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

Подождите немного. Документ загружается.

Chapter 3 Scanning Electron Microscopy 235

Figure 3–56. Stereo pair of SE micrographs (a and b) of the hydrogel poly-(N-isopropylacrylamide)

(PNIPAAm) in the swollen state recorded at 2 keV with the “in-lens” FESEM. The specimen was rapidly

frozen, freeze dried, and ultrathin rotary shadowed with platinum/carbon (for details see Matzelle et

al., 2002). (c) Red–green stereo anaglyph prepared from (a and b). The tilt axis has a vertical direction.

(d) Red–green stereo anaglyph in a “bird view.” (For parts c and d, see color plate.)

236 R. Reichelt

Figure 3–57. Secondary electron micrograph series of increasing electron energies from 0.5 to 30 keV

from a keratinocyte. The micrographs are recorded with an “in-lens” FESEM. The image contrast

varies signiﬁ cantly with the electron energy. Inhomogeneities in the “leading edge” of the keratino-

cyte, which has a thickness of about 200–400 nm, are most clearly visible at 2 keV. (Micrographs kindly

provided by Dr. R. Wepf, Beiersdorf AG, Hamburg, Germany.)

Chapter 3 Scanning Electron Microscopy 237

characterization of polymers (Berry, 1988; Butler et al., 1995; Brown and

Butler, 1997; Sawyer and Grubb, 1996).

4 Scanning Electron Microscopy at Elevated Pressure

The scanning electron microscopic investigation of specimens must

meet several requirements, which were mentioned in previous sec-

tions. To sum it up, it can be said that specimens (1) have to be compat-

ible with the low pressure in the specimen chamber (∼10

−3

Pa in

conventional SEM and 10

−5

–10

−4

Pa in ﬁ eld emission SEM), (2) have to

be clean, i.e., the region of interest has to give free access to the primary

beam, (3) need sufﬁ cient electrical conductivity, (4) need to be resistant

to some extent to electron radiation, and (5) have to provide a sufﬁ cient

contrast. In a narrower sense, only metals, alloys, and metallic com-

pounds fulﬁ ll those requirements. Numerous preparation procedures

mentioned in Section 2.4 were developed in the past and are still in the

process of improvement, to provide a sufﬁ cient electrical conductivity

to nonconductive specimens, to remove the water in samples, and to

replace it or to rapidly freeze it in a structure-conserving manner.

Nevertheless, there was and still is enormous interest in investigating

specimens in their genuine state.

Thirty years ago Robinson (1975) proposed examining any uncoated

insulating specimen in the SEM at high accelerating voltages in the

specimen chamber, which had been modiﬁ ed to contain a small residual

water vapor environment. It appeared that the presence of the water

vapor sufﬁ ciently reduced the resistance of the insulator so that no

charging effects were detected in backscattered electron micrographs.

Danilatos (1980) developed an “atmospheric scanning electron micro-

scope” (ASEM), which later was called an “environmental scanning

Figure 3–58. Secondary electron micrograph pair of the cuticula of a leaf recorded at electron energies

of 0.4 (a) and 30 keV (b) with an “in-lens” SEM. The low-energy image contains information only from

the surface whereas the 30-keV image also reveals information about structural features below the

surface, e.g., new spores, which are not visible in (a). (Micrographs kindly provided by Dr. R. Wepf,

Beiersdorf AG, Hamburg, Germany.)

238 R. Reichelt

electron microscope” (ESEM®) (Danilatos, 1981) and is now a registered

trademark. To enable the investigation of water and water-containing

specimens in their native state at stationary conditions a minimum pres-

sure of water vapor of about 612 Pa is required at 0°C (cf. Figure 3–59).

Stationary conditions in the specimen chamber of the SEM can be

accomplished by controlling the water vapor pressure p in close vicinity

of the specimen as well as the specimen temperature T such that the p–T

values always correspond to points on the solid p–T graph in Figure 3–

59. For example, at 20°C a water vapor pressure as large as about 2330 Pa

is required for stationary conditions. p–T values below the solid graph,

e.g., 300 Pa at 0°C (Figure 3–59), corresponds to a relative humidity of

less than 100%, thus representing nonstationary conditions.

How can stationary conditions be reached during imaging of a wet

sample in the specimen chamber of an SEM? Figure 3–60 shows the

cross section of the ESEM, which permits investigations at pressures

sufﬁ cient for stationary conditions. Basically, the electron beam pro-

pagates in the column as in a conventional SEM until it reaches the ﬁ nal

aperture. Then, since the pressure increases gradually as the electrons

proceed toward the specimen, the electrons undergo signiﬁ cant scatter-

ing on gas molecules until they reach the specimen surface.

