ISO/DIS 16887:2026(en)

ISO/TC 202/SC 3

Secretariat: JISC

Date: 2026-01-03

Microbeam analysis — Analytical electron microscopy —

Guidelines for transmission electron microscopy specimen preparation by lift-out method using focused ion beam system

© ISO 2026

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of the requester.

ISO copyright office

CP 401 • Ch. de Blandonnet 8

CH-1214 Vernier, Geneva

Phone: +41 22 749 01 11

Email: copyright@iso.org

Website: www.iso.org

Published in Switzerland

Contents

Foreword 4

Introduction 5

1 Scope 6

2 Normative references 6

3 Terms and definitions 6

4 Equipment for FIB system 11

4.1 Four functions of the stand-alone FIB system 11

4.2 Improve functionality by integrating with SEM 12

4.3 Characteristics of Ga liquid metal ion sources (LMIS) 13

4.4 Aspects as a scanning ion microscopy 14

4.5. Two roles of gas injection system 14

5 Operating conditions for TEM specimen preparation using FIB fabrication 14

5.1 Common steps in FIB fabrication 14

5.2 Formation of protective deposition film 16

5.3 Rough fabrication process 17

5.4 Extraction of lamellar specimen using manipulator 18

5.5 Concept of invention of TEM half-grid 19

5.6 Finishing fabrication 20

6 Preliminary beam alignment before FIB fabrication 21

6.1 Refinement process of LMIS tip 21

6.2 Beam alignment and stage setting 21

7 Applicable materials 21

7.1 Single crystal Si 21

7.2 Semiconductor materials other than single crystal Si 21

7.3 Metals and Alloys 22

7.4 Non-conductive materials and nano materials 22

7.5 Soft materials and biomaterials 22

Annex A 23

FIB fabrication procedure for single Si (001) wafer 23

A.1. Principle 23

A.2. Fabrication Procedure 23

Annex B 26

TEM observation results for single Si (001) wafer 26

B.1 Quality of TEM specimen 26

B.2 High resolution lattice image 26

Bibliography 27

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).

ISO draws attention to the possibility that the implementation of this document may involve the use of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this document, ISO [had/had not] received notice of (a) patent(s) which may be required to implement this document. However, implementers are cautioned that this may not represent the latest information, which may be obtained from the patent database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.

Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 202, Microbeam analysis, Subcommittee SC 3, Analytical electron microscopy.

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.

Introduction

Several types of transmission electron microscopes (TEM) are widely used for microstructure characterization techniques in the field of material science, and application fields such as manufacturing process development support and semiconductor production line. The facilities are installed in several types of laboratories in commercial companies and national laboratories and universities. With the advancement of nanotechnology, focused ion beam (FIB) systems have become widespread as TEM specimen preparation equipment and are used in various ways in a wide range of application areas, from semiconductors to metals and ceramics, and from organic to composite materials.

Conventional TEM specimen preparation methods have been using electropolishing for conductive materials, and argon ion beam milling for non-conductive ones. Although these techniques are excellent methods for preparing thin specimens, it is difficult to selectively make thinned specimens for TEM in site-specific regions. Since the FIB process uses a fine ion beam to modify any sample, it has high potential to make TEM specimens from a cite-specific small specimen region. However, due to the different milling rates and different reactivity of different materials with accelerated ion beams, TEM specimen preparation by the FIB processing method still has various challenges.

There are many different types of FIB systems in the market. This document describes guidelines for common specimen preparation procedures that do not depend on the characteristics of these equipment types. In the historical development of FIB equipment, however, the first FIB equipment used for TEM specimen preparation was a stand-alone FIB system, followed by a combined FIB-SEM system in which the ion beam column is integrated with a scanning electron microscope (SEM) chamber and the latter FIB-SEM system has become common in large numbers in the market. Detailed discussion of TEM, SEM and FIB theory and operation is beyond the scope of this document and can be found in references [1-3].

The ion gun of the FIB system has several characteristics and differences. FIB equipment using a Liquid Metal Ion Source (LMIS) of Ga ions is the most widely used for TEM specimen preparation. In 1999, FIB milling techniques for TEM specimen preparation was firstly reviewed by Giannuzzi and Stevie.^[4] In recent years, FIB equipment with ion sources based on different principles have emerged both on the low and high current side. The low-current side includes FIB using Gas Field Ion Source (GFIS), and such FIB using Helium and Neon ions is also applied for various biomolecules.^[5] The high-current side includes FIB using Inductively Coupled Plasma (ICP) ion sources or Electron Cyclotron Resonance (ECR) ion source, which can form ion beams with high currents as high as μA, and its application as a FIB for large area fabrication for several materials.^[6,7] With the development of FIB technology using such a variety of ion species, GFIS is mostly used for special applications, and plasma ion sources are applied for rough milling for a large area. This document describes Ga-FIB systems only, however, the process detailed herein may be applicable to other ion source systems.

With regard to preparing TEM specimen from desired micro area, the lift-out method has been developed, in which a target area of several tens of micrometres is extracted. There are two types of methods to extract the micro areas: in situ and ex situ lift-out methods. The in situ lift-out method is used to pick up the target micro area in a vacuum with a manipulator mechanism installed in the FIB equipment, while the ex situ lift-out method is used to remove the target in air outside the FIB equipment after finishing it inside the FIB equipment. The ex situ lift-out procedure is well suited for high throughput applications such as a field of semiconductor process line. As a generic method used in general laboratories and analytical departments, this document is mainly describing the guidelines of the in situ lift-out method. It is noted that the basic procedure of ex situ lift-out method is similar to the in situ one.

Microbeam analysis — Analytical electron microscopy — Guidelines of specimen preparation for transmission electron microscope by lift-out method using focused ion beam processing

This document specifies guidelines on how to prepare reproducible and reliable lamella specimens using the FIB lift-out method to observe a micro area of interest in materials using TEM.

