A History of Scanning Electron Microscopy Developments

8/22/2019 A History of Scanning Electron Microscopy Developments

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A history of scanning electron microscopy developments: Towardswet-STEM imaging

A. Bogner a,b,*, P.-H. Jouneau a,c, G. Thollet a, D. Basset b, C. Gauthier a

a Groupe dEtudes de Metallurgie Physique et de Physique des Materiaux, UMR CNRS 5510, INSA de Lyon, Batiment B. Pascal,

7 Avenue Jean Capelle, 69621 Villeurbanne Cedex, Franceb Total France, Centre de Recherche de Solaize, BP 22, 69360 Solaize Cedex, France

c CEA Grenoble, DRFMC/SP2M, Laboratoire dEtudes des Materiaux par Microscopie Avancee, 15 Rue des Martyrs, 38054 Grenoble, France

Abstract

A recently developed imaging mode called wet-STEM and new developments in environmental scanning electron microscopy (ESEM)allows the observation of nano-objects suspended in a liquid phase, with a few manometers resolution and a good signal to noise ratio. The idea

behind this technique is simply to perform STEM-in-SEM, that is SEM in transmission mode, in an environmental SEM.

The purpose of the present contribution is to highlight the main advances that contributed to development of thewet-STEMtechnique. Although

simple in principle, the wet-STEM imaging mode would have been limited before high brightness electron sources became available, and needed

some progresses and improvements in ESEM. This new technique extends the scope of SEM as a high-resolution microscope, relatively cheap and

widely available imaging tool, for a wider variety of samples.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: Electron microscopy; STEM-in-SEM; Transmission mode; Scattered electrons; Environmental scanning electron microscopy; ESEM

1. First steps in scanning electron microscopy

In scanning electron microscopy (SEM), a fine probe of

electrons with energies typically up to 40 keV is focused on a

specimen, and scanned along a pattern of parallel lines. Various

signals are generated as a result of the impact of the incident

electrons, which are collected to form an image or to analyse

the sample surface. These are mainly secondary electrons, with

energies of a few tens of eV, high-energy electrons back-

scattered from the primary beam and characteristic X-rays. This

section reviews the most important steps that have allowed

using such rich physical interaction in a practical tool, making

the SEM the powerful instrument it is today in materials and

life-science.The history of electron microscopy began with the

development of electron optics. In 1926, Busch studied the

trajectories of charged particles in axially symmetric electric

and magnetic fields, and showed that such fields could act as

particle lenses, laying the foundations of geometrical electron

optics (Oatley, 1982 and references therein). Nearly at the same

time, the French physicist de Broglie introduced the concept of

corpuscule waves. A frequency and hence a wavelength wasassociated with charged particles: wave electron optics began

(Hawkes, 2004 and references therein). Following these two

discoveries in electron optics, the idea of an electron

microscope began to take shape.

In 1931, independently of the material wave hypothesis

put forward by de Broglie several years earlier (1925), Ruska

and his research group in Berlin, were working on electron

microscopy. They were disappointed learning that even with

electrons a wavelength would limit the resolution. But they

found using de Broglie equation that electron wavelengths were

almost five orders of magnitude smaller than the wavelength of

light used in optical microscopy. It was thus considered thatelectron microscopes still could prove a better resolution than

light instruments, and no reason existed to abandon this aim. In

1932, Knoll and Ruska tried to estimate the resolution limit of

the electron microscope. Assuming the resolution limit formula

of the light microscope was still valid for material waves, they

replaced the light wavelength by the electrons wavelength at an

accelerating voltage of 75 kV. A theoretical limit of 0.22 nm

resulted, a value experimentally reached only 40 years later.

Although these calculations proved it was possible to reach a

better-than-light-microscope resolution when working at high

www.elsevier.com/locate/micronMicron 38 (2007) 390401

* Corresponding author. Tel.: +33 4 72 43 61 30; fax: +33 4 72 43 85 28.

E-mail address: [email protected] (A. Bogner).

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doi:10.1016/j.micron.2006.06.008
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magnifications (Ruska, 1986), many technical limits had to be

to overcome. Ruska and Knoll tried to implement Buschs lens

formula experimentally. Their work resulted in the construction

of the first transmission electron microscope (TEM) in 1931,

with a magnification of 16 (Haguenau et al., 2003).

Knoll built a first scanning microscope in 1935. However,

as he was not using demagnifying lenses to produce a fine

probe, the resolution limit was around 100 mm because of the

diameter of the focused beam on the specimen. In 1938, von

Ardenne clearly expressed the theoretical principles underlying

the scanning microscope, as we know it today. Because it was

difficult to compete with TEM in resolution achieved for thin

specimens, the scanning microscopy development was oriented

more toward observing the surface of samples. The first true

SEM was described and developed in 1942 by Zworykin, who

showed that secondary electrons provided topographic contrast

by biasing the collector positively relative to the specimen. One

of his main improvements was using an electron multiplier tube

as a preamplifier of the secondary electrons emission current.

He reached a resolution of 50 nm, which was still consideredlow in comparison with the performance obtainable in TEM

(Goldstein et al., 2003).

Indeed, many microscopists in the TEM community were

seeing only limited applications for an instrument having a

lower resolution than a TEM. Fortunately many scientists and

technologists quickly recognised the SEM ability to yield three-

dimensional information from the surfaces of bulk specimens

over a large range of length-scales.

In 1948, Oatley began to build an SEM based on Zworykins

microscope. Following this development, Smith (1956) shown

that signal processing could be used to improve micrographs.

He introduced nonlinear signal amplification, and improved thescanning system. Besides, he was also the first to insert a

stigmator in the SEM to correct lens cylindrical imperfections.

