Biomedical Engineering Reference
In-Depth Information
3.4.3.1
Scanning Electron Microscopy
Scanning electron microscopy ( SEM ) provides high-resolution topographic information
with little specimen preparation. SEM cannot differentiate carbon, nitrogen, and oxygen
so provides little chemical information about biomass unless higher atomic weight ele-
ments are present in the specimen. In SEM, a narrow beam of electrons is scanned
across a specimen while the intensity of reflected or ejected electrons provides the
image. SEMs require a vacuum and some means to dissipate the charge of electrons
that lodge in the specimen. Traditionally, charge was dissipated by coating specimens
with conductive materials. Microscopes that tolerate higher pressure, called variable
pressure or 'atmospheric' SEMs, were developed to image specimens in a more nat-
ural state, and have the added advantage that ionized gas surrounding the specimen
often carries away accumulated charge. With a tiny aperture over the specimen to
limit evaporation rate, some variable pressure SEMs can image wet specimens at room
temperature.
Another option that allows SEM of wet biomass is the cold stage, which keeps the
specimenverycold(downto
120 C). At this temperature, ice-filled specimens can
be imaged under high vacuum, and the temperature, pressure, and time on the stage
can be adjusted to allow a controlled amount of freeze drying of the specimen surface.
Additionally, specimens analyzed at cryogenic temperatures typically show less evidence
of beam damage (136).
Biomass specimens may benefit from low voltage (0.5-5 keV) SEM operation
( LV S E M ), which often affords good image contrast on uncoated specimens and
minimizes charging and damage (137). LVSEM can also be used to optimize the
difference in secondary electron emission between polymeric materials of different
composition (138). LVSEM images represent only a shallow surface layer because the
penetration depth of these electrons is limited (137).
Energy dispersive X-ray analysis ( EDX ) uses X-rays stimulated by SEM electrons
to make maps of elemental distribution. Resolution is typically a few microns, and
sensitivity is far better for heavy atoms than for oxygen and carbon (137). Gases in the
specimen chamber also scatter electrons, so variable pressure or 'environmental' EDX
analyses almost always has more background than measurements at high vacuum.
3.4.3.2
Helium Ion Microscopy
A helium ion microscope is analogous to an SEM except that helium ions, rather than
electrons, bombard the specimen. Helium ion microscopes provides a brighter imag-
ing beam with better spatial resolution and different elemental sensitivity than SEM
(139). Spatial resolution (approximately 0.5 nm) is partly the result of a much smaller
beam/substrate interaction volume for helium than for electrons. Image contrast mech-
anisms are different than in SEM, which can provide new opportunities for image
enhancement.
Although high-atomic-number ions (such as cesium ions) can damage specimens by
sputtering, the low-atomic-number helium ions have low sputtering probability. As
with an SEM, images can be generated from backscattered ions, whose probability of
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