Materials usable for TEM specimen preparation by FIB processing are not only semiconductor materials such as Si but also metals and alloys, inorganic materials such as ceramics, as well as hard and soft biological materials, where the latter often require special sample preparation and modified FIB process.

Among several types of FIB systems such as plasma FIB and other types, this document is applicable to make TEM specimen of a site-specific lamella by the FIB equipment using Ga-LMIS source, either in a stand-alone FIB or in a FIB-SEM system. Since the basic guidelines in the ex situ lift-out method are the same as those in the in situ lift-out one, this document mainly describes that of the in situ lift-out method.

The basic concept of the present guidelines is as guidelines for automated TEM specimen preparation using FIB fabrication process.

It should be noted other preparation techniques for producing ultra-thin specimens for TEM using FIB are possible by a conventional method such as H-Bar shape, but beyond the scope of this document. For specific cases where the lamella lift-out preparation is unsuitable, readers are referred to the relevant scientific literature.

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 17025:2017, General requirements for the competence of testing and calibration laboratories

ISO Guide 35:2017, Reference materials -Guidance for characterization and assessment of homogeneity and stability

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

accelerating voltage

electrical potential difference between the cathode and the anode, which accelerates ions in an ion gun

3.2

aperture

opening in a thin metal disk or strip used to limit the size of the beam

3.3

beam tails

the outer most edges of the convergent Ga ion beam

Note 1 to entry: beam tails result in a slight angular deviation in milling when a sample is perpendicular to the ion source.

3.4

bend contour

type of diffraction contrast which arises from the local variation of the excitation error in Bragg diffraction condition due to the bending of the specimen

[SOURCE: ISO 15932:2013, 6.1.2]

3.5

box milling

to process a volume of a defined rectangular shape by FIB milling

3.6

Bright field image

image formed using only the non-scattered beam, selected by observation of the back focal plane of the objective lens and using the objective aperture to cut out all diffracted beams

[SOURCE: ISO 15932:2013, 5.5]

3.7

channelling contrast

contrast of formed by secondary electron, backscattered electron or backscattered ion yield differences that depends on the relative orientation of crystalline lattice and incident charged particles

3.8

charge up

situation in which a poorly conductive material is irradiated by an ion or electron beam, causing an accumulation of ion charge on the surface of the specimen - that results in a static electrification phenomenon that reduces the emission of secondary electrons (3.46) and other electrons

3.9

CVD

chemical vapor deposition

a technology for producing high-quality solid thin films and coating by an atomic level chemical reaction

Note 1 to entry: Inorganic thin films are deposited on various substrates.

3.10

combined FIB-SEM

incorporation of an ion beam column and electron beam column focusing both onto the identical interaction point, both beams might be used for imaging, chemical vapor deposition (3.9), and lithography, and the ion beam might be used for milling additionally

3.11

composite materials

material composed of different substances and not of a single substance

Note 1 to entry: Materials are different composition with different mechanical properties such as hardness and strength, and others.

3.12

condenser aperture

opening in a thin metal disk or strip that is used in a fixed or moveable manner in a condenser lens system in order to select a certain portion of the beam

3.13

cross section view

to observe the cross-sectional microstructure of the area of interest

3.14

curtaining

aperiodic stripes on a surface produced by FIB milling

3.15

damage layer

abnormal area where the original structure of the near-surface layer of materials is disturbed by FIB milling process

3.16

deposition gas

inorganic or organic precursor gas to deposit a protected layer on a specimen surface in FIB system

3.17

dislocation

crystallographic defect caused by displacement of atomic arrangement in crystal structure

Note 1 to entry: Dislocation loops are introduced as a damage layer (3.15) of metal and alloy.

3.18

electron lens

basic component of an electron optical system, using an electrostatic field to change the trajectories of the electrons passing through it

[SOURCE: ISO 22493:2014(3.1.3)]

3.19

electrostatic lens

electron lens (3.18) employing an electrostatic field formed by a specific configuration of electrodes

[SOURCE: ISO 22493:2014(3.1.3.1)]

3.20

ex situ lift-out method

method to pick up TEM lamella made by FIB milling (3.22) outside the FIB equipment

Note 1 to entry: A lamellar specimen is attached with a glass rod by electrostatic force and extracted from the bulk material.

3.21

Eucentric position

stage position where tilting the stage results in negligible movement of the specimen from the field of view

3.22

FIB milling

material removal by a beam of ions (usually gallium) focused through a set of electrostatic lenses (3.19) to create a small spot on the substrate

Note 1 to entry: The beam removes material from the substrate through physical sputtering. The beam spot can be scanned across the surface to create a pattern.

[SOURCE: ISO/TS 80004-8:2020, 8.3.10]

3.23

flushing operation

operation of an overvoltage on an ion gun to remove a surface layer of contamination from the ion tip

3.24

Ga ion damage

state in which surface crystal structure becomes amorphous, defect structure including dislocation loops or lattice defects, by Ga ion sputtering

Note 1 to entry: Ga ion mixing occurs in a surface layer of the material, or Ga ion penetrates the grain boundary such as Al alloys.

3.25

H-bar shape

one of traditional FIB fabricated TEM specimen shape, in which centre part of a thin bar material is fabricated by FIB milling to be an electron transparent membrane with the shape of H-bar

3.26

ICP

Inductively Coupled Plasma

a type of plasma source made by several types of gas by excitation to a higher energy level

3.27

ion source/ ion gun

when used in the field of microscopy, the part with a mechanism that generates ions to be formed into a beam by downstream optical elements

3.28

ion milling

technique of removing material from a specimen by sputtering (3.49) by a beam of energetic ions

3.29

ion channelling

physical process occurring in crystalline materials of greater ion penetration along directions of low atomic density

3.30

in situ lift-out method

picking up site-specific regions from the material, using a manipulator (3.36) or similar sample transfer tools inside the FIB chamber under scanning electron or scanning ion microscope (3.45) observations

3.31

J-Cut operation

cutting method used to remove a lamella specimen from the material

Note 1 to entry: It is called J-Cut or U-Cut because of its shape.

