Hawkes P.W., Spence J.C.H. (Eds.) Science of Microscopy. V.1 and 2

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Chapter 3 Scanning Electron Microscopy 215

possibly, such as applications in which high emission currents (cf. Table

3–1) or high electron beam current stability are indispensable. Because

vacuum conditions in FESEMs are more strict concerning the pressure

in the specimen chamber (at least one order of magnitude less than in

CSEM) as well as the content of gaseous hydrocarbons and hydrocar-

bons at the specimen, some specimens may not meet the requirements

for cleanness and very low partial pressure. However, if high-

resolution FESEM is applied instead of CSEM, more information about

the specimen will be obtained due to the higher resolution as soon as

the magniﬁ cation used exceeds approximately 10,000× to 20,000×. That

means that lateral resolutions requiring magniﬁ cations clearly beyond

about 20,000× belong to the dedicated domain of high-resolution

SEM.

The following few applications selected from an almost unlimited

quantity should domonstrate the strength of HRSEM in several ﬁ elds

of research. It is clearly beyond the scope of this section to discuss in

this context speciﬁ c details about the specimens and imaging

techniques.

Figure 3–40a shows the secondary electron micrograph of a regular

protein surface layer of a bacterial cell envelope. The specimen was

unidirectional shadowed with an ultrathin tungsten layer leaving an

uncoated region behind the latex bead. Comparison of the regular

structure of the HPI layer in the coated and the uncoated region shows

that the contrast in the uncoated area is signiﬁ cantly lower than in the

coated region, though the resolution of structural details is very similar

as veriﬁ ed by the related power spectra. This example also demon-

strates that coating with the ultrathin very ﬁ ne-grain metal ﬁ lm does

not mask ﬁ ne structural features. On principle, a similar resolution can

also be obtained with nonregular organic specimens, however, it

remains more difﬁ cult to quantify unambiguously the resolution

obtained.

An extremely important application of HRSEM, as yet unrivaled by

other surface imaging techniques, is the localization of molecules on

surfaces by immunolabeling techniques (for review see, e.g., Grifﬁ th,

1993; Hayat, 1989/1991, 2002; Polak and Varndell, 1984; Verkleij and

started more than 20 years age (de Harven et al., 1984; Gamliel and

Polliack, 1983; Hicks and Molday, 1984; Molday and Maher, 1980;

Walther and Müller, 1985, 1986; Ushiki et al., 1986). Since then efforts

microscopy in terms of localization precision, contrast, and SNR (e.g.,

Albrecht et al., 1988; Hermann et al., 1991; Hirsch et al., 1993; Simmons

et al., 1990). While the colloidal gold can be localized directly in the

BSE image, the precision of the indirect localization of the antigen

depends on the type of labeling and the size of the colloidal gold and

ranges from less than 5 to about 30 nm (Baschong and Wrigley, 1990;

Müller and Hermann, 1990). Figure 3–41b demonstrates the unambig-

uous detection of immunogold-labeled calcium-binding birch pollen

allergen Bet v4 in birch pollen using 10 nm colloidal gold (Grote et al.,

1999b). The BSE micrograph shows the topographic features and the

were made to optimize the technique of immuno-scanning electron

Leunissen, 1989). The use of HRSEM for immunoelectron microscopy

216 R. Reichelt

small colloidal gold beads as sharp bright spots due to the material

microscopy has been established as a trusted technique and, with the

commercial availability of high-quality gold probes (available in sizes

ranging from 1 to 40 nm), is used in many electron microscopic labo-

ratories for various studies (e.g., Apkarian and Joy, 1988; Erlandsen

et al., 1995; Grote et al., 2000; Müller and Hermann, 1992; Ris and

Malecki, 1993; Yamaguchi et al., 1994).