In 1960, Everhart and Thornley greatly improved the

secondary electron detection. A new detector was created with

a positively biased grid to collect electrons, a scintillator to

convert them to light, and a light-pipe to transfer the light

directly to a photomultiplier tube (Goldstein et al., 2003).

In 1963, Pease and Nixon combined all of these improve-

ments in one instrument: the SEM V with three magnetic lenses

and an EverhartThornley detector (ETD). This was the

prototype for the first commercial SEM, developed in 1965

the Cambridge Scientific Instruments Mark I Stereoscan

(Breton, 1999). The SEM we are using today are not verydifferent from this first instrument.

2. What makes the SEM a high-resolution technique?

2.1. Lens aberrations and source brightness limit the

resolution in SEM

In the SEM, electron optics is used to demagnify the size of

the electron source, usually a small tungsten tip, to form the

smallest possible probe. The demagnification is achieved using

a series of condenser lenses, and a final objective lens

also known as the probe-forming lens. This last lens

provides the final demagnification and focuses the electron

beam on the surface. At high magnification, the image

resolution is roughly equal to the width of this probe. It is

limited by a few key parameters: aberrations of the lenses,

especially the objective lens because it works at large

convergence angles, the brightness of the electron source

(Hawkes, 2004), and the interaction volume, especially when

the samples are not very thin.

Aberrations, that are lens imperfections, limit the ability of

focusing the beam, and therefore blur the image. The difficulty

arises from two main aberrations. Due to spherical aberration,

rays travelling far from the optical axis are focused more

strongly than those close to the axis. To reduce this effect, an

objective lens aperture is used to limit the angle of the outer rays

through the lens. Chromatic aberration also blurs the image

since electrons with slightly different wavelengths are focused

more or less strongly. Electron beams with narrow energy

distribution are used to limit this effect. Today, the energy

spread of the beam in a microscope is typically less than 2 eV

for a thermal source and less than 1 eV for field-emissionsources.

The second key parameter limiting the resolution is the

conservation of brightness throughout the microscope column.

It means it is impossible to decrease the size of the electron

probe (by additional optical demagnification) without decreas-

ing the current at the same time. When the electron flux

becomes to low in the probe, the poor signal to noise ratio limits

the resolution.

Two main approaches exist to improve the SEM resolution:

decrease the lens aberrations, or increase the source bright-

ness. Optical aberrations may be reduced by improving lens

design, and by developing electro-optical correcting devices.Much work is currently being carried out on this subject. But

in recent years, most of the progresses in SEM resolution have

resulted from the development of high brightness sources. The

early thermionic sources, still in use in low cost instruments,

are now superseded by field emission and Schottky electron

guns, which exhibits a higher brightness and a better

monochromaticity, as shown in Table 1. Their use leads to

the emergence of the high-resolution SEMs in the 1980s

(Joy, 1991).

2.2. Different technologies of electron sources in the

electron microscope

The first and most widespread electron source in electron

microscopy was the thermionic gun. It consists of a tungsten

filament bent into a V-shaped hairpin, with a tip radius of about

100 mm. This tip spontaneously emits thermionic electrons

when electrically heated to a temperature around 2700 K. The

thermionic electron source presents the advantages to be

relatively inexpensive, and to require a relatively low vacuum.

However, the lifetime of W-cathodes is limited to about 100 h

by evaporation of the cathode material, which result in a failure

when part of the wire becomes too thin. In thermionic LaB6electron guns developed later, the emitting material has a lower

work function, meaning the same amount of electrons can be

A. Bogner et al. / Micron 38 (2007) 390401 391


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emitted at a lower heating temperature. The emitter is a tiny

block of LaB6 single-crystal, about 100 mm in diameter and

about 0.5 mm long, polished to present a 1 mm tip radius.

Depending on the sharpness of the tip, this electron gun exhibits

510 times higher brightness and a 10 times longer lifetime

than a tungsten filament.

The second main technology of electron sources, originally

developed in the 1930s, is based on field emission. A field-

emission gun is a wire with a sharp point, supported by ahairpin. The tip emits electrons by quantum mechanical

tunnelling process when it is brought into close proximity with

a positively biased extraction electrode. Two types of field-

emission sources exist: cold field emission (CFE), and thermal

field emission (TFE), including Schottky field emission (SFE).

In CFE, the high electric field causes electrons to tunnel

through thepotential barrier andleave thecathodewire at room

temperature. Electrons are emitted from a virtual source with

an area of only a few nanometers in diameter. TFE are similar

in principle but operate at a higher temperature. This helps to

keep a clean tip and to reduce noise and instability. Finally, in

SFE, a thin ZrO2 layer is deposited on the flattened tip from asmall dispenser to further reduce the work function. SFE is in

fact a thermionic source but with a brightness comparable to

CFE (cf. Table 1). The main benefit of these field-emission

sources is a higher brightness, of the order of 109 A/cm2 sr at

20 keV. As previously said, brightness is a fundamental limit

for the resolution in the SEM, as it determines the current

available at a given probe size, and sets the recording time per

pixel of the image. Despite this obvious advantage, the

development of field-emission sources took time. Many

technical barriers had to be overcome increasing the

instrument cost. An additional delay may have also been

caused by the great success of the early thermionic SEM

instruments (Pawley, 1997). Besides the high brightness, field-emission guns show another important advantage: the virtual

source is small, usually in the 525 nm range. The optics

required to demagnify this probe for high-resolution observa-

tions is therefore simplified in comparison with the case of

thermionic emitters, where the effective source size is of the

order of 5 mm. Lower demagnification contribute to reduce

lens aberrations. Moreover, the FEGhas a small energyspread,

as low as 0.35 eV for a thermal or Schottky FEG, to compared

with 1.5 eV or more for a thermionic emitter. While chromatic

aberration does not really limit the resolution at high energies

(>10 keV), it is a major factor of improvement for low-energy

imaging (Joy, 1991).

