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    Micron 38 (2007) 390401

    A history of scanning electron microscopy developments: Towards

    wet-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 the wet-STEM technique. 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

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

    E-mail address: Agnes.Bogner@insa-lyon.fr (A. Bogner).

    0968-4328/$ see front matter # 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.micron.2006.06.008

    time, the French physicist de Broglie introduced the concept of

    corpuscule waves. A frequency and hence a wavelength was

    associated 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 that

    electron 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

    mailto:Agnes.Bogner@insa-lyon.frhttp://dx.doi.org/10.1016/j.micron.2006.06.008

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

    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 thediameter 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 considered

    low 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 the

    scanning 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 very

    different 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-emission

    sources.

    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 electronswhen 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) 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

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

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

    hairpin. 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 the potential barrier and leave the cathode wire 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 a

    small 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 reducelens aberrations. Moreover, the FEG has a small energy spread,

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

    with 1.5 eVor 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-energyimaging (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 the

    late 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 by

    rastering 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 specimens

    are 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) 390401 393

    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. The image is formed by 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 contrast

    enhancement, 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 potential

    advantage 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

    Fig. 1. Illustration of the topbottom effect according

    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): due to the absence 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 these

    limitations 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 indium

    cluster 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).

    to Golla-Schindler et al. (Golla-Schindler, 2004).

  • A. Bogner et al. / Micron 38 (2007) 390401394

    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 then

    be 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

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

    figuration (Golla-Schindler, 2004).

    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 electronincident 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-detector

    distance, 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-

    field images can be acquired through the collection of transmitted

    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 the cover to define the collection angle a for bright-

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

    Microscopy and Microanalysis from Cambridge University Press.

  • A. Bogner et al. / Micron 38 (2007) 390401 395

    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.

    or scattered electrons, respectively. This setup is implemented for

    example in the FEI high vacuum STEM holder.

    3.2.3. Transmitted electrons/photons conversion via a

    scintillator

    STEM-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 presented in Fig. 4. It used a 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 presents

    advantages, 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 vacuum

    electron 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 was called

    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 by

    Electroscan, 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).

  • A. Bogner et al. / Micron 38 (2007) 390401396

    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.

    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 potential

    applications are described in numerous recently published

    articles, in particular by Donald (2003).

    Fig. 6. Dependences of the amplification on the pressure for various primary-beam e

    (d) nitrogen, and (e) helium. Figures courtesy of Bradley Thiel (Fletcher et al., 19

    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 around

    3 8C, within a chamber environment of water vapour at apressure in the region of 4.5 Torr, leading to improved

    nergies and for five gases: (a) water vapour, (b) carbon dioxide, (c) nitrous oxide,

    97) and IOP Publishing.

  • A. Bogner et al. / Micron 38 (2007) 390401 397

    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. Saturated vapour pressure curve for water. Image courtesy of D.J. Stokes

    (Stokes, 2001) and Wiley.

    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 in

    the 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 XL

    30 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 a

    micropipette.

    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

  • A. Bogner et al. / Micron 38 (2007) 390401398

    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.

    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 waterlayer 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 mmallow 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 case

    of 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 on

    the 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 the

    ESEM 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,

  • A. Bogner et al. / Micron 38 (2007) 390401 399

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

    dark-field mode.

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

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

    imaging.

    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 liquidlayer (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

    Fig. 11. Chocolate colloidal clay in water imaged

    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 0.1 mm 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 scattering

    of 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 to

    be 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

    successfully observed in wet-STEM. Fig. 13 presents an image of

    bacteria of the species Pseudomonas syringae, acquired at a

    magnification of 50 000. The contrast of the bacteria is very

    in wet-STEM annular dark-field imaging mode.

  • A. Bogner et al. / Micron 38 (2007) 390401400

    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 the

    concentration C2 > C1.

    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,

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

    field imaging mode.

    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 in

    competition 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

  • A. Bogner et al. / Micron 38 (2007) 390401 401

    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.

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    A history of scanning electron microscopy developments: Towards wet-STEM imagingFirst steps in scanning electron microscopyWhat makes the SEM a high-resolution technique?Lens aberrations and source brightness limit the resolution in SEMDifferent technologies of electron sources in the electron microscope

    The use of STEM mode in SEMSTEM modeSTEM-in-SEM detection methodsTransmitted electrons/secondary electrons converter and Everhart-Thornley detector (Golla etal., 1994; Golla-Schindler, 2004; Vanderlinde, 2005)Direct collection of transmitted electrons by a solid-state detectorTransmitted electrons/photons conversion via a scintillator

    Environmental SEM developmentWet-STEM imaging: observing a thin liquid film in transmission modeHistoryExperimental setup of wet-STEMBenefits of STEM-in-SEM mode

    Applications of wet-STEM imagingSummary and outlookAcknowledgmentsReferences

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