The interior structure of biological specimens is accessible by HRSEM,

if samples are rapidly frozen and opened by cryofracturing or cryoul-

Figure 3–40. Secondary electron micrograph of a regular protein surface layer [hexagonally packed

intermediate (HPI) layer (Baumeister et al., 1982)] of Deinococcus radiodurans recorded with an “in-lens”

FESEM at 30 kV (a). The specimen was unidirectional shadowed (see arrow) at an elevation angle of 45°

with a 0.7-nm-thick tungsten layer leaving an uncoated region behind the latex bead. The power spectra

of a coated (b) and an uncoated (c) region of the HPI layer reveal the resolution obtained (outermost dif-

fraction spots are indicated and the corresponding reciprocal values of resolution are given). The con-

trast in the uncoated region is about 15–20% of that from the coated region. [Micrograph kindly provided

by Dr. R. Wepf; from Reichelt (1995); with kind permission of GIT Verlag, Darmstadt, Germany.]

contrast. However, for more than a decade immuno-scanning electron

Chapter 3 Scanning Electron Microscopy 217

tramicrotomy. After partial freeze-drying and double-layer coating of

the block face, the specimen can be directly analyzed in the cryo-SEM

(Echlin, 1971; Hermann and Müller, 1993; Walther et al., 1995). Figure

3–42 shows for comparison the BSE micrograph of the cryosectioned

and the cryofractured block face of high-pressure frozen yeast cells

(Walther and Müller, 1999). At low magniﬁ cation the cryosectioned

block face appeared very ﬂ at in the image (Figure 3–42a), whereas the

cryofractured face exhibits the typical rough fracture pattern (Figure

3–42c). At high magniﬁ cation (Figure 3–42b and d) the cytoplasm

appeared densely packed with different classes of particle. Particles as

small as 25 nm can be visualized clearly. It is important to mention that

the typical artifacts of cryosections such as compression and crevasses

are not visible on the block face.

Both strengths of the HRSEM, namely the high resolution and the

high depth of focus, are required to resolve surface structures at the

nanometer scale on tilted surfaces randomly oriented. One typical

example with submicrometer-sized crystalline zeolite particles is shown

in Figure 3–43. The HRSEM is the tool most suited to characterize the

habit of the individual particles as well as to visualize the ﬁ ne surface

structure such as growth steps of terraces (see, e.g., González et al.,

2004). The growth step-edge height, usually in direct relation to the unit

cell dimension and important in understanding the crystal growth

mechanism of this novel microporous material, cannot be measured

precisely by HRSEM, but the atomic force microscope (AFM) enables

measurement of direct height with subnanometer resolution. Thus, the

combination of the two high-resolution surface-imaging methods

HRSEM and AFM is strongly advisable to obtain more complete

information as demonstrated by different applications (e.g., González

et al., 2004; Huang et al., 2004; Keller and Chih-Chung, 1992; Lian et al.,

2005; Stracke et al., 2003; Wang et al., 2004; Z. X. Zhao et al., 2005).

Figure 3–41. Secondary electron micrograph from a birch pollen at low magniﬁ cation (a) and the

backscattered electron micrograph at high magniﬁ cation recorded with an “in-lens” FESEM at 10 kV.

Immunolabeling of the calcium-binding birch pollen allergen Bet v4 in dry and rehydrated birch

pollen was performed using 10-nm colloidal gold (for more details see Grote et al., 1999a,b). The BSE

image shows superimposed topographic and material contrast. The colloidal gold beads are unam-

biguously detected in the BSE micrograph as tiny bright spots.

218 R. Reichelt

HRSEM is also a very valuable tool for the evaluation of mechanical

properties of structural materials. For example, most structural materi-

als are strengthened by ﬁ ne particles of second phases usually having

diameters less than 500 nm. The strengthening effect is primarily gov-

erned by the mean size, the size distribution, and the volume fraction

of the particles. Both HRSEM and AFM allow for the precise determi-

nation of the mean size, size distribution, and volume fraction of the

particles as demonstrated by Fruhstorfer et al. (2002). Figure 3–44

shows the SEM micrograph (Figure 3–44a) and the AFM topograph

(Figure 3–44c) of the electrolytically polished surface of the superalloy

NIMONIC PE16 with the protruding caps of the second phase parti-

Figure 3–42. BSE micrographs of high-pressure frozen yeast cells recorded in an “in-lens” cryo-

FESEM at 10 kV. (a and b) Block face after cryosectioning and (c and d) block face after freeze facturing.

