Tools for the Characterization of Biomass at the Nanometer Scale - The Nanoscience and Technology of Renewable Biomaterials - page 78

Cluster ions produce greater useful signal intensity and sputter rate while limiting damage
and penetration depth. Cluster ions may be used for analysis at low ion current (static
SIMS) or can systematically remove layers from the specimen at higher ion currents
than gallium, as shown on poly(methyl methacrylate) (146). Because many fragments
have no charge, a second ion beam can be used to ionize fragments (144). Winograd
reviewed the recent development of cluster ion mass spectrometry (147). Some focused
ion guns allow SIMS imaging. An Au 3 ion source provides a high-brightness beam with
a 200-nm spot size, whereas C 60 and gallium ions have been focused to about 2 µ mand

50 nm, respectively.
Specimen bombardment with C 60 may produce a larger spot size than other ions but
has many desirable properties. C 60 produces little roughening during erosion experiments
and has larger usable mass range and sensitivity than do gallium ion beams. C 60 ions
have increased secondary ion yields and fewer low-mass fragments but no increase in
damage with ion energy up to 120 keV (148). Langmuir-Blodgett films sputtered with
C 60 ions produced close to two orders of magnitude more characteristic secondary ions
than gallium (149), apparently from increased sputter yield. Carbohydrate films doped
with peptides bombarded with C 60 ions produced high-quality time-of-flight secondary
ion mass spectra, even with ion doses 100 times greater than those used for gallium
bombardment (150).
The combination of SIMS and AFM is useful because SIMS produces chemical images
and AFM provides topology and other material properties. Zhu used this combination
to examine penetration of gold atoms through alkanethiolate self-assembled monolayers
(151). Wucher used this pair of techniques to produce a three-dimensional representation
of a 300-nm-thick peptide-doped carbohydrate film with a combination of imaging and
etching (152).

3.4.4 Transmission Electron Microscopy

Transmission electron microscopy ( TEM ) is analogous to transmission light microscopy
except that electrons, rather than photons, are passed through the specimen. TEM can
provide images with subnanometer resolution and so has been extensively used by biol-
ogists for nearly 50 years. The most serious limitations of TEM are that specimens must
resist electron damage and usually have thickness less than approximately 100 nm so
that only single-electron scattering events are likely.
Carbon replicas have long been a standard means of studying biomass with TEM
(153-155). For example, the highest resolution images of cellulose fibril morphology
were produced by carbon replicas of developing wood cells at a stage prior to the
incorporation of lignin (156). In this example, cells were frozen quickly to avoid ice
crystal formation and cleaved at 150 ◦ C. The exposed surfaces were coated with carbon

and shadowed with platinum; then the biomass was dissolved with concentrated sulfuric
acid and the replica examined by TEM.
TEM specimens are also made by cutting 30- to 60-nm sections of material. Before
cutting, samples are embedded in resin, which holds the specimen together. Of all spec-
imen preparation techniques, embedding in ice by fast freezing and keeping at

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100 ◦ C
throughout specimen preparation and imaging produces TEM specimens most similar to
the original biomass.

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