The electron–gas interaction is discussed in detail by Danilatos

(1988). According to this study the average number of scattering events

per electron n can be approximated by

n = σ

L/kT (4.1)

where σ

represents the total scattering cross section of the gas molecule

for electrons, L is the electron path length in gas, and k is the Boltzmann

constant. These approximations hold for Λ >> L, where Λ represents the

4000

3000

Solid

Liquid

Vapor

2000

p [Pa]

1000

–5 10

T [°C]

15 20 25 3005

Figure 3–59. Phase diagram of water. Solid line, 100% relative humidity (satu-

rated vapor conditions); dashed line, 50% relative humidity. (Data from Lax,

1967.)

Chapter 3 Scanning Electron Microscopy 239

Figure 3–60. Schematic cross section of the ﬁ rst commercial Electroscan Environmental SEM (ESEM

)

showing the vacuum and pumping system. Two pressure-limiting apertures separate the electron

optical column from the specimen chamber. Differential pumping of the stage above and between the

two pressure-limiting apertures ensures the separation of high vacuum in the column from low vacuum

in the specimen chamber. The differential pumping of two stages and optimum arrangement of the

pressure-limiting apertures can work successfully to achieve pressures up to 10

Pa in the specimen

chamber. [From Danilatos, 1991; with kind permission of Blackwell Publishing Ltd., Oxford, U.K.]

GUN

CHAMBER

ION

PUMP

manual valve

Valve

Gauge

RP1

RP2

RP3

SPECIMEN CHAMBER

EC2

EC1

regulator

valve

V10

V11

VENT

AUX IL IARY GAS

WATER VAPOR

V13

V4V5

VENT

V12

DIF

mean free path of a beam electron in the gas. According to Eq. (4.1) the

average number of collisions increases linearly with the gas pressure p

and the path length in the specimen chamber. Furthermore, n depends

via the scattering cross section on the type of gas molecules and on the

temperature. When the beam electrons start to be scattered by the gas

molecules, the fraction of scattered electrons is removed from the focused

beam and hit the specimen somewhere in a large area around the point

of incidence of the focused beam. The scattered electrons form a “skirt”

around the focused beam, which has a radius of 100 µm for a pathlength

of 5 mm (conditions: E

= 10 keV, water vapor pressure = 10

Pa) (Danila-

tos, 1988). Using a phosphor imaging plate, the distribution of unscat-

240 R. Reichelt

tered beam electrons and the scattered “skirt” electrons was directly

imaged by exposure to the electron beam for a speciﬁ ed time (Wight and

Zeissler, 2000). Related to the electron beam intensity within 25 µm, the

“skirt” intensity as a function of the distance from the center drops to

15% at 100 µm, 5% at 200 µm, and 1% at 500 µm (conditions: E

= 20 keV,

water vapor pressure = 266 Pa, l = 10 mm) (Wight and Zeissler, 2000). The

signals generated by the electrons of the skirt originate from a large area,

which contributes to the background, whereas the unscattered beam

remains focused to a small spot on the specimen surface, although its

intensity is reduced by the fraction of electrons removed by scattering.

The resolution obtainable depends on the beam diameter and the size of

the interaction volume in the specimen, which is analogous to the situa-

tion in conventional and high-resolution SEM, i.e., the resolving power

of ESEM can be maintained in the presence of gas.