3.32

lattice defect

crystallographic defect due to the irregularity in the atomic arrangement in the crystal

3.33

lattice image

high resolution TEM image formed by the interference of transmitted and diffracted beams from a very thin specimen and which represents the periodic lattice structure of the specimen

3.34

LMIS

liquid metal ion source

emitters in which the ion source (3.27) is a metal or alloy that is liquid near room temperature

3.35

ML

machine learning

kind of artificial intelligence and computer technology using statistical methods and algorithms to discover new insights in data mining

3.36

manipulator

needle-shaped operating device that can move freely in an equipment to pick up a lamella and to transfer it to a suitable holder

Note 1 to entry: This holder can be a special fixture designed to fit into the TEM holder on which the specimen is placed after FIB fabrication.

3.37

Micro-sampling

a method of extracting a TEM lamella sample from a small area by manipulating a manipulator in the FIB system, which is a type of in situ lift-out method, and is the name of the technology that first introduced manipulate manipulation into FIB processing technology

3.38

multi-beam FIB system

FIB system equipped with more than two beams in combined FIB-SEM system

Note 1 to entry: Three charged particles columns such as Ga ion, electron, and argon ion are combined in the same housing.

3.39

nanomaterials

material with any external dimension in the nanoscale or having internal structure or surface structure in the nanoscale

[SOURCE: ISO/TS 80004-8:2020, 3.4]

3.40

plan view

to observe the planar microstructure of the area of interest using a TEM lamella cut parallel to the planar sample structures

3.41

plasma ion source

ion source (3.27) from which ions are extracted from a gas in a plasma state

3.42

protective film/protective coating

film which protects a substrate material

3.43

redeposition

phenomenon of accretion of material components removed by FIB milling on the specimen surface

3.44

SEM

scanning electron microscope

instrument that produces magnified images of a specimen by scanning its surface with a focused electron beam

[SOURCE: ISO 16700:2016, 3.1]

3.45

SIM

scanning ion microscope

microscope producing magnified images of a specimen by scanning its surface with an ion beam and detecting secondary electrons (3.46) from the specimen

3.46

secondary electron

atomic shell electrons ejected by energy transfer from an incident primary electron, may be emitted from the sample surface

Note 1 to entry: Its energy is small, 100 eV or less.

3.47

secondary electron detector

detector for detecting secondary electrons (3.46) using a scintillator and a photomultiplier

Note 1 to entry: It is widely used in generic scanning electron microscopes (3.44) and others.

3.48

specimen stage

device, located in the specimen chamber, which enables the specimen to be appropriately mounted, navigated and held in place

[SOURCE: ISO 22493:2014, 4.5.1]

3.49

sputtering

phenomenon in which the surface atoms are removed from a specimen by bombardment with energetic particles such as ions

3.50

stand-alone FIB

FIB equipment with single ion column

3.51

TEM half-grid

sample carrier designed to hold TEM lamellas produced by the in situ lift-out method, to fit into the holder on which the specimen is placed for TEM observation after FIB fabrication

Note 1 to entry: The shape is generally based on a half-moon shape of φ3 mm TEM sheet grid.

Basic FIB equipment has a housing configuration similar to that of a general scanning electron microscope (SEM). As in the scanning electron microscope, an ion beam generated by an ion source is focused onto the specimen surface using electrostatic lenses, while the ion beam is scanned by an electrostatic deflector to modify the specimen surface using the sputtering principle. Secondary electrons and secondary ions are emitted from the area where the focused ion beam is irradiated, and by synchronizing the ion beam scanning with the detection of emitted secondary electrons or ions, a scanning ion microscope image can be obtained on a computer monitor. Figure 1 shows a schematic diagram of a stand-alone FIB equipment.

Key

1 FIB column

2 LMIS

3 accelerator

4 electrostatic lens

5 condenser aperture

6 secondary electron detector

7 gas injection system

8 manipulator system

9 specimen

10 specimen stage

11 computer

12 monitor/scanning ion microscope image

Figure 1 — Equipment configuration of stand-alone FIB system

The FIB gun column mainly consists of an ion source (LMIS), accelerating units, electrostatic lenses, and apertures. The accelerated ion beam is focused by electrostatic lenses, and the amount of ion beam currents is controlled by an aperture size mounted below the condenser lens.

By introducing various precursor gases into the FIB equipment using the gas injection system, a film deposited from gas components can be formed locally where the ion beam is irradiated, which is utilized as a protective film on the surface microstructure during ion beam fabrication. The presence of the protection layer also results in a smoother milled cross-section. The principle to make a protective film is a kind of CVD, and deposition film is also used for the adhesion to attach a manipulator needle tip to the lamellar specimen. The manipulator system is integrated in the same FIB system in order to lift out a lamellar chunk.

Thus, the FIB equipment has mainly four functions:

i) milling of specific locations on the specimen by sputtering

ii) scanning ion microscope imaging

iii) gas deposition system for protective films and manipulator tip attachment

iv) manipulator unit for in situ lift-out method. Note that a different unit is used for ex situ lift-out one

These four functions are the same for the FIB-SEM system with dual guns, which will be explained next.

