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

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Chapter 3 Scanning Electron Microscopy 205

In life sciences, application of SEM mainly for morphological studies

is also widespread and started when commercial SEMs became avail-

able in the late 1960s (see, e.g., Pfefferkorn and Pfautsch, 1971). However,

in life sciences SEM is used less than TEM. Table 3–8 provides a survey

of the specimens and information that can be obtained by SEM. Engi-

neered biomaterials and tissues are becoming increasingly important

Figure 3–33. Secondary electron micrograph of the material of white lines,

dividing the opposite lanes on roads, used to reﬂ ect the light from the head-

lights of cars. Some of the light-reﬂ ecting glass spheres are damaged or

snatched off (their original positions can be recognized by sphere-like inden-

tations). For safety reasons protruding sharp-edged particles are embedded

in the material (e.g., at the bottom left corner) to generate a high friction

between the tire and the white line. The sample is sputter coated with gold.

(Micrograph kindly provided by Rudolf Göcke, Institut für Medizinische

Physik und Biophysik, Münster, Germany.)

Figure 3–34. Secondary electron (a) and backscattered electron (b) micrograph of carbonized fossil

remains of a fern bot embedded in clay. The sample is coated with a very thin carbon ﬁ lm. The contrast

in the SE image is caused by the topography, whereas the BSE image shows a distinct atomic number

contrast. The carbonized fossil remains appear dark due to the lower mean atomic number surrounded

by bright areas of clay. (Micrographs kindly provided by Rudolf Göcke, Institut für Medizinische

Physik and Biophysik, Münster, Germany.)

206 R. Reichelt

in biomedical practice and it has become clear that cellular responses

to materials depend on structural properties of the material at both the

micrometer and nanometer scale. SEM is one of several methods for

controlling material properties on both of these scales and thus it is

increasingly used to study those materials.

Scanning electron microscopy can be used for comparative morpho-

logical studies of tissues as demonstrated by the application in cardio-

vascular surgery to detect endothelial damage caused by skeletonization

(Rukosujew et al., 2004). In cardiovascular surgery, the radial artery is

increasingly used for myocardial revascularization because of its pre-

sumed advantageous long-term patency rates. The vessel can be har-

vested as a pedicle or skeletonized. The SEM reveals the endothelial

morphology (cf. Figure 3–35), and thus allows comparison of the skele-

tonization technique with pedicle preparation using either an ultrasonic

scalpel or scissors.

Table 3 –8. SEM applications on specimens from life sciences.

Specimen Information

Bones, teeth, dentin, cartilage, At the specimen surface:

hairs, ﬁ ngernails, toenails Morphology; ultrastructure, pathological alterations of

ultrastructure, microstructure, roughness; cracks; ﬁ ssures;

fractures; elemental composition

Inside the specimen:

Three-dimensional microstructure; cracks; ﬁ ssures; elemental

composition

Biominerals, e.g., gallstone, At the specimen surface:

kidney stone, tartar, Morphology; microstructure, cracks; ﬁ ssures; fractures; grain

calciﬁ cation size and shape; size and shape of small particles; elemental

composition

Inside the specimen:

Grain size and shape; microstructure; cracks, ﬁ ssures;

material inclusions; cavities; elemental composition

Soft tissues cells, bacteria At the specimen surface:

Morphology; topography (three-dimensional); roughness;

ultrastructure; pathological alterations of ultrastructure; size

and shape of cells and bacteria; elemental composition

Inside the specimen:

Ultrastructure; pathological alterations of ultrastructure;

elemental composition

Biomaterials, implants, Morphology; biocompatibility; biostability; ultrastructure of

prostheses and degradation mechanisms at the bone–implant interface;

mineral apposition; cell and tissue apposition; adsorption

behaviors of ﬁ brinogen, albumin, and fresh plasma on

implants for the cardiac–vascular systems; fault diagnosis of

prostheses; failure analysis after loading tests in simulator;

wear of prostheses; surface erosion of prostheses after use

State-of-the-art preparation and image analysis techniques are required to take full advantage of the capabilities

of SEM.

Chapter 3 Scanning Electron Microscopy 207

Investigation of implants is a strongly growing ﬁ eld, where scanning

electron microscopy is an indispensable tool. Figure 3–36 shows an

example from ophthalmology where a technique for knotting a suture

to the haptic of an intraocular lens is used for its ﬁ rm ﬁ xation onto the

sclera to avoid postoperative dislocation. Because of the large depth of

focus and the distinct topographic contrast in the secondary electron

micrograph, which conveys a pseudo three-dimensional and detailed

view, the SEM allows checking as to the extent to which the haptic and

suture can be damaged by knotting. Moreover, the quality of knots can

be studied.