Today, thanks to these new field-emission sources, resolu-

tions on the order of 12 nm are routinely achieved for

commercial instruments operating between 1 and 30 keV.

3. The use of STEM mode in SEM

3.1. STEM mode

The STEM concept was described by von Ardenne in thelate 1930s: he was the first to perform a STEM mode

experiment in 1938, by adding scan coils to a transmission

electron microscope (Goldstein et al., 2003). However, the

STEM did not develop at that time due to a lack of electronics

and adequate electron sources. In 1960s, interest in STEM was

revived by Crewe and coworkers with the development of the

cold field-emission electron source and the optimization of

electron-optical components, culminating in the first visualiza-

tion of single heavy atoms in the electron microscope in 1971.

In a dedicated STEM, the electron optics are designed to

produce an atomic-size beam of electrons that illuminates a

small area on the surface of the specimen. Images are formed byrastering the subnanometer probe over the surface and

collecting electrons that were transmitted through the sample

(unlike in SEM where secondary electrons are used). A STEM

image may be considered as a collection of individual

scattering experiments. The STEM can be considered a low

dose technique, in comparison with fixed-beam TEM. Various

types of signals discriminated in scattering angle and/or energy

loss yield different structural and chemical information, and

may be captured simultaneously. Quantitative analysis may be

performed, with no limitation of the solid angle and the energy

loss interval over which scattered electrons may be collected. In

fact, nearly all electrons coming through the specimen can be

collected by at least one of the detectors. Biological specimensare difficult to see in the transmission electron microscope

because of their low contrast. A solution is to increase contrast

by staining, i.e. heavy atoms addition, however a question arises

whether the observed image features are actually artifacts.

Dark-field STEM imaging is a direct approach that has been

developed to circumvent the need for staining. It is for this

reason, and for low dose techniques requirements, that STEM

was deliberately aimed at biological applications (Colliex and

Mory, 1994).

Although STEM can be performed in a dedicated instrument

specifically designed for the technique, it is more often

developed as a hybrid technique performed on modified SEMs

A. Bogner et al. / Micron 38 (2007) 390401392

Table 1

Comparison of different electron sources at 20 kV (Goldstein et al., 2003)

Source Brightness

(A/cm2 sr)

Lifetime

(h)

Virtual source

size

Energy spread

DE (eV)

Beam current

stability (%/h)

Tungsten hairpin 105 40100 30100 mm 13 1

LaB6 106 2001000 550 mm 12 1

Cold field emission 109 >1000 1000 1000 1530 nm 0.31.0 $1


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or TEMs, microscopes more user friendly and widely available

(Tracy and Alberi, 2004).

A STEM system added to a standard SEM is often designated

as low-voltage STEM, term referring to the 2030 kV regime,

i.e. low relative to typical TEM operating energies. It is near the

lower limit of energies that will provide sufficient transmission

through the sample. As in a SEM, the beam focuses on a small

spot that scans over the sample. Theimage is formedby mapping

some signal intensity synchronously with the scan. As in a TEM,

image information is extracted from electrons that have passed

through a thin sample.

Using the transmission mode in a scanning electron

microscope, both contrast and resolution are improved due

to the lower accelerating voltages, which increase the cross-

sections and reduce the interaction volume of the incident

electron beam (Tracy and Alberi, 2004; Golla-Schindler,

2004). Indeed in transmission electron microscopy, high

electron energies result in a scattering contrast produced by the

narrow cone of transmitted electrons scattered through small

angles and limited by the objective aperture. For contrastenhancement, the electron energy must be reduced and/or in

the case of low atomic number elements the sections stained

with heavy metal compounds. However, low-voltage fixed-

beam TEM is limited by the strong decrease in transmission

with decreasing electron energy and by chromatic aberrations

from more frequent energy losses. Low-voltage scanning

transmission electron microscopy can be an interesting

alternative. The transmission mode in SEM has an advantage

of avoiding chromatic aberration. As there is no projection

lens, no image deterioration occurs due to chromatic

aberrations, even in the case of inelastic interactions at low

voltages. As already suggested by von Ardenne, the potentialadvantage of transmission-SEM indeed lays in the fact that

electrons are not required to pass through a lens after crossing

the specimen. The spread of velocities caused by absorption in

the specimen therefore do not give rise to chromatic aberration

induced loss of resolution, as it does in a conventional

transmission microscope: it is thus possible to examine a

thicker specimen (Oatley, 1982). It is an argument also cited by

Merli et al.(2004): dueto theabsence of image-forming lenses,

the energy loss and the large scattering angles do not affect the

resolution as in transmission electron microscopy.

In low-energy STEM, the specimen response will be

proportional to the average scattering power of atoms which

will give rise to massthickness contrast, or absorption contrast.

For a good Z-contrast, it is necessary to use large scattering

angles in transmission. In the transmission mode of SEM, the

aperture can be increased to obtain a high transmission and

signal-to-noise ratio.

SEM remains one of the most flexible tools for high-

resolution imaging (resolution down to 1 nm), however 1 nm

resolution is sometimes not achieved when characterizing

ordinary samples, due to limitations arising from the nature of

beamsample interactions when secondary electrons signal is

used. STEM-in-SEM is one method that can overcome theselimitations and enable imaging with high resolution (Vander-

linde, 2005). As the samples investigated are thin in comparison

with SEM, the interaction volume is rather small. For this

reason, in STEM mode, the resolution is only limited by the

spatial broadening of the electron probe at the exit surface of the

sample, whereas structures at the entrance surface are resolved

with a resolution of approximately same as the diameter of the

electron probe. This beam broadening phenomenon is called

the topbottom effect (Golla-Schindler, 2004). For illustra-

tion, Fig. 1 shows the investigation of small indium cluster on a

formvar film with a polystyrene sphere placed on the indium

layer. This test structure can be investigated with the indiumcluster above the polystyrene sphere (top object) or below

(bottom object) in STEM-in-SEM, mode called transmission-

SEM by the authors (Fig. 1a and b) or in TEM (Fig. 1c and d).


