Biomedical Engineering Reference
In-Depth Information
If the wavelength of the elastic light scattering is carefully selected so as to be outside the
major absorption areas due to water and hemoglobin and if the diffusely scattered light is
measured as a function of angle of incidence, there is potential for this approach to aid in
pathologic diagnosis of disease.
Inelastic Raman spectroscopic scattering has been utilized over the past few decades
initially by physicists and chemists. Raman spectroscopy has become a powerful tool for
studying a variety of biological molecules, including proteins, enzymes and immunoglobu-
lins, nucleic acids, nucleoproteins, lipids and biological membranes, and carbohydrates, but
with the advent of more powerful laser sources and more sensitive detectors, it has also
become useful as a diagnostic and sensing tool. The phenomenon of Raman scattering is
observed when monochromatic (single wavelength) radiation is incident upon the media.
In addition to the elastic scatter of the transmitted light, a portion of the radiation is inelas-
tically scattered. Thus, some of the incident light of frequency
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exhibits frequency shifts
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, which is associated with transitions between rotational, vibrational, and electronic
levels that are specific to a particular analyte of interest. Most studies utilize the Stokes type
of scattering bands that correspond to the
scattering. Therefore, the Raman bands
typically used are those shifted by the interaction with the analyte to longer wavelengths
relative to the excitation wavelength.
As with infrared spectroscopic techniques, Raman spectra can be utilized to identify
molecules, since these spectra are characteristic of variations in the molecular polarizability
and dipole moments. Raman spectroscopy can be considered as complementary to absorp-
tion spectroscopy because neither technique alone can resolve all of the energy states of a
molecule. In fact, for certain molecules, some energy levels may not be resolved by either
technique. Due to the anharmonic oscillator model for dipoles, overtone frequencies exist
in addition to fundamental vibrations. It is an advantage of Raman spectroscopy that the
overtones are much weaker than the fundamental tones, thus contributing to simpler spec-
tra as compared to absorption spectroscopy.
One advantage to using Raman spectroscopy in biological investigations is that the
Raman spectrum of water is weaker and therefore, unlike infrared spectroscopy, only mini-
mally interferes with the spectrum of the solute. Thus, the spectrum can be obtained from
aqueous solutions at reasonable path lengths. However, the Raman signal is also weak,
and only recently, with the replacement of slow photomultiplier tubes with faster CCD
arrays as well as the manufacture of higher power near infrared laser diodes, has the tech-
nology become available to allow researchers to consider the possibility of distinguishing
normal and abnormal tissue types as well as quantifying blood chemicals in near real time.
In addition, investigators have applied statistical methods such as partial least squares
(PLS) to aid in the estimation of biochemical concentrations from Raman spectra.
As with elastic scatter, Raman spectroscopy has been used for both diagnostics and mon-
itoring. The diagnostic approaches look for the presence of different spectral peaks and/or
intensity differences in the peaks due to different chemicals present in, for instance, cancer-
ous tissue. For quantifiable monitoring it is the intensity differences alone that are investi-
gated. In tissue, one problem is the high fluorescence background signal as a result of
autofluorescence incurred in heavily vascularized tissue due to the high concentration
of proteins and other fluorescent components. Instrumentation to excite in the NIR
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