Microbeam analysis — Analytical electron microscopy — Selected-area electron diffraction analysis using a transmission electron microscope

ISO 25498:2010 specifies the method of selected-area electron diffraction (SAED) analysis using a transmission electron microscope (TEM) to analyse micrometer and sub-micrometer sized areas of thin crystalline specimens. Such specimens can be obtained in the form of thin sections from a variety of metallic and non-metallic materials, as well as fine powders, or alternatively by the use of extraction replicas. The minimum diameter of the selected area in a specimen which can be analysed by this method depends on the spherical aberration coefficient of the objective lens of the microscope and approaches 0,5 mm for a modern TEM. When the diameter of an analysed specimen area is smaller than 0,5 mm, the analysis procedure can also be referred to ISO 25498:2010 but, because of the effect of spherical aberration, some of the diffraction information in the pattern can be generated from outside of the area defined by the selected-area aperture. In such cases, the use of microdiffraction or convergent beam electron diffraction, where available, might be preferred. The success of the selected-area electron diffraction method relies on the validity of indexing the diffraction patterns arising, irrespective of which axis in the specimen lies parallel to the incident electron beam. Such analysis is therefore aided by specimen tilt and rotation facilities. ISO 25498:2010 is applicable to acquisition of SAED patterns from crystalline specimens, indexing the patterns and calibration of the diffraction constant.

Analyse par microfaisceaux — Microscopie électronique analytique — Analyse par diffraction par sélection d'aire au moyen d'un microscope électronique en transmission

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Publication Date
16-May-2010
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9599 - Withdrawal of International Standard
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16-Mar-2018
Completion Date
16-Mar-2018
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INTERNATIONAL ISO
STANDARD 25498
First edition
2010-06-01
Microbeam analysis — Analytical
electron microscopy — Selected-area
electron diffraction analysis using a
transmission electron microscope
Analyse par microfaisceaux — Microscopie électronique analytique —
Analyse par diffraction par sélection d'aire au moyen d'un microscope
électronique en transmission
Reference number
ISO 25498:2010(E)
ISO 2010
---------------------- Page: 1 ----------------------
ISO 25498:2010(E)
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ii © ISO 2010 – All rights reserved
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ISO 25498:2010(E)
Contents Page

Foreword ............................................................................................................................................................iv

Introduction.........................................................................................................................................................v

1 Scope......................................................................................................................................................1

2 Normative references............................................................................................................................1

3 Terms, definitions and symbols ..........................................................................................................1

4 Principle..................................................................................................................................................2

4.1 Spot diffraction pattern.........................................................................................................................2

4.2 Kikuchi pattern ......................................................................................................................................5

4.3 Polycrystalline specimen......................................................................................................................6

5 Equipment ..............................................................................................................................................7

6 Specimens..............................................................................................................................................7

7 Reference materials ..............................................................................................................................7

8 Experimental procedure .......................................................................................................................8

8.1 Instrument preparation .........................................................................................................................8

8.2 Procedure for acquirement of selected-area electron diffraction patterns.....................................8

8.3 Determination of diffraction constant Lλ ..........................................................................................10

9 Measurement and solution of the SAED patterns............................................................................11

9.1 Selection of the basic parallelogram.................................................................................................11

9.2 Indexing diffraction spots...................................................................................................................12

10 The 180° ambiguity..............................................................................................................................13

11 Uncertainty estimation........................................................................................................................13

11.1 Factors affecting accuracy.................................................................................................................13

11.2 Calibration with a reference material ................................................................................................14

Annex A (informative) Interplanar spacing of pure Au and Al .....................................................................15

Annex B (informative) Spot diffraction patterns of single crystals with body-centred cubic (BCC,

face-centred cubic (FCC) and hexagonal close packed (HCP) structure ......................................16

Bibliography......................................................................................................................................................28

© ISO 2010 – All rights reserved iii
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ISO 25498:2010(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 25498 was prepared by Technical Committee ISO/TC 202, Microbeam analysis, Subcommittee SC 3,

Analytical electron microscopy.
iv © ISO 2010 – All rights reserved
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ISO 25498:2010(E)
Introduction

Electron diffraction techniques are widely used in transmission electron microscopy (TEM) studies.