Fig. 1. Illustration of the topbottom effect according to Golla-Schindler et al. (Golla-Schindler, 2004).


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Improved sharpness of the indium cluster can be obtained by

using the top object and the transmission-SEM mode (Fig. 1a).

Due to the parallel illumination in TEM the best object is the

bottom object, but the resolution of the indium cluster is limited

by the chromatic aberration of the post specimen objective lens

(Fig. 1c). Therefore, for TEM investigations thinner sections

and increased accelerating voltages are necessary to improve

the attainable resolution (Fig. 1d).

Today, every microscope manufacturer offers a STEM

detector for SEM. In addition with the success related to SEM

ease of use and availability when compared to TEM, it is indeed

recognised that STEM-in-SEM extends the usefulness of the

SEM (Woolf et al., 1972). On one hand, thicker samples can be

observed thanks to large collection angles, and high contrast is

results from the use of low voltages. On the other hand, high

resolution is available thanks to limited interaction volume and

chromatic aberration.

3.2. STEM-in-SEM detection methods

In the present article, we are interested in the transmission

mode performed in SEM. Several detection strategies for

transmitted 1030 keV electrons exist, using several types of

STEM detectors.

3.2.1. Transmitted electrons/secondary electrons converter

and EverhartThornley detector (Golla et al., 1994; Golla-

Schindler, 2004; Vanderlinde, 2005)

Various devices have been developed using the following

idea, that does not require the development of a new detector:

transmitted electrons (TE) are converted to secondary electrons

(SE) using a plate below the sample: the converter. SEs can thenbe detected by using a conventional EverhartThornley

detector (ETD). This is one of the configurations exhibiting

the advantage of TV-rate image.

This principle is applied in the configuration used by Golla

et al. presented in Fig. 2. In their device, a cylindrical shield

stops the reflected secondary electrons, so that only

transmitted or scattered electrons can be collected by the

ETD. An electron trap also prevents large angle scattered

electrons from reaching the ETD. An adjustable aperture

determines the collection angle for the bright-field mode or the

angle segment for the dark-field mode.

3.2.2. Direct collection of transmitted electrons by a solid-

state detector

Transmitted electrons can also be directly collected by a

semiconductor detector. In this case, electrons reaching the

detector are physically detected as they create electronhole

pairs in the silicon diode ($2700 pairs by an 10 keV electron

incident on the detector), resulting electrical charge is collected

from the biased pn junction of the detector.

The configuration described in Fig. 3 has been used by Merli

et al. Transmitted electrons are collected using the detector

normally used for backscattered electrons (BSE) but placed just

below the sample (Merli and Morandi, 2005). There are several

positions for the detector: (1) by placing the center of the

detector on the optical axis and varying the specimen-detectordistance, it is possible to select the angular distribution for dark-

field imaging (Fig. 3a); (2) by removing the center of the

annular detector from the optical axis and defining with an

aperture a portion of the detector itself, it is possible to define

the angular range of transmitted electrons producing the bright-

field image (Fig. 3b).

It should be noted that in the case of a dipolar detector, two

diodes are placed on the optical axis. In this way, bright- or dark-

fieldimages can be acquiredthroughthe collection of transmitted


Fig. 2. Scheme of a STEM-in-SEM system: TE/SE converter and ETD con-

figuration (Golla-Schindler, 2004).

Fig. 3. Merli et al. STEM-in-SEM detection conditions: (a) BSE annular

detector centered on the optical axis for dark-field imaging; (b) off-axis annular

detector with an aperture on thecover to define the collection anglea for bright-

field imaging. Scheme courtesy of Merli et al. (Merli and Morandi, 2005) and

Microscopy and Microanalysis from Cambridge University Press.


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or scattered electrons, respectively. Thissetup is implementedfor

example in the FEI high vacuum STEM holder.

3.2.3. Transmitted electrons/photons conversion via a

scintillatorSTEM-in-SEM has become an established technique because

it extends the capabilities of the SEM and offers considerable

advantages in signal processing and analysis when compared to

the TEM (Woolf et al., 1972). The detection strategy of Woolf

et al. in their transmission stage for the scanning electron

microscope is presentedin Fig.4. It useda scintillator/light-pipe

combination. After their path through the sample, electrons are

converted to photons by a scintillator and pass through a light-

guide. The signal is amplified afterwards by a photomultiplier.

This is a relatively expensive standard detector system, but it

allows TV-rate imaging, and the selection of diffracted beams.

Each of the three precedent detection strategies presentsadvantages, but practically a type of detector is often adopted

because of financial or availability reasons.