The detection of BSE, CL, and X-rays is to a great extent analogous to

the detection in a conventional SEM, because these signals can pene-

trate the gas sufﬁ ciently (Danilatos, 1985, 1986). However, the situation

is completely different for the detection of SE. The conventional Ever-

hart–Thornley detector would break down at elevated pressure in the

specimen chamber. However, the gas itself can be used as an ampliﬁ er

in a fashion similar to that used in ionization chambers and gas propor-

tional counters. An attractive positive voltage on a detector will make

all the secondary electrons drift toward it. If the attractive ﬁ eld is sufﬁ -

ciently large, each drifting electron will be accelerated, thus gaining

enough energy to cause ionization of gas molecules, which can create

more than one electron. This process repeating itself results in a signiﬁ -

cant avalanche ampliﬁ cation of the secondary electron current, which

arrives at the central electrode of the environmental secondary electron

detector (ESD) (Danilatos, 1988). The avalanche ampliﬁ cation works

best only in a limited pressure range and can amplify the SE signal up

to three orders of magnitude (Thiel et al., 1997). Too high pressure in the

specimen chamber makes the mean free path of the electrons very

small and a high electric ﬁ eld between specimen and detector is required

to accelerate them sufﬁ ciently. Too low pressure in the chamber results

in a large mean free electron path, i.e., only a few ionization events take

place along the electron path from the specimen to the detector, thus the

avalanche ampliﬁ cation factor is low. The new generation of ESD, the

gaseous secondary electron detector (GSED), which consists of a 3-mm-

diameter metallic ring placed above the specimen, provides better dis-

crimination against parasitic electron signals. Both the ESD and GSED

are patented and are available only in the ESEM.

However, the ionization of gas molecules creates not only electrons

but also ions and gaseous scintillation. The latter can be used to make

images (Danilatos, 1986), i.e., in that case the imaging gas acts as a

detector. This principle is used in the patented variable pressure sec-

ondary electron (VPSE) detector. Nonconductive samples attract posi-

tive gas ions to their surface as negative charge accumulates from the

electron beam, thus effectively suppressing or at least strongly reduc-

ing charging artifacts (Cazaux, 2004; Ji et al., 2005; Tang and Joy, 2003;

Thiel et al., 2004; Robertson et al., 2004). The gas ions can affect or even

reverse the contrast in the GSED image under speciﬁ c conditions, e.g.,

Chapter 3 Scanning Electron Microscopy 241

at specimen regions of enhanced electron emission, where the rate of

electron–ion pairs increases (Thiel et al., 1997). The highly mobile elec-

trons generated by electron–gas interaction are removed from the gas

by rapid sweeping to the GSED, which in turn causes an increased

concentration of positive ions during image acquisition due to different

electric ﬁ eld-induced drift velocities of negative and positive charge

carriers in the imaging gas (Toth and Phillips, 2000).

However, imaging of wet, soft specimens can be hampered by the

effect of surface tension (Kellenberger and Kistler, 1979), which may

ﬂ atten and hereby deform the specimen. Obviously, this is a mislead-

ing situation demonstrating that “environmental conditions” do not

necessarily guarantee structural preservation.

As mentioned above, about 612 Pa is the crucial minimum pressure for

wet specimens. In addition to the ESEM, which enables imaging with SE

at pressures up to about 6500 Pa, numerous variable pressure SEM

(VPSEM), high pressure SEM, and low vacuum SEM (sometimes the

abbreviation LVSEM is used, which cannot be distinguished from the

low-voltage SEM) became commercially available. The water vapor pres-

sure in the specimen chamber of those SEM is typically at maximum

300 Pa, i.e., below the crucial value of 612 Pa, which is not sufﬁ cient for

imaging of wet specimens at stationary conditions. To separate the speci-

men pressure of maximum 300 Pa from the high vacuum in the column

only one pressure-limiting aperture is sufﬁ cient. For imaging at pressures

in the range from 250 to 300 Pa backscattered electrons are utilized.

Very recently, Thiberge et al. (2004) demonstrated scanning electron

microscopy of cells and tissues under fully hydrated atmospheric condi-

tions using a small chamber with a polyimide membrane (145 nm in thick-

ness) that is transparent to beam and backscattered electrons. The

membrane protects the fully hydrated sample from the vacuum. BSE

imaging at acceleration voltages in the range of 12–30 kV revealed struc-

tures inside cultured cells and colloidal gold particles having diameters

of 20 and 40 nm, respectively. Another interesting experimental setup is

the habitat chamber designed to keep living cells under fully hydrated

atmospheric conditions as long as possible and to reduce the exposure time

to the lower pressure in the ESEM below 2 min (Cismak et al., 2003).

Scanning electron microscopy at elevated pressure is increasingly

used in very different ﬁ elds. Apart from variations in the pressure and

chamber gas a heating stage (maximum temperature about 1500°C)

allows changes in the specimen temperature. For example, chemical

reactions such as corrosion of metals, electrolyte–solid interactions,

alloy formation, and the degradation of the space shuttle ceramic

shields by increasing oxygen partial pressures at high temperatures

are possible with micrometer resolution. The onset of chemical reac-

tions that depend on various parameters can by studied in detail.

Insulators, including oil and oily specimens, can be directly imaged.