Applications include phase identification, determination of the crystalline lattice type and lattice parameters,

crystal orientation and the orientation relationship between two phases, phase transformations, habit planes

and defects, twins and interfaces, as well as studies of preferred crystal orientations (texture) etc. While

several complementary techniques have been developed, e.g. microdiffraction, convergent beam diffraction

and reflected diffraction, selected-area electron diffraction (SAED) is the most frequently employed. Selected-

area electron diffraction allows direct analysis of small areas of the sample (fine layers, grains, precipitates,

etc.) and is routinely performed on thin specimens of a variety of crystalline materials. The SAED is also a

supplementary technique for acquisition of high resolution images, microdiffraction or convergent beam

diffraction studies. The information generated is widely used in the studies of structure/property relationships

as well as for inspection and quality control purposes.

This International Standard explains the mechanism of the diffraction pattern formation, the practical aspects

of SAED operation and the indexing of the patterns.
© ISO 2010 – All rights reserved v
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INTERNATIONAL STANDARD ISO 25498:2010(E)
Microbeam analysis — Analytical electron microscopy —
Selected-area electron diffraction analysis using a transmission
electron microscope
1 Scope

This International Standard specifies the method of selected-area electron diffraction (SAED) analysis using a

transmission electron microscope (TEM) to analyse micrometer and sub-micrometer sized areas of thin

crystalline specimens. Such specimens can be obtained in the form of thin sections from a variety of metallic

and non-metallic materials, as well as fine powders, or alternatively by the use of extraction replicas. The

minimum diameter of the selected area in a specimen which can be analysed by this method depends on the

spherical aberration coefficient of the objective lens of the microscope and approaches 0,5 µm for a modern

TEM.

When the diameter of an analysed specimen area is smaller than 0,5 µm, the analysis procedure can also be

referred to this International Standard but, because of the effect of spherical aberration, some of the diffraction

information in the pattern can be generated from outside of the area defined by the selected-area aperture. In

such cases, the use of microdiffraction or convergent beam electron diffraction, where available, might be

preferred.

The success of the selected-area electron diffraction method relies on the validity of indexing the diffraction

patterns arising, irrespective of which axis in the specimen lies parallel to the incident electron beam. Such

analysis is therefore aided by specimen tilt and rotation facilities.

This International Standard is applicable to acquisition of SAED patterns from crystalline specimens, indexing

the patterns and calibration of the diffraction constant.
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/IEC 17025, General requirements for the competence of testing and calibration laboratories

3 Terms, definitions and symbols
For the purposes of this document, the following terms and definitions apply.
3.1
(h k l)
Miller indices of a specific set of crystalline planes
3.2
{h k l}
Miller indices, which denote a family of crystalline planes
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ISO 25498:2010(E)
3.3
[u v w]
Miller indices of a specific crystalline direction or a zone axis
3.4
(u v w)
Miller indices, which denote a family of crystalline directions
3.5
interplanar spacing
hkl

perpendicular distance between consecutive planes of the crystalline plane set (h k l)

3.6
(u v w)*
indices of a plane in the reciprocal lattice

NOTE The normal of the reciprocal plane (u v w)* is parallel to the crystalline zone axis [u v w].

3.7
reciprocal vector
hkl
vector in the reciprocal lattice

NOTE The reciprocal vector g is normal to the crystalline plane (h k l) with its magnitude inversely proportional to

hkl
interplanar spacing d .
hkl
3.8
hkl

vector from centre 000 (the origin) to the diffraction spot h k l in a diffraction pattern

See Figure 1.
3.9
eucentric position

specimen position at which the image exhibits minimal lateral motion resulting from specimen tilting

4 Principle

When an energetic electron beam is incident upon a thin crystalline specimen in a transmission electron

microscope, a diffraction pattern will be produced in the back focal plane of the objective lens. This pattern is

magnified by the intermediate and projector lenses, and displayed on a viewing screen. This pattern can also

be displayed on a monitor if the TEM is equipped with a TV or charge-coupled device (CCD) camera system.

4.1 Spot diffraction pattern

The diffraction pattern of a single crystal appears as an array of 'spots', the basic unit of which is characterized

by a parallelogram. A schematic illustration of a spot diffraction pattern is shown in Figure 1. Each spot

corresponds to diffraction from a specific set of crystal lattice planes in the specimen, denoted by Miller

indices (h k l). The vector R is defined by the position of the diffracted spot h k l relative to position on the

hkl

pattern corresponding to the transmitted beam, i.e. the centre-spot 000 of the pattern. It is parallel to the

normal of the reflecting plane (h k l). The magnitude of R is inversely proportional to the interplanar spacing

hkl

d of the diffracting plane (h k l) (References [1], [2], [3] and [4] in the Bibliography). In the context of this

hkl
R , R ,()RR− ()RR+
International Standard, vectors and are simplified as R ,
hk l hk l hk l hk l hk l h k l
111 222 222 1 11 111 2 2 2

R , R and R , respectively. The included angle between vectors R and R is denoted by γ *.