4. Environmental SEM development

The origin of environmental SEM is directly linked to the

high vacuum needed in electron microscopes, that introduce

restrictions on the way that certain specimens are prepared and

imaged. Very early in the history of electron microscopy,

studies were related to the possibilities of imaging specimens

in a more natural state. In the 1950s, experiments concerned

differentially pumped, aperture-limited TEMs, or creation of

environmental chambers. The separation of high vacuumelectron gun chamber from a gaseous specimen chamber via

open diaphragms (PLA for pressure limiting apertures) was

first used in several high-voltage TEM. Research by Danilatos

and Robinson in the 1970s led to the first SEM capable of

maintaining a relatively high pressure, removing the need to

dry and coat the specimens (Danilatos, 1991). The term

environmental SEM was introduced in 1980. As mentioned

in Section 1, during the first development steps of SEM, the

established TEM community was very suspicious. In the same

way, the SEM community was not convinced when the first

environmental SEMs appeared (Stokes, 2003). The introduc-

tion of gas in an electron microscope is completely opposed to

the generally established ideas of a clean high vacuum. It was

generally perceived that a gaseous environment would

deteriorate the resolution by an assumed broadening of the

electron beam and exclude the SE detection mode of imaging

because highly positive bias of the ETD induces arcing in the

presence of gas. To convince the microscopy community, these

two main problems i.e. reduced resolution, and exclusion of

SE detection had to be resolved with the: (1) determination

that the electron probe diameter remained small at elevated

pressure; (2) invention of the gaseous detection device (GDD),

where the principle of gaseous ionization produced by the

signal-gas interaction is used for imaging; this GDD wascalled

GSED (for gaseous secondary electron detector) to insist on

the fact that it is possible to detect secondary electrons in

ESEM (Danilatos, 1991). Over more than two decades,

Danilatos showed tenacity and developed the ESEM instru-

ment to a point where the rest of the scientific community and

manufacturing world could further benefit. ESEM became a

stable instrument. By the late 1980s, the first commercial

environmental scanning electron microscopes (ESEMs byElectroscan, a company created for the purpose of gaseous

microscope manufacturing) were being produced, opening up

a world of possibilities for observing untreated specimens. A

few years later in 1996, a traditional electron microscope

manufacturer Philips FEI took over the operation to further

commercialize the instrument. Other manufacturers have in

the meantime followed with low-vacuum systems reaching

100 Pa, the level of pre-ESEM technology, probably due to

patent restrictions (ESEM Research Laboratory, Australia).

The ESEM has rapidly gained acceptance by the scientific,

technical and industrial community as shown by the large

number of publications arising from its use.To summarize, ESEM differs from conventional SEM

mainly by the presence of a gas in the specimen chamber.

Samples are thus not viewed under high vacuum but under a

deteriorated or low vacuum. This is possible thanks to a

special design of electron optics column that allows

differential pumping: the column is divided into different

pressure zones separated by pressure limiting apertures

(Danilatos, 1993). The presence of gas, or environment around

the sample that inspired the term environmental SEM, can

play two main roles. The first, common for environmental as

well as low-vacuum SEMs,1 is electronic. The gas acts as an

electrical charge conductor avoiding sample charging and

facilitates signal detection. This first role is described in Fig. 5:collisions between electrons and gas molecules create positive

ions that can balance the accumulation of negative charges on

the surface of specimens, and cascade electrons that help to

amplify the signal collected by the gaseous secondar electrons

detector (GSED). The second role, more specific to environ-

mental SEMs, is thermodynamic, i.e. the gas is a conditioning

medium, preventing evaporation of liquids from a sample

(Thiel and Toth, 2005).


Fig. 4. Scheme of Woolf et al. transmission stage for the SEM, including a

scintillator. Scheme courtesy of David Joy (Woolf et al., 1972) and IOP

Publishing.

1 ESEM is a trademark from FEI Company, but other constructors have

developed their low-vacuum SEM too.


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With this specialized form of electron microscopy a wide

range of insulating materials, oils, liquids, etc., can be observed

at equilibrium or during in situ evolutions. The potentialapplications are described in numerous recently published

articles, in particular by Donald (2003).

In this paper, we discuss liquid specimens, most of the time

aqueous. Water vapour is the most common gas used in ESEM,

due to its efficiency in signal amplification, and its useful

thermodynamic properties leading to good quality images and

specimen stability.

Fig. 6 is an example concerning the amplification signal of

water, compared with four other gases: carbone dioxide, nitrous

oxide, nitrogen, helium (Fletcher et al., 1997). The authors

conclude that water vapour gives quantitatively the largest

maximum amplification of all the cases considered in their

study (variable accelerating voltage and partial pressure). It

should also be noted that the maximum of these amplification

curves is around 45 Torr for water.

Concerning aqueous specimen stability, it is important to

consider the thermodynamic saturated vapour pressure (SVP)

curve for water as reproduced in Fig. 7 (Stokes, 2003). It can

be seen that the pressure range up to around 10 Torr meets the

SVP when the specimen temperature is lowered to a few

degrees Celsius from ambient. Typically, a water-cooled

Peltier stage is employed to keep the specimen at around3 8C, within a chamber environment of water vapour at a

pressure in the region of 4.5 Torr, leading to improved


Fig. 5. A schematic of the amplification process due to collisions between the

secondary electrons and gaseous molecules. Image courtesy of D.J. Stokes

(Stokes, 2003) and the Royal Society of U.K.

Fig. 6. Dependences of the amplification on the pressure for various primary-beam energies and for five gases: (a) water vapour, (b) carbon dioxide, (c) nitrous oxide,

(d) nitrogen, and (e) helium. Figures courtesy of Bradley Thiel ( Fletcher et al., 1997) and IOP Publishing.


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specimen stability and good quality images. It is also essential

to ensure that the specimens natural moisture is preserved

during the initial pumpdown of the chamber. It is therefore

usual to perform a sequential pumpdown such that the air inthe chamber is successively replaced with water vapour, and

that evaporation from and condensation onto the sample are

minimized (Cameron and Donald, 1994).

ESEM also extends the possibilities of SEM in term of the

wide variety of samples and of states that can be characterized.

However, in the case of liquids, classical wet mode in ESEM

only allows the observation of the surface, which is often very

smooth so that very little information about the sample can be

collected. For this purpose, the wet-STEM imaging mode

described hereafter may be suitable for aqueous samples, or

other liquids.