Systems combining a FIB column in the same housing as the SEM column are being widely used. The SEM function enables the specimen observation before, during and after milling, with less ion damage compared with the stand-alone FIB system, as there is no need to use ion beam imaging to monitor the milling progress. This is extremely useful to precisely determine the endpoint of milling. Furthermore, its functionality can be utilized to construct 3D images of material microstructures by a serial sectioning operation. Figure 2 shows a diagram of the equipment configuration of the combined FIB-SEM system.

An important aspect of the combined FIB-SEM is that the FIB and the SEM columns are tilted towards each other at a fixed angle. This angle θ is typically between 50 and 90 degrees.

Additionally, it is convenient to position the sample at the coincident point of the SEM electron beam and FIB ion beam. At this position it is possible to generate secondary electron or secondary ion images without any movement of the field of view. The system has been developed as a multi-beam FIB system.

Note: It may be necessary to apply some SEM beam shift to minimise any movement of the field of view.

Key

1 FIB column

2 electron gun column

3 secondary electron detector

4 gas injection system

5 manipulator system

6 specimen

7 specimen stage

8 computer

Figure 2 — Equipment configuration of a combined FIB-SEM arrangement

The most widely used ion source for FIB equipment is the Liquid Metal Ion Source (LMIS), with Ga⁺ being the most used ion species by far. With an atomic weight of 69,7 Ga is sufficiently heavy to achieve a good sputtering rate for milling compared to Ar ion. Ga is also a metal with a low melting point of 303 K, and its vapor pressure is very low at about 0,13 Pa. There are two types, one is a capillary type in which metal tubing is used and the other of the needle type in which a droplet of Ga, liquid at room temperature, forms a reservoir from which Ga flows along the needle shaft to wet the tip of the needle. The needle type provides the most stable ion release. A schematic diagram of the LMIS is shown in Figure 3. By applying the electric field, the liquid Ga forms the so-called Tayler cone at the needle tip. Due to the extremely small tip radius of the cone, the electric field at the tip apex is so strong that Ga atoms are ionized and extracted from the cone to be accelerated downstream to selectable energy by applying a respective voltage to an extractor electrode.

Key

1 taylor cone formed by electric field

2 needle

3 filament

4 storage of Ga ion source

5 extracting electrode

6 direction of electric field

Figure 3 — Schematic diagram for LMIS using Ga ion source

Instead of the LMIS, other ion sources equipped with high-frequency inductively coupled plasma have been developed, resulting in different types of FIB systems with ion beams of Xe⁺, Ar⁺, O⁺, N⁺, etc. While Ga⁺ FIBs produce beam currents of up to 100 nA, Xe⁺ FIBs can produce maximum beam currents in the order of 2,5 μA. Comparing beams with the same energy and current, the beam diameter of such so-called Plasma FIBs is generally larger, and its beam profile is less well defined than with a Ga⁺ LMIS FIB. Because the milling rate is more than 10 times faster, Plasma FIBs are more efficient in removing large amounts of material than Ga FIBs. However, the latter are more efficient and more precise in preparing lamellas due to its tighter beam profile.

Scanning ion microscopy is a unique function of the FIB system. Since the scanning ion microscope (SIM) image is characterized by a strong ion channelling contrast, it can be used for qualitative grain orientation imaging, which is useful for identification of target areas for lamella preparation in metal and alloy samples.

Although mostly SEM images are used for microstructural observation in FIB-SEM systems, SIM images sometimes represent microstructures more clearly and stand-alone FIB systems are still being used in this regard.

However, when the SIM image is acquired, the specimen surface is being etched by the ion beam, and if a sufficiently intense ion beam is selected, the etching causes microstructural changes, and an artifact-free scanning ion microscope image cannot be obtained. Therefore, surface microstructure modification must be taken into consideration care under SIM observation. To minimizes surface damage during SIM imaging, much smaller beam currents should be used, while medium to large beam currents will be used for cross-sectioning and lamella preparation, with the exception of small low energy beam currents often being used for the final thinning and polishing steps of lamella preparation.

Since FIB imaging erodes surface structures of the sample, it is necessary to form a deposition layer to protect the target area for TEM lamella preparation of the surface structures. A deposition layer also serves as a cutting mask that reduces milling artifacts and helps to achieve a smooth cross-section. The dominant such artifact is referred to as “curtaining (also called “waterfall effect”), due to its vertically striated appearance. The gases used for metal deposition in this process are generally organometallic crystals that vaporize at relatively low temperatures, such as W(CO)₆. The vaporized gas is injected onto the specimen surface, where a Ga ion beam causes a gas-phase chemical reaction, depositing an amorphous tungsten (W) layer on the target area. Other gaseous precursors for carbon (C), platinum (Pt), silicon oxide (SiO_x), and gold (Au) are commercially available. As another function, this deposition film is highly adhesive and is used to make a bond between the manipulator and the lamella specimen, and to mount it on a TEM.

In both stand-alone FIB and combined FIB-SEM systems, the specimen surface is adjusted to be perpendicular to the direction of the ion beam gun, because the Ga ion beam milling is performed from the vertical direction to the surface. Although the stage setting angle is different as shown in Figures 1 and 2, the entire FIB fabrication sequence to make TEM lamellas is described in terms of a common concept, as indicated by Figure 4. They are called by the lift-out method, or the micro-sampling one.

Key

1 formation of surface protective film at a target area

2 rough fabrication around a target area

3 bottom cut of the target area and a bridge formation (J-cut operation)

4 inserting a manipulator and attaching its tip to the lamella, and cutting the bridge

5 lamella is lifted out

6 mounting the lamella on TEM half-grid by deposition gas

7 finishing fabrication of the lamella to certain thickness

8 polishing to obtain high quality TEM specimen

9 gas injection system

10 protection deposition layer

11 bridge section formed

12 needle shaped manipulator tip

13 cutting the bridge section

14 deposition layer for bonding

15 added protective deposition layer (optional)

Figure 4 — Total flow of FIB fabrication process to make TEM specimen

The sequence of processing steps shown in Figure 4 is described with a specimen for cross-sectional microstructural observation. A similar procedure is used to prepare a plane-view observation specimen, but the shape of the area to be processed is different. Also, the way in which the specimen is placed on the grid after being picked up changes. The difference between cross section and plan view is described in a later section.

