Biomedical Engineering Reference
In-Depth Information
of non-conductors. With the electron accelerating voltage
lowered to approximately 1 keV, charge accumulation is
not as critical and metallization is not required. Low-
voltage SEM has been used to study platelets and phase
separation in polymers. Also, the environmental SEM
(ESEM) permits wet, uncoated specimens to be studied.
The primary electron beam also results in the emission
of X-rays. These X-rays are used to identify elements with
the technique called energy-dispersive X-ray analysis
(EDXA). However, the high-energy primary electron
beam penetrates deeply into a specimen (a micron or
more). The X-rays produced from the interaction of these
electrons with atoms deep in the bulk of the specimen can
penetrate through the material and be detected. There-
fore, EDXA is not a surface analysis method.
The primary use of SEM is in image topography. SEM
for this application is well elaborated in the literature.
penetration depth = 1-5
m
sample must be inimate contact with crystal
μ
A
source
detector
liquid flow cell
ATR crystal
solid sample
B
penetration depth = 1-1005Å
sample must be on a specular mirror
detector
source
C
penetration depth = 15
μ
m (poorly defined)
sample is often rough
Infrared spectroscopy
detector
source
Infrared spectroscopy (IRS) provides information on the
vibrations of atomic and molecular species. It is a standard
analytical method that can reveal information on specific
chemistries and the orientation of structures. Fourier
transforminfrared (FTIR) spectrometry offers outstanding
signal-to-noise ratio (S/N) and spectral accuracy. However,
even with this high S/N, the small absorption signal asso-
ciated with the minute mass of material in a surface region
can challenge the sensitivity of the spectrometer. Also, the
problem of separating the vastly larger bulk absorption
signal from the surface signal must be addressed.
Surface FTIR methods couple the infrared radiation
to the sample surface to increase the intensity of the
surface signal and reduce the bulk signal (
Allara, 1982;
Leyden and Murthy, 1987; Urban, 1993; Dumas
et al.
,
1999
). Some of these sampling modes, and their char-
acteristics, are illustrated in
Fig. 3.1.4-12
.
The attenuated total reflectance (ATR) mode of sam-
pling has been usedmost often in biomaterials studies. The
penetration depth into the sample is 1-5
m
m. Therefore,
ATR is not highly surface sensitive, but observes a broad
region near the surface. However, it does offer the wealth
of rich structural information common to infrared spectra.
With extremely high S/N FTIR instruments, ATR stud-
ies of proteins and polymers under water have been
performed. In these experiments, thewater signal (which is
typically
99%
or more of the total signal) is subtracted from
the spectrum to leave only the surface material (e.g.,
adsorbed protein) under observation.
Another infrared method that has proven immensely
valuable for observing extremely thin films on reflective
surfaces is infrared reflection absorption spectroscopy
(IRAS),
Fig. 3.1.4-12
. This method has been widely ap-
plied to self-assembled monolayers (SAMs), but is
Fig. 3.1.4-12 Three surface-sensitive infrared sampling
modes: (A) ATR-IR, (B) IRAS, (C) diffuse reflectance.
applicable to many surface films that are less than 10 nm
in thickness. The surface upon which the thin film re-
sides must be highly reflective and metal surfaces work
best, though a silicon wafer can be used. IRAS gives in-
formation about composition, crystallinity and molecular
orientation. IRS is one member of a family of methods
called vibrational spectroscopies. Two other vibrational
spectroscopies, sum frequency generation (SFG) and
Raman, will be mentioned later in the section on newer
methods.
Scanning tunneling microscopy, atomic
force microscopy, and the scanning
probe microscopies
STM and atomic force microscopy (AFM) have de-
veloped from novel research tools to key methods for
biomaterials characterization. AFM has become more
widely used than STM because oxide-free, electrically
conductive surfaces are not needed with AFM. General
review articles (
Binnig and Rohrer, 1986; Avouris, 1990
;
Albrecht
et al
., 1988
) and articles oriented toward bi-
ological studies with these methods (
Hansma
et al.
,
1988
;
Miles
et al.
, 1990
;
Rugar and Hansma, 1990; Jandt,
2001
) are available.