5. Wet-STEM imaging: observing a thin liquid film in

transmission mode

It actually refers to the STEM-in-SEM applied to environ-

mental SEM, benefiting from the improved FE SEM

performance in STEM mode and low-vacuum techniques.

5.1. History

Bultreys and Thollet were the pioneers of the wet-

STEM imaging mode a few years ago. During one of their

experimental collaborations in the GEMPPM laboratory in

Lyon, they placed a TEM grid on a Peltier stage of an FEI XL30 FEG ESEM, and relocated the dipolar detector usually

used for backscattered electrons detection below the grid: the

same configuration used in the high vacuum STEM detector,

already commercially available in FEI XL series SEMs, but

adapted for the use on a cooling stage.

A series of investigations were conducted by Bogner et al.,

examining the experimental procedure and signal detection

conditions as well as the influence of gas purge, type of grids,

sample-detector distance, annular dark-field detection (Bogner

et al., 2005).

The wet-STEM instrument in our laboratory is described as

follows.

5.2. Experimental setup of wet-STEM

As the presented imaging mode allows the observation of

wet samples in transmission mode, the term wet-STEM is

self-explanatory. The device, described in Fig. 8, is placed in an

FEI XL 30 FEG ESEM. A copper grid is placed on a TEM

sample holder, and positioned on a Peltier cooling stage. A

small amount of liquid containing particles or floating objects

(organic, inorganic, liquid or solid) is dropped on the grid with amicropipette.

As in the conventional wet mode of ESEM and in relation

with the work described in Cameron and Donald (1994), we use

an optimized pump down sequence in order to prevent

evaporation from, and condensation onto the sample droplet.

Classical ESEM detectors are also available enabling to

control of the sample surface in SE mode and in BSE mode,

using the gaseous SE detector (GSED), and the gaseous

backscattered electron detector (GAD), respectively. This is

very helpful for example to control the presence of liquid until

the thickness is adequate for transmission imaging to perform

transmission observations and to detect whether objects are

submerged in water.When the required partial pressure of water is reached,

pressure and temperature can be adjusted to evaporate a small

amount of water if the considered sample is aqueous from

the droplet. It allows obtaining a water layer thin enough such

that the incident electrons can pass through it, and can be

collected to form a STEM image. Films of wet samples are

thinned in situ in the ESEM chamber, their thickness depends

on the quantity of water evaporated from the initial droplet. As

evaporation is an endothermic reaction, it is then possible to

follow it by checking the difference between the setting

temperature and the measured one. Then, the thickness of the

film is kept constant thanks to an equilibrium water pressure


Fig. 8. The wet-STEM device at the GEMPPM laboratory: (a) Peltier stage; (i)

incident convergent electron beam; (b) SEM mount maintaining the TEM grid;

(c) solid-state annular detector.

Fig. 7. Saturatedvapour pressurecurvefor water. Image courtesyof D.J. Stokes

(Stokes, 2001) and Wiley.


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using the (P, T) water diagram (presented in the precedent

section). For instance, a water pressure of 5.3 Torr is required at

a sample temperature of 2 8C, so that objects remain in a water

layer with constant thickness. By controlling the sample

temperature through the Peltier stage, and using water vapour

as the imaging gas at a controlled pressure, samples can be kept

above their saturated vapour pressure during all the experiment.

When the required thickness is obtained, the incident

electron beam is focused on the droplet, and passed through the

liquid layer and floating nano-objects. The signal is then

collected by a detector, usually used for the collection of

backscattered electrons, but in the present configuration located

below the sample. Holey carbon coated TEM copper grids

placed with the carbon layer faced pointing down enable copper

squares to play the role of retention basins. In the carbon layer,

holes of typical diameter ranging from less than 1 to 20 mm

allow maintaining overhanging liquid films on very small areas.

It is important to note that adequate initial droplet parameters

i.e. volume and solid content enable to control the amount of

nano-objects on the grid. It is possible, for instance in the caseof a latex emulsion sample, to choose to image only a

monolayer of particles if required. Due to the initial

concentration of the suspension, when the droplet of smallest

volume that can be dropped contains more objects than for a

monolayer on the grid, a dilution step with distilled water can

be performed before placing the liquid on the grid.

As discussed above, several detection strategies can be

implemented for transmitted electrons detection in an SEM.

The present device is based on the direct collection of electrons

passed through the sample by a solid-state detector (two semi-

annular detectors A and B). For the setup usually used in STEM

detectors for FEI SEMs, the incident electron beam arrives onthe border of diodes A and B, and bright- or dark-field images

can be produced with the collection of transmitted or scattered

electrons, respectively. In the latter case, only a small area of

the sample is above the border of diodes A and B; so that we do

not have the choice of the imaging mode (bright- or dark-field)

for the other areas. In the present study, we choose another

possibility: annular dark-field imaging conditions can be

obtained if the dipolar detector is placed on the optical axis so

that the direct transmitted electron beam is not collected and

only scattered beams are detected on a ring constituted by both

of the diodes A and B. Using this method, a more important part

of the scattered electrons available is used to form an image and

higher-contrast images can be obtained. Consequently, all theESEM micrographs presented in the following section have

been obtained using the annular dark-field configuration. The

second advantage of this configuration, using the summed

signal, is that imaging conditions are not linked to the area of

the sample imaged. Moreover, in our experimental setup, the

distance between the sample and the detector has been

experimentally investigated in order to optimize the contrast.

Best results have been obtained with a distance of about 7 mm,

corresponding to collection angles between 349 and 820 mrad

for the dark-field mode. Nevertheless, the optimal distance

between the grid and the detector depends on the sample

composition and thickness, and should be a variable parameter.