The only difference between the procedure shown in Figures 4 and 5 is the additional cut at the left side of the lamella in Figure 4. As this side of the lamella is already cut free, the subsequent undercut has a J-cut operation. In Figure 5, the cut at the left end of the lamella is not done, instead, a U-shaped undercut operation is used. Apart from that, the workflow is the same.

Key

1 rough fabrication around a target area

2 bottom and side cut of the target area and a bridge formation (U-cut operation)

3 inserting a manipulator and attaching its tip to a lamella, and cutting the bridge

4 lamella is lifted out

5 needle shaped manipulator tip

6 bridge section formed

7 cutting the bridge section

Figure 5 — Partial flow of FIB fabrication process with a focus on speed (U-cut operation)

The FIB processing procedures shown in Figure 4 and Figure 5 are both conceptual diagrams. In the case of the combined FIB-SEM system, the protective film is formed on the sample surface in step 1 with the ion beam, where the sample stage is tilted to a position perpendicular to the ion gun. Alternatively, the protection layer can be deposited with the electron beam. In this case the sample stage is in horizontal position. When the rough processing is finished and it comes to the step of cutting the bottom, the sample stage is returned to the horizontal position again, and the bottom is cut with an ion beam from an oblique angle. The only reason why the workflow in Figure 5 is faster is that the additional cut at the left end of the lamella in Figure 4 takes a little extra time.

Since the FIB process is a technique using the sputtering phenomenon to fabricate a surface, the target area for TEM specimen is always protected from the ion beam irradiation. The formation of a protective film is very important, and a thickness of about 1 μm is usually recommended for the surface protective layer. The morphology of the deposition layers is different from the pure components of gases such as C, W and Pt atoms. The structure of the deposition film formed by various gases is mostly amorphous, but in general, the amorphous deposition film has a strong protective function.

With the combined FIB-SEM systems, the surface structure can be observed in the SEM image, so there is no sputter damage, but with a stand-alone FIB system, the surface structure is always observed by the scanning ion image, so even if a weak ion beam is used, it is important to remember that the surface structure is always being sputtered. If the TEM sample is to be prepared while maintaining the surface layer, it is important to pre-coat the region of interest with carbon or another suitable deposition. The thickness of the carbon deposition layer to protect against ion beams during observation of the surface structure should be at least 0,1 μm.

Another important role of the protective film is to use it to locate the position during the FIB fabrication. The position of this protective film is used as a reference point to adjust the subsequent FIB processing.

Since samples often drift while being processed by FIB, FIB systems often have drift correction functions. Since the sample stage is often tilted at more than 50 degrees, the drift correction function is particularly important for the FIB-SEM system. The markings used for the drift correction function are often added using FIB processing adjacent to the protective film, as shown in Figure 6. The user is free to decide the form of this drift correction marking. The marker is mainly used for automatic lamella milling. It is not required if the lamella milling steps are done manually, as in this case the user will correct in between the milling steps for any sample drift.

Figure 6 — Schematic diagram showing protective film and marking for drift correction function

The surface protective coating formation process described above assumes that the surface layer of the area where the TEM sample is prepared is relatively flat. This is because if the surface layer is not flat, curtaining will occur at the cross-section processing area during FIB processing. However, when attempting to lift out TEM specimens from areas with severe surface irregularities, such as metallic fracture surfaces, various pretreatments may be necessary. One method involves levelling the surface by filling in the pitted areas with a conductive coating. After that, a protective film is formed at the targeted location.

After the protective film on the surface layer of the target area is formed, the surrounding area is fabricated by FIB processing. The lamella chunk is milled from three directions, and only one portion is left unprocessed as a bridge, as shown in Figure 7a. The processing area can be defined on the monitor screen as a rectangle or trapezium, and multiple areas can be fabricated automatically by combining these rectangles or trapeziums. This is sometimes called box milling. The fabricated areas 1, 2, and 3 shown in the figure are milled individually or continuously by an auto processing sequence built into the FIB system. Since these procedures vary depending on the FIB equipment used, the best processing method is the one that requires minimum fabrication time. Another method is to box process areas on both sides of the protective film only, as shown in Figure 7b. In this case, only a thin area remains under the protective film, so the sample is tilted and the thin area is fabricated by a U-shape at the end.

Key

1 fabrication area, normally boxed-shape

2 fabrication area, normally staircase shape

3 fabrication area, boxed-shape connecting between area 1 and 2

4 base material

5 protective deposition layer

Figure 7 — Schematic diagram of rough fabrication area

During this rough fabrication process, a high-current beam can be used when large areas are trench milled, but the closer to the target area to be extracted, the smaller current ion beam is used to minimize the roughness of the fabricated surface. 10 to 60 nA, or more are appropriate for the high current level. For the clean-up steps close to the target area, less than 10 nA down to the order of pA is recommended.

As a general processing shape to mill a lamella chunk, box-shaped shapes (see Figure 7, Keys 1 and 3) are set as the processing pattern, and the staircase shaped area (see Figure 7, Key 2) is configured to minimize the fabrication time for the large boxed area. In order to avoid the redeposition on the fabricated flesh surface, it is recommended that the direction of a scanning ion beam is towards the processed surface, as indicated by arrows in Figure 7. The milling time is depended on the selected volume for the rough fabrication process, and the processing time is approximately 30 minutes or less for Si material.