Microtechnology and “microelectromechanical systems” (MEMS)

are additional ﬁ elds in which SEM is used as a tool for monitoring

processes, detecting defects, or measuring sizes and distances, e.g., in

micromachines and micromechanical or micromachining processes

(see, e.g., Ishikawa et al., 1993; Aoyagi, 2002; Hernandez-Lopez et al.,

2003; Khamsehpour and Davies, 2004).

The acquisition of quantitative data about the third dimension

(stereo, 3D) of surfaces and interior specimen structures was previ-

ously mentioned (see Sections 2.14, 2.15, and 2.4). In general, this

requires digital image analysis, speciﬁ c instrumentation for the SEM

(e.g., specimen stage, detectors), and special specimen preparation

(e.g., ultramicrotome, IBSC, FIB). Recently, interesting applications of

3D morphometry for accurate dimensional and angular measurements

of microstructures (Minnich et al., 1999, 2000) and of volumetric mea-

surements (Chan et al., 2004) were shown using stereopaired images

and digital image analysis.

Figure 3–35. Secondary electron micrograph of the cross-sectioned radial artery at low magniﬁ cation

(a) and of the endothelial cells at medium magniﬁ cation (b). The vessel was critical point dried and

sputter coated with gold.

208 R. Reichelt

Figure 3–36. Secondary electron micrographs of a poly(methyl methacrylate) (PMMA) intraocular

lens (IOL) with knots from 10-0 polypropylene (Prolene) on the haptic recorded at different magniﬁ ca-

tions. (a) Whole IOL with the haptic and the ﬁ xated sutures; (b–e) Detailed views to the haptic and

knots, respectively, showing some minor damage. The IOL is sputter coated with gold and imaged at

25 kV.

Chapter 3 Scanning Electron Microscopy 209

3 Field Emission Scanning Electron Microscopy

The diameter of the electron beam at the specimen surface sets a fun-

damental lower limit to the signal localization and, therefore, also to

the resolution, which can potentially be obtained. As discussed in

Section 2.2 and shown in Figures 3–13 and 3–14, the SE and BSE are

emitted from a surface area, which commonly is much larger than the

beam diameter at the specimen surface. The large emitting area is

caused by multiple elastic and inelastic electron scattering events

within the excitation volume, whose size depends on the specimen

composition and energy of the beam electrons. Only the SE1 and BSE1

generated as the beam enters the specimen carry local information,

while the SE2 and BSE2 carry information about the larger region sur-

rounding the point of beam entrance (cf. Figure 3–14). High-resolution

information can be obtained from SE1 and BSE1 generated by an elec-

tron probe with a diameter at the specimen surface of about 1 nm or

even less. A probe of that small size can be achieved by using ﬁ eld

emission electron sources, electromagnetic lenses with low aberration

coefﬁ cients (cf. Eqs. 2.7, 2.8, and 2.10), and both highly stabilized accel-

eration voltage (cf. Eq. 2.8) and objective lens current. High-resolution

scanning electron microscopy at conventional acceleration voltages—

that is 5–30 kV—will be treated in Section 3.1. Alternatively, high-

resolution information, in principle, can also be achieved when the

excitation volume is reduced to a size similar to the SE1 and BSE1 emit-

ting area by using low-energy beam electrons. By deﬁ nition, electrons

below 5 keV are considered low-energy beam electrons and, conse-

quently, scanning electron microscopy at low energies is called scan-

ning low-energy electron microscopy or low (acceleration)-voltage

scanning electron microscopy (LVSEM). This type of scanning electron

microscopy will be treated in Section 3.2. However, the majority of

commercial high-resolution SEMs are capable of operation at both con-

ventional energies, i.e., from 5 to usually 30 keV, and at low energies,

i.e., below 5 keV down to usually 0.5 keV.

3.1 High-Resolution Scanning Electron Microscopy

3.1.1 Electron Guns

Three different types of electron guns are suitable sources for high-

the so-called Schottky emission cathode (SEC). The characteristic

parameters of the different electron guns are listed in Table 3–1.

