 General
For calibrating the magnification of an image, wherever possible, choose a CRM that is produced in
accordance with ISO Guide 34 and certified in accordance with ISO Guide 35.
When a suitable CRM is not available, an RM produced in accordance with ISO Guide 34 may be used.
5.2 Requirements for CRM/RM
Ensure that the chosen CRM/RM
⎯ is stable with respect to vacuum and repeated electron-beam exposure,
⎯ is aligned to a low-index zone axis along the electron optical axis, if the specimen region is a single
crystal,
⎯ provides a good contrast and clear interface for the periodic structure in the TEM image,
⎯ can be cleaned to remove contamination without causing mechanical/electrical damage or distortion,
⎯ has a smooth surface on both sides and identical thickness for a super-lattice structure, at least within the
area used for the calibration process,
⎯ has an associated valid calibration certificate.
NOTE Single crystal specimens of pure elements used for calibration do not need a calibration reference certificate.
5.3 Storage and handling
The CRM/RM shall be stored in a desiccating cabinet or in a vacuum container.
To ensure minimal handling of the actual CRM/RM, it may be permanently mounted on a specimen holder or
a specimen cartridge.
The CRM/RM should be carefully handled without causing damage during the handling.
Check the contamination and deterioration of the CRM/RM, as these may affect calibration. Do not use the
CRM/RM if it is damaged or grossly contaminated.
Check the calibration of the CRM/RM at intervals by comparing its calibration values with those of other
CRMs/RMs; record the results. The frequency of verification may depend on the nature and usage of the
CRM/RM.
The CRM/RM shall be used for calibration purposes only.
6 Calibration procedures
6.1 General
Parameters that influence the magnification of a TEM may cause systematic errors. These are listed in
Annex A.
A major factor that influences the reproducibility of the calibration is the magnetic hysteresis of the electro-
magnetic lens. It is necessary to minimize its influence by adopting the procedure described below in the
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ISO 29301:2010(E)
same sequence each time, especially related to the direction of magnification setting (higher to lower, or lower
to higher). Also, the specimen height and focus setting will influence the reproducibility of the calibration.
To obtain the value of the uncertainty within the laboratory, it is necessary to repeat the calibration procedure
periodically.
The selection of the CRM/RM depends on the magnification range being used and the accuracy required. For
the purpose of this International Standard, ensure that the uncertainty and repeatability of calibration is better
than ±5 % and 98 %, respectively.
The flowchart of the calibration procedure is shown in Annex B.
6.2 Mounting CRM/RM
At the time of mounting the specimen, ensure that handling of the CRM/RM is carried out in accordance with
5.3.
Mount the CRM/RM in accordance with the instructions provided by the TEM and the CRM/RM manufacturers.
Check that the CRM/RM is securely fixed on the specimen holder or specimen cartridge so that it does not
move from its mounting. This enables any image degradation caused by vibration to be minimized.
Check that the height of the specimen in the specimen holder is at the position recommended by the TEM
manufacture's instructions, in order to keep the eucentric condition.
It is desirable to use a double-tilt or tilt-rotate specimen holder for aligning the crystal orientation of the
specimen to the optical axis.
6.3 Setting TEM operating conditions for calibration
Set the operating condition of the TEM according to the following procedures to ensure, as far as possible,
use of the same conditions.
−4
a) Check that the degree of vacuum in the TEM column is lower than 10 Pa and stable.
b) The high voltage shall be applied and an appropriate time be allowed for it to stabilize.
NOTE Oil-filled 100 kV tanks take about 2,5 h; gas-filled tanks take about 45 min. Higher voltage instruments
are normally operated with the high voltage continually applied, therefore a stabilization period is not usually required.
c) Use an anti-contamination device, if needed.
d) Select a specimen region of interest (ROI) for the calibration which is clean and free from damage,
ensure the eucentric height of the ROI and adjust the height of the ROI, if necessary.
e) In order to minimize the effect of the magnetic hysteresis of the lenses, set the magnification of the TEM
to the target value for calibration according to the same sequence, for example, adjust higher
magnification than the target magnification at first, then set the target magnification after that.
f) Set the excitation of the objective lens to the desired reproducible value; the standard condition is
recommended.
g) Adjust the specimen height to focus the magnified image projected on the fluorescent screen, the TV
monitor or the personal computer (PC) screen.
NOTE If the TEM in question is not equipped with a specimen-height control function, this procedure can be
omitted.
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ISO 29301:2010(E)
h) Correct astigmatism at a slightly higher magnification than the target value and adjust the accelerating
voltage centre. For example, if the target calibration is ×100k, set the magnification in the range
×150k ~ ×200k for alignment.
i) Switch the observation mode of the TEM to the selected-area electron-diffraction (SAED) mode or the
convergent-beam electron-diffraction (CBED) mode from the image mode. Also, make sure that the
objective aperture is removed.
NOTE For the SAED mode, it is necessary to insert a selected-area aperture over the area of interest of the
specimen in order to project a selected-area electron-diffraction pattern on the viewing device (fluorescent screen/TV
monitor/PC screen).
j) Adjust the condenser lens system to provide nearly parallel illumination conditions.
k) Align a low-index zone axis of the crystal parallel to the optical axis (i.e. zone-axis illumination), if the
specimen is a single crystal, see Figure 2.
a) Off-axis condition b) Zone-axis condition
Figure 2 — Difference of diffraction pattern by crystal orientation
l) Insert the objective aperture, centring it about the electron optical axis. Also, switch the observation mode
of the TEM back to the image mode.
m) Return the magnification to the target value of calibration, and set the excitation current of the objective
lens to the standard exciting condition again.
n) Apply a relaxation function to relax the magnetic hysteresis of the objective lens, if the TEM has it.
o) Adjust the specimen height to focus the magnified image roughly.
NOTE If the TEM in question is not equipped with a specimen-height control function, this procedure can be
omitted.
p) Adjust the fine focus by varying the exciting current of the objective lens.
NOTE If necessary, it is possible to use the Image Wobbler function for focusing the image.
q) Turn off the auto-focus correction function to the optimum under-focus condition linked with the Image
Wobbler function, if the TEM is equipped with this function.
r) Adjust the illumination condition of the condenser lens system (spot size and brightness) with reference to
t
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