The bottom cut operation to extract a lamella is a unique one for the rough fabrication process, and timing and its procedure is different among FIB systems. In both cases of a stand-alone FIB and a combined FIB-SEM system however, the sample stage is tilted by ϕ degree. The concept is explained by Figure 8, which is a cross-section view of the sample including a lamella roughly fabricated.

Generally, in the case of the stand-alone FIB, it is done at an inclination of Φ = 45 degrees. In the case of the combined FIB-SEM, on the other hand, the direction of the ion gun is inclined about 52 to 58 degrees to the vertical direction, so this bottom cut is performed by returning the sample stage to horizontal. Therefore, the angle of Φ is 52-58 degrees.

Key

1 cross section view of sample after the rough fabrication around the target area

2 cross section view showing the Ga ion beam direction

3 scanning Ga ion beam direction for bottom cutting, including J-Cut and U-Cut operation

4 bridge section left behind to prevent a lamella from falling off during the undercut process

Figure 8 — Schematic diagram showing characteristic geometry of bottom cutting

In the in situ lift-out technique, the defining operation is to pick up the lamella specimen using a manipulator and mount it on a TEM half-grid. Deposition gas is used several times on this process, so careful operation of the manipulator is recommended to avoid colliding with the deposition gun nozzle.

It is noted here that the lamellar specimens for cross-sectional and plan-view operations are mounted in different directions on the TEM half-grid. Figure 9 shows a simple case for the cross-sectional view operation. When the manipulator is bonded on the lamellar surface by the deposition gas, it is difficult to determine if the tip of the manipulator has contacted the specimen surface. The touching of the tip can be observed in the high-resolution SEM and FIB images. If this is not available, and if the specimen or protective film is a conductive material, an electrical signal can be used to confirm the touching. As an alternative method, technology development is underway to use the machine learning (ML) to determine the timing of contact from SIM or SEM images.

Key

1 lamella specimen has been picked up by a manipulator

2 mounting a lamella on a TEM half-grid

3 manipulator operating in a FIB system

4 deposition layers for protecting and bonding

Figure 9 — Schematic diagram for mounting cross-sectional specimen on TEM half-grid

In the case of a plan-view operation, a lamella specimen is mounted on a TEM half-grid from a different direction, as seen in Figure 10. The TEM half-grid is tilted 90 degrees in the FIB system, and a plan-view specimen is glued to it, and then a manipulator is cut. The TEM half-grid is reattached in the original direction for FIB processing, and the protective layer is formed again on the top lamellar surface.

Key

1 plan-view lamella specimen picked up by a manipulator

2 mounting a lamella on a TEM half-grid

3 reset TEM half- grid and addition protective film on top surface

4 manipulator operating in a FIB system

5 deposition layer formed previously

6 deposition layer added on top lamella surface

Figure 10 — Schematic diagram for mounting plan-view specimen on TEM half-grid

A variety of TEM half-grids are available, including copper (Cu), molybdenum (Mo), nickel (Ni), silicon (Si) and others. It is recommended to select a TEM grid material that is compatible with the target TEM lamella specimen, in order to prevent reactions caused by unexpected material components during the finish FIB processing. It is also recommended that the surface morphology of a TEM half-grid is smooth and well flat. Since commercial grid surfaces are not always smooth, it is often required that the grid surface has been pre-smoothed by FIB fabrication before mounting the TEM lamella on it.

Attention to the TEM half-grid shape itself is also recommended to improve the operability as a TEM specimen. TEM specimens are placed on a stage with 3 mm in diameter with tweezers or other tools, which often leads to the specimens being dropped during operation. If a lift-out lamella is on the top of the half-grid, the lamella often breaks first when the grid is dropped. In order to prevent it, concepts of shaping the grid are recommended, as indicated by Figure 11. Figure 11 is not true to scale, since the specimen size is 10 μm in dimension.

Concept 1 is to form taller sections around the lamellar sample so that the bottom edge is contacted first when falling. Concept 2 is to additionally create a space on the underside of the specimen to prevent redeposition. Concept 3 includes concepts 1 and 2 and aims to prevent redeposition by hollowing out the lamellar specimen during additional milling to remove further damage layer on the lamellar.

Key

1 concept 1 with guarding pillars near a specimen position

2 concept 2 with a gap on the underside of the sample

3 concept 3 is side attaching at a grid post to prevent redeposition under additional milling

4 lamella specimen

5 TEM half grid

Figure 11 — Various concepts of TEM half grid shape to protect a lamellar specimen

Finishing FIB fabrication is the most important process in preparing good quality TEM specimens. If the surface protective film has become thin in the previous processes, it is recommended that the surface deposition film is formed again before the finishing process.

The direction of a scanning ion beam is also from towards the processing surface, as seen in arrows in previous Figure 7. In comparison with the rough fabrication process, a small ion beam current is used for processing. It is recommended that the beam current of finish milling is less than 1 nA until 100 nm in thickness, and during further thinning process, the beam current is further decreased, and at the same time, the accelerating voltage itself may be lowered. There are several kinds of fine tuning conditions for the FIB finishing fabrication under 100 nm in thickness, in respect to the materials and the object for the TEM observation.

The suitable lamella thickness is also different among several methods for the TEM observation. The average thickness for the conventional TEM observation is from 50 to 100 nm. However, thinner specimen less than 50 nm are required for atomic level observations using HRTEM and STEM, so it is important to provide a lamella without Ga ion damage layer. In addition to achieving thin specimen thickness, uniformity of specimen thickness is also required in the EDS and EELS measurement using TEM and STEM methods.