5.3. Benefits of STEM-in-SEM mode

By using low voltages, which lead to improved contrasts,

and thanks to the absence of a projection lens when compared to

TEM, thicker samples can be imaged using the present STEM

mode. For example, an estimation of the thickness of water that

can be passed through with an incident electron voltage of

30 kV is presented in Fig. 9. It has been estimated using an

evoluted Monte Carlo Model applied by SAMx1 in their

Hurricane software. This powerful tool has been specially

adapted so that it is possible to consider transmitted electrons

and to store them as a function of their energy and their

scattering angle. Here, transmission is defined as the ratio

between the number of electrons scattered with angles from 349

to 820 mrad and the number of the incident electrons. It can be

shown that transmission occurs in as much as several microns

of water. Although it is not within the scope of the present

article, it should be pointed out that this special extension of

Hurricane is also very helpful to progress in wet-STEM images

contrast interpretation.Theoretically, the resolution of wet-STEM mode should be

better than seen in ESEM wet mode, due to the properties of the

thin sample (STEM-in-SEM a high-resolution mode in SEM

because there is no interaction volume, only topbottom effect:

see Section 3), and that contrary to TEM, electrons do not have

to pass through a lens after their path through the sample (see

Section 3 also). Unfortunately in both cases (wet mode in

classical reflection ESEM, and wet-STEM), we have observed

that the resolution is limited by the mechanical vibrations

partially due to the cooling flow of the Peltier stage and to the

vacuum system.

We are limited to relatively high beam energies (2530 kV)when compared to the range typically used in SEM (a few eV

to 30 kV). Indeed, in environmental or low-vacuum

SEMs, the electrons leaving the specimen are significantly

retarded by their collisions with gas molecules reducing their

energies. The use of a TE/photons conversion via a scintillator,

as proposed by KE company in the Centaurus detector, could

be used to explore other imaging conditions in wet-STEM,


Fig. 9. Evaluation of the scattering transmission through a water layer at 30 kV

with Hurricane (SAMx1) Monte Carlo simulation: scattered electrons are

collected from 349 to 820 mrad.


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such as using lower. This detector is very sensitive at low

energies (0.5 keV) and offers the advantage of TV-rateimaging.

As a feature of STEM-in-SEM imaging mode, wet-STEM

mode improves contrast and resolution. This technique also

improves volume information in comparison with classical wet

mode in ESEM, where only the surface of liquids can be

observed.

6. Applications of wet-STEM imaging

Using the wet-STEM imaging mode described previously, a

wide variety of nm- to mm-scale objects suspended in a liquid

layer (not only water) can be investigated (Bogner et al., 2005).The present imaging conditions correspond to annular dark-

field mode, using very large collection angles. An acceleration

voltage of 30 kV has been chosen to optimize resolution and

contrast.

An image of aqueous suspension of gold nanoparticles is

presented in Fig. 10. This image highlights the high resolution

of wet-STEM imaging: particles with diameter of 20 nm are

well resolved; the resolution is estimated as low as 5 nm. Au

particles exhibit a very high bright contrast, as expected when

considering their high atomic number.

Even objects constituted of lighter elements induce an

observable contrast in wet-STEM using annular dark-field

detection. Fig. 11 presents images of colloidal clay called

chocolate referring to the colour of the aqueous solution.

Clay platelets typical sizes are 0.1mm 0.5 mm 2 mm.

These objects can be observed using a cryo-TEM, but exhibit

little contrast when they are disposed as to be imaged through

their thinner thickness. Wet-STEM imaging mode corre-

sponds to a scattering contrast: whether the flattest face of a

platelet is parallel or perpendicular to the grid, the scattering of

electrons will occur in a different local thickness. In wet-

STEM, perpendicular platelets show brighter contrast than

others because of the important local thickness acting for the

scattering of electrons, and some lamellar structures are

observed; even parallel platelets exhibit a good contrast.

Contrast interpretation is not always trivial, as suspensions

often contain several additives, that may modify the scatteringof electrons, or the sample sensitivity to the electron beam.

Fig. 12 presents carbon nanotubes in two different solutions: (1)

water with two different concentrations of sodiumdodecylsul-

fate (SDS) surfactant; (2) pure ethanol. Micrometric holes

present in the carbon layer of the grid are observed as dark

regions. In Fig. 12a, carbon nanotubes are well distinguished,

and some brighter contrasts correspond to superpositions, or

heavy atomic number particles resulting from the carbon

nanotubes synthesis. In Fig. 12b, carbon nanotubes are also well

distinguished but the presence of water introduces a fuzziness,

and some bright nm-scale features are present. The latter

objects are understood as surfactant clusters, and are found tobe larger and more numerous in a solution containing more

surfactant: Fig. 12c.

Biological specimens, typically low in atomic number and

sensitive to beam damage and dehydration, have also been

successfullyobservedin wet-STEM. Fig. 13 presents an image of

bacteria of the species Pseudomonas syringae, acquired at a

magnification of50 000. The contrast of the bacteria is very


Fig. 10. Au particles suspended in water, and imaged in the wet-STEM annular

dark-field mode.

Fig. 11. Chocolate colloidal clay in water imaged in wet-STEM annular dark-field imaging mode.


11/12

good without preliminary staining. Moreover, volumic informa-

tion is obtained as double membrane structure is distinguished.

Others types of samples have been imaged in wet-STEM

(Bogner et al., 2005) such as mini-emulsions, latices,

bacteriophages, particles in oil, etc. In fact, the term wet

is restrictive in comparison with the effective imaging

possibilities of the wet-STEM imaging mode: provided that

the liquid is compatible with the microscope, a thin layer of

nonaqueous liquids can also be studied, its stability only

depending on its saturated vapour pressure.