2 2−1 1+2 1 2
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ISO 25498:2010(E)

Because the centre-spot is often very bright, it is often difficult to determine the exact centre of the pattern.

Therefore, a practical procedure is to establish the magnitude of R by measuring the distance between

hkl

the spots h k l and hk l on the diffraction pattern and dividing by two, i.e. . Figure 2

RR=+()R
hkl hkl
2 hk l

shows an example of the SAED pattern where the magnitude of R , R and R is obtained from ()R + R,

1 2 2−1 11
1 1
()R + R and ()RR+, respectively.
22 21−−21
2 2

Figure 1 — Schematic spot diffraction pattern from a single crystal, the basic parallelogram

constituted by the diffraction spots h k l , h k l , (h + h , k + k , l + l ) and central spot 000

1 1 1 2 2 2 1 2 1 2 1 2

Figure 2 — Example of SAED spot pattern showing the basic parallelogram constituted by R and R

1 2
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ISO 25498:2010(E)

The relationship between the interplanar spacing d and the magnitude R for a reflecting plane (h k l) can

hkl hkl
be approximately expressed as (Reference [4] in the Bibliography)
hkl
LRλ=×d⎢⎥1(−× =R ×d1−∆) (1)
hklhkl ⎜⎟ hklhkl
where
L=×fM×M is the diffraction camera length,
opi
f is the focal length, in millimetres, of the objective lens in the microscope,
M is the magnification of the intermediate lens,
M is the magnification of the projector lenses,

λ is the wavelength, in nanometres, of the incident electron beam which is dependent

upon the accelerating voltage and can be given by Equation (2) (Reference [2] in the

Bibliography):
1,226
λ(nm) = (2)
VV(1+×0,978 8 10 )
where V is the accelerating voltage, in volts, of the TEM,

Lλ is the diffraction constant (or camera constant) of the transmission electron microscope operating

under the particular set of conditions. This parameter can be determined from the diffraction pattern

of a crystalline specimen of known lattice parameters (refer to 8.3).

For most work using a TEM, the value of ∆ in Equation (1) is usually smaller than 0,1 % and hence a more

simplified equation may be used, namely
R ×=dLλ (3)
hkl hkl

This relation can be understood through the Ewald sphere construction, which is illustrated in Figure 3. For the

derivation of the above equation, refer to the textbooks (References [2] to [6] in the Bibliography).

The use of Equation (3) requires measuring the length of R . Since, as mentioned earlier, the location of the

hkl

pattern centre may not be easily determined, it is recommended that the distance measurement taken,

2 R , be from the h k l diffracted spot to the hk l spot on the pattern. This is equivalent to a diameter

hk l 1 1 1 111
111

measurement on the ring pattern from a polycrystalline specimen. To obtain the interplanar information, the

measured distance 2 R is halved and Equation (3) applied.
hk l
111

If the camera constant is known, the interplanar spacing d of plane (h k l) can be calculated. The included

hkl

angle between any two vectors R and R can also be measured on the diffraction pattern. This is

hk l hk l
111 222

equal to the angle between the corresponding crystalline planes (h k l ) and (h k l ).

1 1 1 2 2 2

Since diffraction data from a single pattern will provide information on a limited number of the possible

diffracting planes in a specimen area, it is necessary to acquire additional diffraction patterns from the same

area (or from different grains/particles of the same phase). This requires either the tilting of the specimen or

the availability of differently oriented grains or particles of the same phase.

Acquire a second diffraction pattern from another zone axis from the same area by tilting the specimen so that

the two patterns contain a common spot row (also see 8.2.10). Index the diffracted spots, and then select

three non-planar spots in the two patterns to constitute a reciprocal lattice, which, if the spots correspond to

low values of Miller indices, may define the primitive unit cell of the crystal lattice. Therefore, crystal lattice

parameters can be determined and the orientation of the grain or particle in the thin specimen can also be

calculated.
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ISO 25498:2010(E)
Key
1 incident beam
2 specimen
3 Ewald sphere
4 reciprocal plane
5 diffracted beam
6 diffraction pattern
7 transmitted beam

Figure 3 — Ewald sphere construction illustrating the diffraction geometry in TEM

4.2 Kikuchi pattern

When a specimen area is nearly perfect but not thin enough, Kikuchi lines may occur. They arise from

electrons scattered inelastically through a small angle and suffering only a very small energy loss being

scattered again this time elastically. This process leads to local variations of the background intensity in the

diffraction pattern and the appearance of Kikuchi lines.