The following items should be noted in the finish milling process for a thickness of 100 nm or less.

i) The curtaining phenomenon occurring by inhomogeneous irradiation of Ga ion beams should be suppressed, in order to observe smooth microstructure.

ii) The thickness of the TEM lamellar should be as uniform as possible toward the depth of a specimen.

iii) A damaged layer introduced at the specimen surface during FIB fabrication processing should be minimized.

Key

1 finish fabrication parallel to the lamella plane

2 finish fabrication to a slight tilt to the lamella plane

3 finish fabrication with slight over tilt

4 TEM lamellar specimen with homogeneous thickness

5 Ga ion beam

6 protected layer

7 lamellar specimen

Figure 12 — Schematic diagram of final fabrication process

Although these artifacts remain a challenge in the preparation of good quality TEM specimens, the following countermeasures are recommended for each of them. The convergent nature of the Ga ion beam and beam tails often results in a non-uniform thickness of the lamella during rough fabrication, however, the thickness of the TEM lamella should be as uniform as possible toward the depth of a specimen. To achieve a uniform specimen thickness, the sample is tilted a few degrees towards the ion beam during final processing, as shown in Figure 12.

The damage layer introduced by FIB processing varies depending on the material to be processed, such as Si, metals, and inorganic materials. In order to remove the damage layer, it is known that the voltage during FIB finishing can be reduced from 30 kV to 5 kV, and even further to a few kV or even below 1 kV. Furthermore, after finishing with the Ga-FIB system, the surface layer can be removed by Ar ion milling at a low acceleration voltage.

To ensure the stable operation of FIB equipment stably, Ga ion beams must be emitted from the tip of LMIS during the FIB processing period for the same specimen. From the characteristics of the typical LMIS structure shown in Figure 3, contaminants are incorporated into the liquid metal at the tip apex.

In order to remove the contaminants, a flushing operation is required to maintain the cleanliness of the tip. The necessity of this operation depends on the vacuum level of the FIB equipment and its ion gun section. It is important to remember that ion sources are gradually depleted, and it must be taken care to ensure a stable ion irradiation rate. The beam specifications of general-purpose FIB machines currently available on the market are mostly 1 pA to 100 nA current and 0,5~40 kV voltage.

In FIB processing, typically different beam currents are used for observation, deposition and milling. It is recommended before the FIB fabrication that all of used beams are adjusted so that the beam profile is as narrow as possible and focused on the specimen surface at the desired current level. In FIB-SEM systems, especially, the electron beam and the ion beam are designed to converge at the same point, so the stage height is adjusted to bring the specimen to the eucentric position at that location.

If this adjustment is not performed accurately, it becomes difficult to mill the exact same location with the FIB as the area observed with the SEM, so careful attention is required.

The FIB processing guidelines presented in this document are mainly applicable when processing semiconductor Si device materials. Automated algorithms for FIB processing developed so far in accordance with these guidelines.

It is well known that the ion sputtering rate depends on the material. For example, the sputtering rate is about twice as high as that of Si, when iron (Fe) is fabricated by Ga ion beams. It is recommended to find out firstly how much the processing speed of the target material differs from that of single crystal silicon, in order to determine optimal machining conditions and times immediately.

Silicon carbide (SiC), a material for power semiconductors, is a hard material, and its processing speed is more than three times slower than that of Si. The Ga-FIB processing method makes it difficult to process thin sections in deep regions, and a combination with high-speed processing techniques such as Xe plasma utilization is useful. The lift-out method for preparing TEM specimens of the specific microarea of interest is applicable to the method described in this document.

The property of damaged layers in compound semiconductors such as gallium arsenide (GaAs) is of the lattice defective type rather than amorphous damaged layers in Si processing. Removal of the damaged layer is required in the final polishing process, and the guidelines for preparing TEM specimens by the lift-out method up to that point are common. In order to reduce the damaged layer, the accelerating voltage for the final milling is decreased to less than 1 kV, and or the Ar ion milling method is used normally.

TEM specimens are prepared by the FIB fabrication method for many metallic materials such as steels, copper alloys, aluminium alloys, and several types of thin film materials. In comparison with the Si material, the damage phenomena introduced during FIB processing are different, for example, dislocation loops are introduced on the surface area of metals. However, the procedure for reproducible preparation of TEM specimens from the local area by the lift out method is the same as for Si. Various methods are known to solve this problem, but this is also a highly material-dependent process of finishing milling, which is not within the scope of this document. It is noted that Ga ions react with Al alloys, causing Ga to penetrate the crystal grain boundaries and causing other types of erosion, so it is highly taken care for the fabrication of Al alloys.

In the case of materials consisting of the lamella structure or having different surface layers such as galvanized steels, the FIB fabrication methods is a good way to make a good site-specific TEM specimen from the cross-sectional microstructure observations. However, based on the material properties of the layered structure and coating materials, the FIB fabrication conditions must be selected carefully, and also a cooling stage of the FIB system should be used for low melting point materials such as Zn, Sn and solders.

Non-conductive ceramics, organic materials, and materials with low melting points were recently tried to make by FIB system to prepare the TEM specimens. It is important to reduce the specimen size to prevent charge-up during the ion milling. The specimen shape is not always of a specific size, like metals and semiconductors but can be a fine powder, needle-like, or various other forms. When FIB fabrication on these characteristic shapes is performed, the tiny specimens can be processed by embedding or forming with conductive resin or other compounds.

It has been considered to be difficult to apply the FIB processing for making a TEM specimen of soft materials and biomaterials. However, the technology for reducing ion dose during FIB processing is progressing rapidly, the establishment of the FIB technology as a TEM specimen preparation method for soft materials and others has been done using the cryogenic FIB fabrication techniques.

1. 
   (Informative)
   
   FIB fabrication procedure for single Si (001) wafer
   1. Principle

The process for preparing a TEM specimen by combined FIB-SEM processing with a cross-sectional observation area of around 5 μm x 5 μm is shown using a Si(001) wafer substrate as the standard sample.