7. Summary and outlook

The history of electron microscopy presented in this article

highlights the extent of SEM applications. SEM is not incompetition with TEM as it allows different imaging modes.

Wet-STEM, i.e. STEM-in-SEM performed in environmental

SEM, has been presented as an powerful imaging technique

developed thanks to general progress in electron microscopy. It

allows straightforward transmission observations of wet

samples constituted of nano-scale objects in a liquid layer.

With the benefits of field emission and STEM mode applied in

SEM, an excellent resolution can be achieved. For example

5 nm was achieved on a resolution test sample of gold

nanoparticles in colloidal suspensions. Thanks to the low

operating voltage of an SEM and large scattering angle

collection, the contrast is enhanced, this result is especially


Fig. 12. Carbon nanotubes: (a) dispersed in ethanol without surfactant; (b) in water with surfactant at the concentration C1; (c) in water with surfactant at theconcentration C2 > C1.

Fig. 13. Bacteria Pseudomonas syringae, imaged in wet-STEM annular dark-

field imaging mode.


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interesting for low atomic number materials. Low-vacuum

technology allows imaging samples in their native state. The

scanning mode allows to image samples at a low dose, that is

important when examining polymers and biological samples.

Finally, the transmission mode gives access to volume

information since that we do not image only the surface of

the liquid drop. Particularly adapted for suspension-type

samples, and others delicate objects with nanometric features,

wet-STEM allows characterizations in materials science as well

as life-science at the nano-scale.

Acknowledgments

We are very grateful to D. Bultreys from FEI Company

(Brussels) for shared experimental sessions and discussions.

We also would like to acknowledge authors for figures reprint

permissions.

References

Bogner, A., Thollet, G., Basset, D., Jouneau, P.-H., Gauthier, C., 2005. Wet

STEM: a new development in environmental SEM for imaging nano-objects

included in a liquid phase. Ultramicroscopy 104, 290301.

Breton, P.J., 1999. From microns to nanometers: early landmarks in the science

of scanning electron microscope imaging. Scanning Microsc. 13-1, 16.

Cameron, R.E., Donald, A.M., 1994. Minimizing sample evaporation in the

environmental scanning electron microscope. J. Microsc. 173, 227237.

Colliex, C., Mory, C., 1994. Scanning transmission electron microscopy of

biological structures. Biol. Cell 80, 175180.

Danilatos, G.D., 1991. Review and outline of environmental SEM at present. J.

Microsc. 162, 391402.

Danilatos, G.D., 1993. Introduction to the ESEM instrument. Microsc. Res.

Tech. 25, 354361.

Donald, A.M., 2003. The use of environmental scanning electron microscopy

for imaging wet and insulating materials. Nat. Mater. 2, 511516.Fletcher, A.L., Thiel, B.L., Donald, A.M., 1997. Amplification measurements of

potential imaging gases in environmental SEM. J. Phys. D 30, 22492257.

Goldstein, J., et al., 2003. Scanning Electron Microscopy and X-ray

Microanalysis, 3rd ed. Plenum Press, New York.

Golla, U., Schindler, B., Reimer, L., 1994. Contrast in the transmission mode

of a low-voltage scanning electron microscope. J. Microsc. 173-3, 219

225.

Golla-Schindler, U., 2004. STEM-Unit measurements in a scanning electron

microscope. In: Proceedings of the European Microscopy Congress in

Antwerpen.

Haguenau, F., Hawkes, P.W., Hutchison, J.L., Satiat-Jeunematre, B., Simon,G.T., Williams, D.B., 2003. Key events in the history of electron micro-

scopy. Microsc. Microanal. 9, 96138.

Hawkes, P., 2004. Recent advances in electron optics and electron microscopy.

Ann. Fondation Louis de Broglie 29-1 .

Joy, D.C., 1991. The theory and practice of high-resolution scanning electron

microscopy. Ultramicroscopy 37, 216233.

Merli, P.G., Morandi, V., Corticelli,F., 2004. Lowenergy scanning transmission

electron microscopy: influence of the electron detection mode. In: Pro-

ceedings of the European Microscopy Congress in Antwerpen.

Merli, P.G., Morandi, V., 2005. Low-Energy STEM of multilayers and dopant

profiles. Microsc. Microanal. 11, 97104.

Oatley, C.W., 1982. The early history of the scanning electron microscope. J.

Appl. Phys. 53 (2).

Pawley, J., 1997. The development of field-emission scanning electron micro-

scopy for imaging biological surfaces. Scanning 19, 324336.Ruska, E., 1986. The development of the electron microscope and of electron

microscopy. Nobel Lecture.

Stokes, D.J., 2001. Characterisation of soft condensed matter and delicate

materials using environmental scanning electron microscopy (ESEM). Adv.

Eng. Mater. 3-3, 126130.

Stokes, D.J., 2003. Recent advances in electron imaging, image interpretation

and applications: environmental scanning electron microscopy. Phil. Trans.

R. Soc. Lond. A 361, 27712787.

Thiel, B.L., Toth, M., 2005. Secondary electron contrast in low-vacuum/

environmental scanning electron microscopy of dielectrics. J. Appl. Phys.

97 (051101), 118.

Tracy, B., Alberi, K., 2004. Adopting low-voltage STEMand automatedsample

prep to perform IC failure analysis. Micromagazine 8793.

Vanderlinde, W.E., November 2005. Ultra-high resolution in the scanning

electron microscope (SEM). In: ISTFA Proceedings.Woolf, R.J., Joy, D.C., Tansley, D.W., 1972. A transmission stage for the

scanning electron microscope. J. Phys. E 5, 230233.


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A History of Scanning Electron Microscopy Developments