The block faces were double-layer coated with 2.5 nm platinum/carbon and subsequently with 5–10 nm

carbon. The cytoplasm from both the sectioned (b) and the fractured (d) sample appears to consist of

particles with variable size. [From Walther and Müller (1999); with kind permission of Blackwell

Publishing Ltd., Oxford, U.K.]

Chapter 3 Scanning Electron Microscopy 219

cles. In contrast to AFM, where corrections were necessary to take into

account the exact tip radius, corrections for the very small electron

probe diameter are not urgently required in HRSEM. The size distribu-

tion function and mean radius of the second phase particles calculated

from HRSEM (Figure 3–44b) and AFM (Figure 3–44d) data are in excel-

lent agreement with those gained earlier by TEM (Nembach, 1996). The

distinct advantages of HRSEM in this application are that micrographs

are readily recorded and the data can be processed without additional

correction procedures.

The characterization of porous materials such as porous silicon or

porous aluminum oxide gains increasing attention because of impor-

tant potential applications (see, e.g., Anglin et al., 2004; Galca et al.,

2003; Pan et al., 2004; Yamazaki, 2004; Z. X. Zhao et al., 2005; Y. C. Zhao

et al., 2005). Among others, HRSEM is an indispensable tool for struc-

tural characterization of porous materials taking advantage of the large

depth of focus and the high resolution obtainable. Figure 3–45 shows

high-resolution SE and BSE micrographs of the surface and cross

section of porous aluminum oxide, which exhibits a network with

randomly distributed, but almost perfectly aligned cylindrical pores

perpendicular to the substrate. The simultaneous imaging of the surface

and the cross section reveals information about the three-dimensional

specimen structure. Under the conditions given the SE mode yields

higher resolution than the BSE mode.

However, the BSE mode is of signiﬁ cant importance if greater infor-

mation depth and material differentiation are required. Figure 3–46

shows SE and BSE micrographs of tem perature-sensitive hydrogels,

based on poly (vinylmethyl ether) (PVME), with ferromagnetic proper-

ties due to incorporated nickel particles used as ferromagnetic ﬁ ller.

The contrast in the SE micrograph (Figure 3–46a) is mainly caused by

Figure 3–43. Secondary electron micrograph of zeolite FAU (faujasite) parti-

cles recorded with an “in-lens” ﬁ eld emission SEM at 10 kV. The particles are

adsorbed to a thin hydrophilic amorphous carbon ﬁ lm and rotary shadowed

with 1.5 nm platinum/carbon (Pt/C) at an elevation angle of 65° and, addition-

ally, unidirectional shadowed with 2 nm Pt/C at an elevation angle of 10°. The

habit, intergrowth of particles, and growth steps at the surface are clearly

visible. (Specimen kindly provided by Dr. G. Gonzaléz, Instituto Venezolano

de Investigaciones Cientíﬁ cas, Caracas, Venezuela.)

220 R. Reichelt

Figure 3–44. Surface of electrolytically polised superalloy NIMONIC PE16. Secondary electron micro-

graph recorded with an “in-lens” FESEM at 10 kV (a) and AFM topograph (c). The related distribution

functions g of the true radii ρ are shown for the HRSEM in (b) and for the AFM in (c). [Adapted

from Fruhstorfer et al. (2002); with kind permission of Taylor & Francis Ltd., http://www.tandf.co.uk/

journals.

the very thin membrane-like PVME, which envelops the nickel parti-

cles, whereas the BSE image (Figure 3–46b) has a strong material con-

trast component due to the nickel particles underneath the PVME

membrane. This new class of hydrogels is of great interest for delivery

of materials at the micro- and nanometer scale.

