Tools for the Characterization of Biomass at the Nanometer Scale - The Nanoscience and Technology of Renewable Biomaterials - page 71

3.4.1.3 Focused Ion Beam Cutting

Focused ion beams (FIBs) are often used in the semiconductor electronics industry, but
the process may be especially useful for cutting specimens with phases of very different
hardness, which is a particular problem when preparing plant cell walls. Typically FIB
cuts specimens with a 100-nm wide-beam of 30-keV gallium ions. In the usual process,
stair-stepped depressions are carved into the specimen with a rastered ion beam on either
side of a thin (

100 nm) section to be studied. Finally, the edges of the thin section are
cut free from the specimen, which is recovered and mounted on a transmission electron
microscopy (TEM) grid (71-73).
While cutting the specimen, these ions also cause a number of artifacts from ion
implantation and heat (74). Specific results depend upon material composition and ion
energy, but the ion implantation depth is usually about 20 nm and atom displacements
are confined to about 30 nm for gallium ions in low atomic number materials. The depth
of the damage layer is strongly dependent upon ion accelerating voltage and the incident
ion milling angle (75). Because almost all the ion kinetic energy is converted to heat
(76), organic and polymeric materials can experience temperatures up to 500 ◦ Catthe
etching site. However, FIB cutting does not necessarily overheat specimens; vitreously
frozen water has been prepared without heat-induced ice crystal formation (77). New
developments in cluster ions, discussed later under 'Secondary Ion Mass Spectrometry,'
promise to produce even less damage in biomass specimens.
Most published examples of FIB involve inorganic semiconductor materials (78, 79)
or inorganic composites (80), but use with soft materials is also possible. Human hair
and housefly eye (81) represent biological specimens. Photographic film (79) and a toner
particle (82) have also been successfully sectioned. The toner particle is an important
example because contrary to microtomy sections, filler within the toner was not damaged
or displaced in FIB sections. FIB is also used to decompose gases such as W(CO) 6 to
tungsten metal at the specimen surface for protection, to minimize charge accumulation,
or to weld a section to a micromanipulator.

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3.4.2 Scanning Probe Microscopies

Scanning probe microscopy ( SPM ) techniques, especially tapping mode atomic
force microscopy (AFM), are the most frequently used techniques for characterizing
nanometer-scale structures. These techniques move a probe along the specimen surface
to determine topography, material properties, or chemical structure. SPM techniques
are reported in well over 10,000 research papers each year, but the results must be
interpreted with caution. Like all microscopy techniques, with enough images it
is possible to find exactly what is of interest, even if it is an artifact of specimen
preparation or not representative.
The early use of AFM by Hanley and Gray et al . to describe wood cell structure
illustrates some of the specimen preparation problems and limitations (83, 84). Cell
structures were subjected to physical and chemical treatment in the preparation process.
Although such methods are commonly used, whether the resulting specimen is an accu-
rate representation of native plant morphology is questionable. The AFM images in the
paper illustrate a problem common to all SPM: the observed surface is a convolution of
the actual surface and the shape of the probe tip.