• 1. Fabrication Procedure
     1. Protection layer

Carbon deposition was carried out on the whole surface of Si(001) wafer with about 20 nm in thickness.

Using FIB deposition system, a carbon deposition film was formed on an area of 10 μm x 2 μm, and 0,7 μm in thickness. The deposition conditions were as follows: acceleration voltage 30 kV, ion current 0,1 nA, and deposition time 4 min.

Figure A.2.1 — Scanning Ion microscopic (SIM) image of the deposition film on Si wafer,

• • 1. Rough fabrication process

The lamella chunk was fabricated by FIB box milling with high current Ga ion beam, as shown in Figure A.2.2(a). The stage was tilted by 53 degrees in the combined FIB-SEM system. The fabrication conditions were as follows; acceleration voltage 30 kV, ion current 30 nA, and about 1 min. As the next, the clean up fabrication has been done by changing the tilting angle to 51,5 degrees, as seen in Figure A.2.2(b). The fabrication conditions were acceleration voltage 30 kV, ion current 10 nA, and about 1 min.

After the rough fabrication, the sample stage was returned to horizontal position, then the bottom of specimen was cut, as shown in Figure A.2.2(c). In order to provide a bridge, the bottom and its side was cut as the J-Cut operation. The manipulator was inserted to the specimen area and attached to the corner of the lamella chunk using W deposition, as seen in Figure A.2.2(d).

Figure A.2.2 — Rough fabrication and bottom cutting process

• • 1. Extraction of lamellar specimen using manipulator

In the in situ lift-out technique, the lamella specimen was picked up using a manipulator and mounted it on a TEM half-grid, as shown in Figure A.2.3(a). The manipulators glued using W deposition was detached from the sample and the stage was tilted again to the parallel direction to the Ga ion beam, as seen in Figure A.2.3(b).

Figure A.2.3 — Lamella attachment to grid by a manipulator and set for final milling

• • 1. Finishing fabrication process

Finishing FIB fabrication was carried out at accelerating voltage of 30 kV, as shown in Figure A.2.4(a) to (c). The Ga ion current was 1 nA from start thickness down to 500 nm, and 300 pA from 500 nm to 250 nm, and 100 pA from 250 nm to 100 nm. The SEM image of the specimen with 100 nm in thickness is shown in Figure A.2.4(d), in which the cross sectional view size is 5,5 μm x 5,5 μm.

Figure A.2.4 — SIM images on finishing process (a)~(c) and SEM image after step (c).

1. 
   (Informative)
   
   TEM observation results for single Si (001) wafer
   1. Quality of TEM specimen

It is good practice to repeat FIB fabrication and TEM observation cycles until it is confirmed that the TEM specimen is thin enough to allow the desired microstructural analysis. Examples of TEM micrographs of single crystal Si after FIB processing conducted by Annex A conditions are shown in Fig. B.1. The TEM observation has been done at the accelerating voltage of 200 kV. The bright field image of the specimen is described in Figure B.1. The deposition layer of carbon film layer is still left on the top area of the Si lamellar. The curved line contrast observed is a bend contour commonly seen in thin-film samples.

Figure B.1 — Overall image of lamellar Si specimen fabricated by FIB milling

• 1. High resolution lattice image

The material is (001) single-crystal Si wafer, and the cross-sectional microstructure in <110> direction is extracted by the in situ lift out method, which is confirmed by the 110 electron diffraction pattern, as seen in Figure B.2(a). Using the same specimen, another high resolution electron micrograph is shown in Figure B.2(b). This is a crystal lattice structure image taken from the <110> pole direction at 200 to 300 nm from the top surface of the sample. Although it depends on the purpose, it is possible to take high-resolution images of Si single-crystal samples processed by FIB at a high acceleration voltage of 30 kV. However, detailed analysis requires a procedure that can remove the damaged layer on the surface.

Figure B.2 — 110 electron diffraction pattern and lattice image of Si lamellar specimen

Bibliography

[1] D. B. Williams, C.B. Carter: Transmission Electron Microscopy, A Textbook for Materials Science, (2009), Springer

[2] J. I. Goldstein, D. E. Newbury, J. R. Michael, N. W.M. Ritchie, J. H. J. Scott, D. C. Joy, “Scanning Electron Microscopy and X-ray Microanalysis” (2018) Springer

[3] L. A. Giannuzzi, F. A. Stevie, “Introduction to Focused Ion Beams”, (2005) Springer

[4] L. A. Giannuzzi and F. A. Stevie, “A review of focused ion beam milling techniques for TEM specimen preparation” Micron, 30(1999) 197-204

[5] G.Hlawacek, A.Golzhauser: Helium Ion Microscopy, Springer, Berlin/Heidelberg, Germany, 2016

[6] N.S.Smith, W.P.Skoczylas, S.M.Kellogg, D.E.Kinion, P.P.Tesch, O.Sutherland, A.Aanesland and R.W.Boswell, “High brightness inductively coupled plasma source for high current focused ion beam applications”, Jounal of Vacuum Science & Technology B (2006) 24(6), 2902

[7] N. Bassim, K. Scott, L. Giannuzzi, MRS Bulletin (2014), 39(4), 317-325

[8] ISO 22493:2014, Microbeam analysis — Scanning electron microscopy — Vocabulary

[9] ISO 15932:2013, Microbeam analysis — Analytical electron microscopy — Vocabulary

[10] ISO 80004‑8:2020, Nanotechnologies –Vocabulary – Part 8: Nanomanufacturing process

[11] ISO 16700:2016, Microbeam analysis — Scanning electron microscopy — Guidelines for calibrating image magnification