As mentioned in Section 2.2.3, the high-resolution “in-lens” FESEM

equipped with an annular dark-ﬁ eld detector is capable of mass mea-

surements on thin specimens (Engel, 1978; Wall, 1979) at a resolution

close to that of a dedicated STEM (Reichelt et al., 1988; Krzyzanek and

Reichelt, 2003; Krzyanek et al., 2004). Mass measurement of molecules

Chapter 3 Scanning Electron Microscopy 221

and molecular assemblies are of great importance in biophysics and

structural biology (for review see, e.g., Müller and Engel, 2001).

Finally, nanotechnology and “nanoelectromechanical systems”

(NEMS) are additional ﬁ elds in which HRSEM is used as a tool for

monitoring processes, detecting defects, or measuring sizes and dis-

tances, e.g., in nanodevices, which will contain nanotubes, nanoparti-

cles, nanowires, and other particles (see, e.g., Aoyagi, 2002; Nagase and

Kurihara, 2000; Nagase and Namatsu, 2004).

3.2 Low- and Very-Low-Voltage Scanning Electron Microscopy

Scanning electron microscopy with electron energies below 5 keV is

usually designated as scanning low-energy electron microscopy

Figure 3–45. High-resolution micrographs of the surface (upper part of image) and the cryosectioned

cross section (lower part of image) of porous aluminum oxide recorded with an “in-lens” FESEM at

8 kV with SE (a) and BSE (b). The specimen is rotary shadowed with 1.5 nm platinum/carbon. The bar

corresponds to 200 nm. (Specimen kindly provided by Drs. C. Blank and R. Frenzel, Institut für Poly-

merforschung Dresden e.V., Dresden, Germany.)

Figure 3–46. High-resolution micrographs of poly(vinyl methyl ether) (PVME) hydrogel with ferro-

magnetic properties ﬁ lled with submicrometer nickel particles in the swollen state. The hydrogel was

rapidly frozen, freeze dried, and rotary shadowed with an ultrathin layer of platinum/carbon. The SE

(a) and BSE (b) micrograph were recorded with an “in-lens” FESEM at 10 kV, (Specimen kindly pro-

vided by Dr. K.-F. Arndt, Institut für Physikalische Chemie und Elektrochemie, Technische Universität

Dresden, Dresden, Germany.)

222 R. Reichelt

(SLEEM) or, related to the acceleration voltage, LVSEM. The energy of

5 keV can be considered as some threshold energy because the monotonic

dependence of the BSE coefﬁ cient on the atomic number breaks below

this (cf. Section 2.2.2). A second prominent energy threshold is at about

50 eV, which corresponds to the electron energy with minimum inelastic

mean free path of electrons in matter (Seah and Dench, 1979). Therefore,

scanning microscopy with electron energies below 50 eV is designated as

scanning very low-energy electron microscopy (Müllerova and Frank,

2003) or, related to the acceleration voltage in the scanning mode, very

low-voltage scanning electron microscopy (VLVSEM).

What is the motivation for low electron energy operation in SEM?

What are the advantages expected at low energies and what are the

inherent disadvantages?

Clearly, almost all of the advantages for working at low energy

derive directly from the energy dependence of the electron–specimen

interaction (see Section 2.2). The advantages include the following:

1. The penetration depth of the impinging electrons decreases with

decreasing energy due to the reduced electron range R [Eq. (2.29)], i.e.,

the excitation volume in the specimen shrinks (cf. Figure 3–13) and the

volume emitting SE2 and BSE2 approaches the volume emitting SE1

and BSE1 (cf. Figure 3–14). As a result the edge effect, i.e., overbrighten-

ing of edges, is strongly reduced or even suppressed completely.

2. The SE yield δ increases because of the reduced electron range

and the SE are generated near the surface, where they can escape (cf.

