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 d’Etudes de Me ´tallurgie Physique et de Physique des Mate ´riaux, UMR CNRS 5510, INSA de Lyon, Ba ˆtiment B. Pascal, 7 Avenue Jean Capelle, 69621 Villeurbanne Cedex, France b Total France, Centre de Recherche de Solaize, BP 22, 69360 Solaize Cedex, France c CEA Grenoble, DRFMC/SP2M, Laboratoire d’Etudes des Mate ´riaux par Microscopie Avance ´e, 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 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 www.elsevier.com/locate/micron Micron 38 (2007) 390–401 * Corresponding author. Tel.: +33 4 72 43 61 30; fax: +33 4 72 43 85 28. E-mail address: [email protected](A. Bogner). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.06.008
12
Embed
A history of scanning electron microscopy developments ... · A history of scanning electron microscopy developments: Towards ‘‘wet-STEM ... of the first transmission electron
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
www.elsevier.com/locate/micron
Micron 38 (2007) 390–401
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 d’Etudes 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 d’Etudes 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
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) 390–401398
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 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 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 top–bottom 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 (25–30 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) 390–401 399
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 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
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) 390–401400
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) 390–401 401
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.
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, 290–301.
Breton, P.J., 1999. From microns to nanometers: early landmarks in the science
of scanning electron microscope imaging. Scanning Microsc. 13-1, 1–6.
Cameron, R.E., Donald, A.M., 1994. Minimizing sample evaporation in the
environmental scanning electron microscope. J. Microsc. 173, 227–237.
Colliex, C., Mory, C., 1994. Scanning transmission electron microscopy of
biological structures. Biol. Cell 80, 175–180.
Danilatos, G.D., 1991. Review and outline of environmental SEM at present. J.
Microsc. 162, 391–402.
Danilatos, G.D., 1993. Introduction to the ESEM instrument. Microsc. Res.
Tech. 25, 354–361.
Donald, A.M., 2003. The use of environmental scanning electron microscopy
for imaging wet and insulating materials. Nat. Mater. 2, 511–516.
Fletcher, A.L., Thiel, B.L., Donald, A.M., 1997. Amplification measurements of
potential imaging gases in environmental SEM. J. Phys. D 30, 2249–2257.
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