Schottky emission cathodes are of the ZrO/W(100) type—also called

ZrO/W(100) thermal ﬁ eld emitter (TFE)—and have a tip radius of

0.6–1 µm (Tuggle and Swanson, 1985). The work function of the TFE is

lowered to about 2.8 eV. In operation the SEC is heated to about 1800 K

and electrons are extracted by a high electric ﬁ eld, which lowers the

potential barrier (Schottky effect). The SEC brightness is about three

orders of magnitude higher and the energy spread of the emitted elec-

trons is about a factor of 2 lower than those for the thermionic W-

resolution SEM: the cold ﬁ eld emission gun (FEG), the hot FEG, and

210 R. Reichelt

cathode. Presently, the SEC in commercial high-resolution SEM is less

frequently an electron source than the FEG.

The FEG usually consists of a very sharp [100] or [321] oriented

tungsten single crystal and two anodes in front, which extract

(ﬁ rst anode) and accelerate or decelerate (second anode) the electrons

by the electric ﬁ eld to a ﬁ nal energy E

= eU (Figure 3–37). Caused by

the small tip radius r, which is in the range of 10 to about 50 nm, the

electric ﬁ eld strength amounts to at least 10

V/c m with an extraction

voltage of approximately 4–5 kV applied between the ﬁ rst anode

and the tip. Due to the high ﬁ eld strength at the tip the width of the

potential barrier is signiﬁ cantly reduced and ﬁ eld emissions take place.

The ﬁ eld emission current density j

is described by the Fowler–Nord-

heim equation

= c

|E|

/Φ exp(−c

3/2

/|E|) (3.1)

where |E| ≈ U

/r, c

and c

depend weakly on |E|, and Φ is the work

function of tungsten. The density j

depends strongly on |E|, and E can

be varied by U

. The so-called cold FEG (CFEG) is operated at room

temperature and generates a current density of typically 2 × 10

A cm

−2

However, after several hours of work adsorbed gas layers have to be

removed by short heating to about 2500 K (ﬂ ashing), otherwise the

emission current becomes very unstable. The distinct advantage of the

cold FEG is the low-energy spread.

The hot FEG (HFEG) is operated at approximately 1800 K, which

increases the energy spread to about twice that from the cold FEG. The

current density is higher than for the cold FEG and typically amounts

to 5 × 10

A cm

−2

. The advantage of the hot FEG is the less noisy emis-

sion current.

Anode

2nd

1st

1-50 kV

–

Figure 3–37. Schematic drawing of the ﬁ eld emission gun with an electrolyti-

cally polished sharp monocrystalline tungsten tip. The hot FEG operates the

tip at high temperature heated by the applied voltage U

. U, acceleration

voltage; U

, extraction voltage. [Adapted from Reimer (1993); with kind

permission of the International Society of Optical Engineering (SPIE),

Bellingham, WA.]

Chapter 3 Scanning Electron Microscopy 211

Field emission guns require ultrahigh vacuum in the order of 10

−8

–

−9

Pa in the gun chamber, which is generated by ion getter pumps.

This means that SEMs equipped with an FEG need a sophisticated and

consequently cost-intensive vacuum system. Another disadvantage of

FEGs compared to the thermionic W-cathode is their signiﬁ cantly

lower short- and long-time beam current stability.

3.1.2 Electron Lenses

Electron lenses are used to demagnify the virtual source size, which

amounts to 3–5 nm for both the cold and hot FEG, and about 20–30 nm

for the SEG. To obtain and electron beam diameter of about 1 nm or

less a demagniﬁ cation of only 10–100× is required in contrast to up to

about 5000× for the thermionic emission triode gun (cf. Section 2.1.2).

To achieve the smallest effective electron probe diameter, the spherical

and the chromatic aberration constants have to be as small as possible

[see Eqs. (2.7), (2.8), and (2.10)]. In the conventional SEM usually large

working distances ranging from about 10 to 40 mm are used. Typical

values of the spherical aberration constant C

are 10–20 mm. Since C

increases strongly with increasing WD (C

∼ WD

) sufﬁ ciently small

values of C

∼ 1–2 mm can be achieved only with very short WD, i.e.,

the focus of the electron beam has to be inside (so-called “in-lens”