Figure 3–15). As a result, the SNR of the SE signal increases with

decreasing energy as low as E

0,m

3. As the SE yield increases, the total amount of emitted electrons

approaches unity (cf. Figure 3–16). Because of the conservation of elec-

tric charge [Eq. (2.40)] the amount of incoming and emitted charges is

balanced and, consequently, the specimen current equals zero. That

means that at this particular electron energy E

no electric conductivity

of the specimen is required. Ideally, imaging of electric insulators

without conductive coating becomes possible. For normal incidence, E

is within the range 0.5–5 keV for most of the materials. E

increases

with the increasing angle of beam incidence θ according to

(θ) = E

(0)/cos

θ (3.2)

where E

(0) = E

(θ = 0) (Joy, 1989), i.e., increases as θ increases.

4. As mentioned above, the monotonic de-pendence of the BSE coef-

ﬁ cient on the atomic number breaks below 5 keV (Reimer and Tollkamp,

1980; Schmid et al., 1983). This behavior enables the material contrast

in the BSE image to be ﬁ netuned by choosing the most suitable electron

energy (Müllerova, 2001).

5. There is a reduced depth of specimen radiation damage (see

Section 2.5). At very low electron energies, say less than 30 eV, the

elastic scattering dominates and radiation damage becomes negligible

(Müllerova and Frank, 2003).

The problems and disadvantages inherent to microscopy at low elec-

tron energy concern both the instrumentation and the specimen include

the following:

Chapter 3 Scanning Electron Microscopy 223

1. Reduced resolution due to chromatic aberration and diffraction

[see Eqs. (2.8)–(2.10)].

2. Stronger sensitivity of the electron beam to electromagnetic stray

ﬁ elds.

3. Special detector strategies required for SE and BSE.

4. Enhanced contamination rate, which can be counteracted by

ultrahigh vacuum.

5. Reduced topographic contrast in SE and BSE micrographs. For

electron energies below 5 keV, the increase of SE yield δ(θ) with

increasing θ [cf. Eq. (2.31)] drops way down to 0.5 keV as shown for

different metals experimentally and by Monte Carlo simulation

(Joy, 1987a; Böngeler et al., 1993). Similarly, the backscattering co-

efﬁ cient η(θ) shows less increase with θ than given by Eq. (2.34),

which is more pronounced at low electron energies (Böngeler et al.,

1993).

6. Reduced material contrast, because the differences of the back-

scattering coefﬁ cient between low and high atomic number material

become smaller (Darlington and Cosslett, 1972; Lödding and Reimer,

1981; Reimer and Tollkamp, 1980).

3.2.1 Electron Lenses

Modern commercial ﬁ eld emission scanning electron microscopes can

operate usually from 30 keV down to 1 keV or even 0.5 keV, i.e., that

energy range covers conventional electron energies and most of the

low-energy region. Improved computer-aided methods enable electron

optical systems to be designed that have high performance within

the whole energy range mentioned above. Compared with the old-

fashioned thermionic gun scanning electron microscopes the aberra-

tion coefﬁ cients of the objective lens were improved dramatically for

modern ﬁ eld emission instruments commercially available: C

was

reduced by a factor of about 30 down to C

= 1.6 mm (Uno et al., 2004),

and C

was reduced by a factor of about 10. With the ultrahigh-

resolution objective lens, the CFEG, improved electrical and mechani-

cal stability, as well as strongly reduced specimen contamination rate,

the resolution obtained with test specimens amounts presently to

0.5 nm (at 30 keV) and 1.8 nm (at 1 keV) (Sato et al., 2000). Those values

are exemplary for the high performance of commercial FESEM over an

energy range from 1 to 30 keV, though obtained with special test speci-

mens. Very recently, a new commercial FESEM became available

equipped with a spherical and chromatic aberration corrector, which

consists of four sets of a 12-pole component that corrects the spherical

and chromatic aberration simultaneously (Kazumori et al., 2004). Using

the spherical and chromatic correction, the resolution obtained with

the test specimen amounts to 1.0 nm (at 1 keV) and 0.6 nm (at 5 keV)

(Kazumori et al., 2004).

