Page 250 - Vibrational Spectroscopic Imaging for Biomedical Applications

226 Cha pte r Ei g h t

resolution which was obtained using multimode optical fiber probes
to high-spatial resolution which was obtained in tip-enhanced Raman
spectroscopy (TERS) using functionalized AFM tips.

8.1 Introduction
The expression “biophotonic” is composed of the Greek syllables
“bios” for life and “phos” for light. The term denotes the scientific
discipline which applies light-based techniques to problems in medi-
cine and life sciences. Biophotonic methods can provide information
about inherent optical properties of cells and tissues, and the presence or
absence of endogenous or exogenous fluorophores. The optical detec-
tion offers advantages in (1) spatial resolution in the sub-micrometer
range whereby Abbe’s detection limit can even be overcome by super
resolution approaches such as stimulated emission depletion in fluores-
1
cence spectroscopy, (2) nondestructivity because photons with
wavelengths from visible to the infrared do not harm cells and tissues,
(3) speed because data acquisition usually takes seconds, and (4)
costs because most instruments are less expensive compared to clini-
cal devices. These advantages are utilized in various areas in life sci-
ences. Absorption, fluorescence, reflection, and bioluminescence
belong to the most prominent techniques. A general drawback of all
optical methods for diagnostic purposes is the low penetration depth
in tissue which depends on absorption and scattering properties.
High absorbance exists in the visible range (350 to 700 nm) mainly
due to hemoglobin of blood and in the infrared range (>900 nm)
mainly due to water and lipids. In the near-infrared region all bio-
molecules show minimal absorption so techniques using this spectral
window take advantage of maximum penetration in the order of 1 to
2 cm. However, this value is still too low, e.g., to localize brain
tumors inside the skull using optical procedures. Therefore, clinical
applications require microscopes or handheld fiber-optic probes
during open surgery, or miniaturized fiber optic probes during minimal
invasive endoscopy.
Raman spectroscopy has been recognized as another powerful
technique for biomedical applications. Its main advantage is that the
method yields a wealth of information about the molecular structure
and biochemical composition without labeling or any other sample
preparation. The last decade brought a tremendous progress in
Raman instrumentation. Improved detection sensitivity and excita-
tion with near-infrared or ultraviolet lasers enabled to record Raman
spectra from biological samples. More bands are resolved in Raman
spectra than in other optical spectra because numerous bands in
biomolecules can simultaneously be excited. This fingerprint capabil-
ity of Raman spectroscopy is applied to characterize and identify tis-
sue, cells, and bacteria. As the spectral contributions of all constituents
such as proteins, lipids, nucleic acids, and carbohydrates overlap to