type) or very close to the objective lens [frequently called “semi-in-

lens” with a snorkel-type conical objective lens (Mulvey, 1974)]. The

chromatic aberration constant C

corresponds approximately to the

focal length of the objective lens for large WD, i.e., also the chromatic

aberration is strongly lowered at a very short WD. The shortest WD of

the “in-lens” type SEM is about 2.5 mm in order to secure a specimen

traverse in the x and y direction perpendicular to the optical axis as

well as specimen tilt angles up to a maximum of |±15°|. Larger tilt

angles obviously require a larger work distance. To obtain the minimum

effective electron probe diameter under these conditions, the optimum

aperture α

opt

has to be used [see Eq. (2.11)]. Presently, the highest reso-

lution obtained with the “in-lens” type FESEM at 30 keV using a test

3.1.3 Detectors and Detection Geometries

The detectors used in ﬁ eld emission scanning electron microscopes

(FESEM) have been described in Section 2.1.3.1. The detection geome-

try depends on the particular type of the FESEM. The instruments

using the conventional specimen position outside the objective lens

(“out-lens”), i.e., the WD is in the range of about 5–30 mm, are com-

monly equipped with an ET detector located laterally above the speci-

men and a BSE detector located centrally above the specimen. The

“semi-in-lens” instruments, where the specimen is outside but

immersed in the ﬁ eld of the objective lens, usually have both the detec-

tor arrangement of the “out-lens” type SEM and the “through-the-lens”

detection, thus combining the advantages of both detection geometries.

The “in-lens” type SEM is restricted to “through-the-lens” detection

(cf. Section 2.1.3.2).

sample amounts to 0.4 nm (Hitachi, 2001).

212 R. Reichelt

3.1.4 Specimen Stages

The purpose of the specimen stage in high-resolution scanning elec-

tron microscopes is of course the same as in conventional SEM, i.e., the

stage has to allow for precise backlash-free movement, tilting, and pos-

sibly rotation of the sample during the investigation. As for conven-

tional SEM, there are optionally special specimen stages available that

allow investigations of the specimen at elevated temperature, during

different types of mechanical deformation, at positive or negative bias,

and last at low temperature. Independent on the special type of speci-

men stage, a higher stability in terms of mechanical vibrations as well

as mechanical or thermal drift is required to avoid any deterioration

of the performance of the high-resolution SEM. The “in-lens”-type

SEMs use side-entry specimen holders, which are almost identical to

the ones used in TEMs (cf. Section 2.1.4). However, the limited space

available in this type of SEM places some restrictions on the specimen

stage for the ultimate resolution of “in-lens”-type FESEM.

3.1.5 Contrast Formation and Resolution

At high beam energy, e.g., 30 keV, the lateral extension of the excitation

volume in the specimen is for carbon approximately 10 µm and for a

high atomic number element such as gold about 1 µm (cf. Figure 3–13).

Secondary and backscattered electrons are emitted from a surface area

of the specimen, which corresponds in size to about the lateral exten-

sion of the excitation volume (cf. Figure 3–14). As discussed in Sections

2.2.1 and 2.2.2, the SE2 and BSE2 represent the majority of the SE and

BSE, respectively, whereas the SE1 and BSE1, both carrying high-

resolution information, represent the minority. Assuming for simplic-

ity an electron beam diameter of 1 nm, the ratio of the lateral size of

the excitation volume and the beam diameter amounts to approxi-

mately 10

for carbon and 10

for gold. By choosing the magniﬁ cation

such that the ﬁ eld of view at the specimen surface approaches the

lateral size of the excitation volume, i.e., related to a 100-mm image size

about 10,000× for carbon and 100,000× for gold, both the SE2 and the

BSE2 contributions will change in response to the features of the ﬁ eld

of view on the size scale of the excitation volume. In contrast to this

the SE1 and BSE1 contributions will change in response to the features

of the ﬁ eld of view approximately on a size scale of the electron beam

diameter. That means that in the course of scanning the electron beam

across the ﬁ eld of view, the SE2/BSE2 contribution only insigniﬁ cantly

varies from pixel to pixel whereas the SE1/BSE1 contribution depends

sensitively on local features as small as the beam diameter. With a

further increase of magniﬁ cation the ﬁ eld of view becomes signiﬁ -

cantly smaller than the lateral size of the excitation volume, conse-

quently the SE2/BSE2 contribution is almost constant over the image.

The changes in the total SE/BSE signal are almost exclusively due to

the SE1/BSE1 component and correspond to the changes in the very

tiny volume where SE1/BSE1 are generated. Figure 3–38 shows an

example of a high-resolution SE micrograph recorded from a test

sample at a magniﬁ cation of 500,000×. The distinct changes in image

intensity reﬂ ect the variation of the SE1 component, which is due to

Chapter 3 Scanning Electron Microscopy 213

the large differences in the atomic number between the carbon and the

Au–Pd particles. This type of test sample is usually used to demon-

strate the performance of SEMs.