The Kikuchi patterns consist of pairs of parallel bright and dark lines, which are parallel to the projection of the

corresponding reflecting planes. The bright (excess) line and dark (defect) line in the Kikuchi pattern are

denoted by K and K , respectively. Therefore, the line pair, K and K , will be perpendicular to

B−hkl D−hkl B−hkl D−hkl

the vector R from the corresponding crystalline plane (h k l). An example of a Kikuchi pattern is given in

hkl

Figure 4, where the bright line K and dark line K pair is superimposed on the spot pattern. The

B−hkl D−hkl

perpendicular distance D between the line pair, K and K , is related to the interplanar spacing d

K−hkl B−hkl D−hkl hkl
and camera constant Lλ by Equation (4).
D ×=dLλ (4)
K-hkl hkl
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ISO 25498:2010(E)

The angles between different Kikuchi line pairs are also equal to the angles between the relevant crystalline

planes. The Kikuchi patterns present the real crystal symmetry of the specimen. They are a very useful aid in

tilting the specimen from one zone axis to another and also in establishing crystal orientation with very high

accuracy (References [6] and [7] in the Bibliography).

Generally, when Kikuchi lines are visible, the specimen-tilting method is preferred to acquire diffraction

patterns with different zone axes from the same area.

Figure 4 — Kikuchi pattern from a steel specimen, D : the distance between the line pair K

K−hkl B−hkl
(bright line) and K (dark line)
D−hkl
4.3 Polycrystalline specimen

The diffraction pattern from a polycrystalline specimen is comprised of a series of concentric rings centred on

the transmitted spot 000. An example of the pattern from a polycrystalline gold (Au) specimen is given in

Figure 5. Each diffracted ring arises from the diffraction beams from differently oriented crystalline planes of

the form {h k l}; each of these having an identical interplanar spacing. From the diameter of each diffraction

ring, the corresponding interplanar spacing d can be calculated using Equation (3). Indices of the diffraction

hkl

rings can be ascribed and then the lattice parameters can also be determined. For the method of indexing ring

patterns refer to that used in X-ray powder diffraction (Reference [8] in the Bibliography).

Figure 5 — Diffraction ring pattern with indices from a polycrystalline Au specimen

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ISO 25498:2010(E)
5 Equipment

5.1 Transmission electron microscope, with double tilt or tilt rotation specimen holder.

5.2 For a TEM not equipped with a TV or CCD camera system, films or imaging plates are necessary for

recording the patterns. When films are used, the following facilities are also required.

5.2.1 Measuring magnifier and protractor, to measure length and plane angle, respectively.

5.2.2 Photographic materials and negative viewer.
5.2.3 Darkroom, with negative developing and fixing outfit.
5.3 Facilities for specimen preparation.
6 Specimens

6.1 Most specimens are prepared as thin foils (References [9] and [10] in the Bibliography). The shape and

external size of the specimen should match that of the TEM specimen holder or alternatively it can be held by

a support grid. Extraction replicas or powdered specimens shall be prepared on the grid with supporting films.

6.2 The selected specimen area shall be thin enough for the electron beam to pass through it and

diffraction patterns can be observed on the viewing screen.

6.3 The surface of the specimen shall be clean, dry and flat without an oxidizing layer or any contamination.

6.4 For those materials that are stable under energetic particle beam bombardment, contamination on the

specimen surface can be avoided or removed by ion beam sputtering or other techniques before the TEM

observation (Reference [10] in the Bibliography).

6.5 Prepared specimens shall be labelled and placed in a special specimen box and preserved in a

desiccator or evacuated container.
7 Reference materials

A reference specimen is required for determining the diffraction constant Lλ of the microscope in electron

diffraction studies. In principle, any thin crystalline foil or powder could be considered as the reference

specimen, provided its crystalline structure and lattice parameters have been acquired accurately and they are

certified and stable under irradiation of the electron beam. The thickness of the crystal foil or powder grain

size should be consistent with the beam energy and the quality of the diffraction pattern (when it is too thick

the pattern will lack sharpness).

Reference materials in common use are polycrystalline specimens made from pure gold (which has a face-

centred cubic (fcc) lattice with parameter a = 0,407 8 nm) or pure aluminium (Al) (fcc structure with lattice

parameter a = 0,404 9 nm). The mass fraction of Au or Al in the reference materials shall be not less than

99,9 %. The reference specimen shall be prepared by evaporating a small piece of Au or Al on a grid with a

supporting film.