Water can also be imaged directly in the ESEM, which allows studies

of wetting and drying surfaces (e.g., de la Parra, 1993; Stelmashenko et

al., 2001; Liukkonen, 1997) and direct visualization of the dynamic

behavior of a water meniscus (Schenk et al., 1998; Rossi et al., 2004).

Figure 3–61 shows an example of dynamic studies of a water menis-

cus between the scanning tunneling tip and a support when the tip is

242 R. Reichelt

Figure 3–61. Time-resolved sequence of secondary electron images recorded with an ESEM®-E3

(ElectroScan Corp., Wilmington, MA). The water meniscus between the hydrophilic tungsten tip

(normal electron beam incidence) and the Pt/C-coated mica (incidence angle of 85°) is clearly visible

(a–d). Due to locally decreasing relative humidity the meniscus becomes gradually smaller until it

snaps off (e). The absence of the meniscus leads to a signiﬁ cant change of shape of the water bead

below the tip [cf. (d) and (e)]. Some water drops are located on the sample in front of and behind the

tip. The sequence was recorded within 11 s and each image was acquired within about 2 s. Experimen-

tal conditions: E

= 30 keV, I

= 200 pA, p

= 1.2 kPa. (From Schenk et al., 1998; with kind permission of

the American Institute of Physics, Woodbury, NY.)

Chapter 3 Scanning Electron Microscopy 243

moved across the sample. The wetting of the tip indicates a hydrophilic

surface, whereas Figure 3–62 clearly indicates a hydrophobic tip

surface.

ESEM studies of the wettability alteration due to aging in crude

oil/brine/rock systems that are initially water wet are of signiﬁ cant

importance in the petroleum industry in understanding the water

condensation behavior on freshly exposed core chips. Surface active

compounds are rapidly removed from the migrating petroleum, thus

changing the wettability and subsequently allowing larger hydropho-

bic molecules to sorb (Bennett et al., 2004; Kowalewski et al., 2003;

Robin, 2001). Furthermore, the ESEM is a powerful tool with which

study the inﬂ uence of salt, alcohol, and alkali on the interfacial activity

of novel polymeric surfactants that exhibit excellent surface activity

due to their unique structure (Cao and Li, 2002).

Environmental scanning electron microscopy disseminates rapidly

among scientiﬁ c and engineering disciplines. Applications range

widely over diverse technologies such as pharmaceutical formulations,

personal care and household products, paper ﬁ bers and coatings,

cement-based materials, boron particle combustion, hydrogen sulﬁ de

corrosion of Ni–Fe, micromechanical fabrication, stone preservation,

and biodeterioration. In spite of the broad applications, numerous con-

trast phenomena are not fully understood as yet. This is illustrated in

Figure 3–63 by a series of SE micrographs recorded at different electron

energies, but otherwise identical conditions. In addition, ESEM inves-

tigations of polymeric and bio logical specimens, which are known

from conventional electron microscopy to be highly irradiation sensi-

tive, are more difﬁ cult because water acts as a source of small, highly

mobile free radicals, which accelerate specimen degradation (Kitching

and Donald, 1996; Royall et al., 2001).

Figure 3–62. Secondary electron image recorded with an ESEM-E3 (Elec-

troScan Corp., Wilmington, MA) from a hydrophobic tungsten tip (normal

electron beam incidence) and a water bead on Pt/C-coated mica (incidence

angle of 85°). The shape of the deformed water surface in the sub micrometer

vicinity of the tip clearly indicates its hydrophobic surface. The spherical object

(black) at the right of the tip in the back is probably a polystyrene sphere and

any resemblance is purely coincidental. (From Schenk et al., 1998; with kind

permission of the American Institute of Physics, Woodbury, NY.)

244 R. Reichelt

Figure 3–63. Secondary electron micrograph series of the starch glycolys D recorded at electron ener-

gies from 30 keV down to 5 keV (see the individual legends below each micrograph) with an ESEM

demonstrating the effect of the electron energy on image contrast. (Micrographs kindly provided by

Fraunhofer-Institut für Werkstoffmechanik, Halle, Germany; the project was supported by the State

Sachsen-Anhalt, FKZ 3075A/0029B.)

Glycolys D 30 kV 500x 15 mm 3Torr

Glycolys D 20 kV 500x 15 mm 3Torr

60 µm

Glycolys D 15 kV 500x 15 mm 3Torr

Glycolys D 10 kV 500x 15 mm 3Torr

Glycolys D 7.5 kV 500x 15 mm 3Torr

Glycolys D 5 kV 500x 15 mm 3Torr