Electrostatic as well as combined magnetic and electrostatic lenses

in LVSEM are a very interesting alternative to the magnetic lenses

mentioned above. Microscopes equipped with this type of objective

lens permit nonconstant beam energy along the column, i.e., the beam

electrons pass the column with high energy and are decelerated to low

energy in the immersion electrostatic lens. First, the magnitude of the

224 R. Reichelt

aberrations of immersion electrostatic lenses corresponds to the high

energy at the entrance side. A more detailed treatment of the estima-

tion of electrostatic lenses is beyond the scope of this section (see, e.g.,

Lencová and Lenc, 1994; Lencová, 1997). Second, the high electron

energy in the column is advantageous because the gun brightness

increases with electron energy [see Eqs. (2.4) and (3.1)] and electromag-

netic stray ﬁ elds result in less deterioration of the electron beam at high

energy. The combined magnetic–electrostatic objective lens (Frosien

et al., 1989) has aberration coefﬁ cients as low as C

= 3.7 mm and C

1.8 mm. Martin et al. (1994) achieved with this lens a resolution of

2.5 nm at 5 keV, 4.0 nm at 1 keV, and 5.0 nm at 0.5 keV.

Very low landing energies of the electrons can be realized with a

retarding-ﬁ eld SEM. There are several retarding-ﬁ eld conﬁ gurations

described in the literature but basically in all of them the specimen is

connected to the adjustable bias supply U

(e.g., Zworykin et al., 1942;

Paden and Nixon, 1968; Zach and Rose, 1988a,b; Munro et al., 1988;

Müllerova and Lenc, 1992). The landing energy of the beam electrons

simply is given by the difference E

− eU

. Using retarding-ﬁ eld SEM,

landing energies of a few electronvolts are achievable and recently

micrographs with reﬂ ected electrons even at 0.5 eV were obtained

(Müllerova et al., 2001).

With the availability of magnetic materials having high coercive

force permanent rare-earth-metal magnets attract attention as replace-

ments for magnetic lens coils (Adamec et al., 1995). Khursheed (1998)

proposed a portable SEM column design, which makes use of perma-

nent magnets. The column of this miniature SEM amounts to a height

of less than 12 cm and is designed to be modular, so that it can ﬁ t onto

different specimen chamber types, and can also be readily replaced.

Focusing of the electron beam onto the specimen can be achieved by

varying the specimen height or by an outer magnetic slip ring on the

objective lens, which controls the strength of the magnetic ﬁ eld on the

axis. Scanning of the beam is performed by deﬂ ection coils, which are

located above and within the permanent magnet objective lens. A high-

resolution miniature SEM with a total height of less than 5.5 cm, pro-

posed by Khursheed (2000), uses a permanent magnet objective lens

that lies outside the vacuum with spherical and chromatic aberration

coefﬁ cients (parameters: E

= 1 keV, WD = 7. 5 m m ) of 0.36 and 0.6 mm,

respectively. These aberration coefﬁ cients are about an order of mag-

nitude smaller than those for conventional SEMs with comparable

working distance conditions.

Miniaturization of the SEM column has advantages such as micro-

lenses with small aberration coefﬁ cients, reducing the inﬂ uence of

electromagnetic stray ﬁ elds and of the electron–electron interaction,

improving the mechanical stability, and reducing the demands on

space for the microscope. Chang et al. (1990) proposed a miniaturized

electron optical system consisting of a ﬁ eld emission microsource and

an electrostatic microlens for probe forming with performance, exceed-

ing that of a conventional system over a wide range of potentials (0.1–

10 kV) and working distances (up to 10 mm). Liu et al. (1996) proposed

another design that has a column length of only 3.5 mm and can be