The low SE yield of low atomic number specimens (cf. Figure 3–17)

such as soft biological objects and polymers limits the resolution due

to the poor SNR. However, the SNR can be improved signiﬁ cantly by

coating the specimen surface with an ultrathin very ﬁ ne-grain metal

ﬁ lm (Peters, 1982) by Penning sputtering or by evaporation in oil-free

high vacuum (cf. Section 2.4). The thickness of such ﬁ lms can be as

small as 1 nm and, as we shall see later, such ultrathin ﬁ lms do not

mask ﬁ ne surface structures. In addition to improving the SNR the

ultrathin coating plays an important role in contrast formation and the

image resolution obtainable. As mentioned earlier, the SE1 arise from

the area directly irradiated by the electron beam and its immediate

vicinity caused by the delocalization of the inelastic scattering in the

order of a very few nanometers (cf. Section 2.2). In the case of the speci-

men coated with an ultrathin metal ﬁ lm the SE1 generation is conﬁ ned

almost exclusively to the ﬁ lm. Figure 3–39a shows schematically the

cross section of an object coated with a continuous metal ﬁ lm of con-

stant thickness. As the electron beam is scanned across the object the

projected ﬁ lm thickness will vary between the nominal ﬁ lm thickness

Figure 3–38. High-resolution secondary electron micrographs of test specimens recorded with the

“in-lens” ﬁ eld emission scanning electron microscope S-5000 (Hitachi Ltd., Tokyo, Japan) at 30 kV (a)

and 1 kV (b). The test specimens were Au–Pd particles on carbon (a) and magnetic tape evaporated

with gold (b). The related power spectra are inserted at the top right. The dashed circles correspond

to (0.6 nm)

−1

(a) and (3.5 nm)

−1

(b), respectively.

214 R. Reichelt

and the maximum, which is several times greater than the nominal

thickness. As shown by Monte Carlo calculations the SE1 yield increases

very quickly with the thickness of the metal ﬁ lm (Joy, 1984). For

example, the Monte Carlo calculations by Joy (1984) reveal for chro-

mium and 20-keV electrons that half of the maximum SE1 yield is

reached for a thickness of 1–1.5 nm only. The dependence of the SE1

yield versus the thickness of a coating ﬁ lm is shown schematically in

Figure 3–39b. It indicates that the increase of the SE1 yield with the

thickness slows down at twice the thickness at half of the maximum

SE1 yield, i.e., the continuous ﬁ lm should be as thin as possible. Monte

Carlo calculations of the SE1 yield for some of the metals suitable for

preparing ultrathin very ﬁ ne-grain metal ﬁ lms show a monotonic

increase with the atomic number (Joy, 1991); thus some further improve-

ment of the SNR may be expected with high atomic number metals.

The ultrathin very ﬁ ne-grain metal ﬁ lm on the sample surface also

improves the BSE1 component signiﬁ cantly, thus improving the SNR

in high-magniﬁ cation BSE micrographs. The BSE1 are very important

for high-resolution SEM because the elastic electron scattering is

strongly localized. The intensity of the BSE1 component increases with

the projected ﬁ lm thickness, i.e., increases with the number of atomic

scattering centers. Since the BSE coefﬁ cient strongly increases with the

atomic number (cf. Figure 3–17), the BSE1 component of the metal ﬁ lm

is signiﬁ cantly larger than the contribution from the coated low-atomic

number specimen. The same is also true for small metal clusters or

small particles at the specimen surface, e.g., such as colloidal gold down

to a minimum diameter of 0.8 nm (Hermann et al., 1991), which can be

identiﬁ ed unambiguously in the high-resolution BSE micrograph.

3.1.6 Selected Applications

Since the achievable resolution is the main difference between the

high-resolution ﬁ eld emission SEM and the conventional SEM, it is

obvious that the high-resolution SEM (HRSEM) can readily handle

almost all of the applications mentioned in Section 2.6. Exceptions are

Layer

Specimen

T(nm)

1234

SE1-yield (arb. units)

Figure 3–39. Schematic cross section of a specimen coated with an ultrathin continuous metal layer

of constant thickness (a). The projected mass thickness of the metal layer varies as the electron beam

is scanned across the specimen. (b) Graph of the SE1 yield versus the thickness of the coating ﬁ lm.