It is also feasible to evaporate a layer of the reference material onto a local surface area of the specimen,

which is to be analysed.
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ISO 25498:2010(E)
8 Experimental procedure
8.1 Instrument preparation

8.1.1 The general working condition of the TEM laboratory shall comply with ISO/IEC 17025.

8.1.2 It is recommended to use the cold finger of TEM before conditioning in order to minimize specimen

contamination.

8.1.3 When the vacuum of the transmission electron microscope is suitable for operation, switch on and

select an appropriate accelerating voltage.
8.1.4 Carry out the axis alignment for the electron optical system.

8.1.5 Place the specimen to be observed and the reference firmly in the double-tilting or tilting-rotation

specimen holder, and insert the holder into the specimen chamber.

A specimen coated with an evaporated layer of reference material can be directly placed in the specimen

holder and inserted into the chamber.

8.1.6 When the SAED patterns need to be related to features observed in the corresponding micrograph

the angle of rotation between the two may need to be determined and compensated for.

The method in common use is to take a diffraction pattern and a micrograph of a molybdenum trioxide crystal

specimen. The rotation angle of the image is then measured on the photographic plates, on which the

micrograph and superimposed diffraction pattern has been recorded. For details of the calibration procedure

refer to the appropriate text books (References [2][5][7] in the Bibliography).
8.2 Procedure for acquirement of selected-area electron diffraction patterns

8.2.1 Obtain a magnified bright field image of the specimen on the viewing screen of the transmission

electron microscope. Adjust the specimen height to the eucentric position so that the image movement is

minimized during tilting of the specimen. The procedure for establishing the eucentric position of the specimen

may be obtained by consulting the manufacturer's operating manual.

8.2.2 Adjust the magnification of the specimen image until details in the specimen can be observed clearly.

A suitable magnification for SAED analysis is usually from about 5,000 to 50,000 times. Focus the image and

correct the astigmatism.

8.2.3 Insert the field limiting aperture (selected-area aperture) and focus the image of this aperture. Focus

the specimen image again. This makes the selected-area aperture plane conjugate with the image plane of

the objective lens.

8.2.4 Switch the microscope to the diffraction mode, focus the image of the objective lens aperture, that is,

make this objective lens aperture coincide with the back focal plane of the objective lens. Return to the bright

field image mode and focus the image again.

8.2.5 Insert the reference (i.e. the calibration standard) and locate at the eucentric position. Choose a

camera length L consistent with the capabilities of the subsequent measuring equipments and then obtain a

diffraction pattern from it. Focus the diffraction pattern and correct any astigmatism carefully to make the

diffraction pattern sharp. Record the diffraction pattern of the reference.

8.2.6 If the reference and test specimen are not in the same specimen holder, withdraw the reference and

insert the test specimen again, without changing the operating conditions and without switching the

microscope off. Again locate it at the eucentric height.

8.2.7 Obtain a focused bright field image of the specimen again with an appropriate magnification.

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ISO 25498:2010(E)

Select an area of interest on the specimen using the field limiting aperture (selected-area aperture). Record

the image of the specimen area. The phase boundary and grain boundaries in the specimen should be kept

away from the selected area when a single crystal grain is analysed.

8.2.8 Switch to the diffraction mode again, withdraw the objective lens aperture and obtain a diffraction

pattern on the viewing screen. Where possible, tilt the specimen slightly so that brightness of the spots in the

diffraction pattern is evenly distributed, or where Kikuchi lines appear; the Kikuchi line pairs are symmetrical

about the pattern centre.

This pattern will then derive from a small index direction in the crystal approximately parallel to the incident

electron beam. This crystal direction will be the zone axis [u v w ], which is the normal of the reciprocal plane

1 1 1

(u v w )*, i.e. the diffraction pattern (References [3] and [4] in the Bibliography).

1 1 1

Adjust (defocus) the second condenser lens current (the brightness knob) to sharpen the diffraction spots,

making them as sharp as possible.

8.2.9 Record the pattern or/and save the original uncompressed pattern in the computer system. Take note

of the reading on the X and Y axis of the specimen-tilting device as X and Y , respectively. Using dark field

1 1
conditions, identify the source of the pattern.

8.2.10 Insert the reference specimen and obtain a second diffraction pattern from the reference. Record this

diffraction pattern (also see 8.3), making sure that the same experimental conditions are used (i.e.

accelerating voltage, lens settings and, especially, the specimen height and camera length L).

8.2.11 Obtain sufficient data for each phase of interest by either of the following procedures.

a) By tilting the specimen. Choose a row of close-spaced diffraction spots collinear with the central

transmitted spot on the diffraction pattern. Align this row with the tilting axis and obtain the second

diffra
...

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