Page 273 - Vibrational Spectroscopic Imaging for Biomedical Applications

Raman Micr oscopy for Biomedical Applications 249

colony count. In addition 12 ± 24 hours are needed for organism iden-
tification and susceptibility testing. As a first differentiation crite-
ria relatively unspecific morphological parameters, such as size,
shape, and color of the bacteria as well as of the colonies, are used
which need to be complemented with expensive and tedious meta-
bolic tests, such as the ability to grow in various media under dif-
ferent conditions, degradation of certain substrates and enzyme
activity. 52
Raman spectroscopy offers a fast and reliable way to obtain
detailed information about the chemical composition in a noninva-
sive manner. The Raman spectra provide complex and detailed
“fingerprint-like” information of the overall molecular composition
of living bacterial cells in an extremely brief time span. An early
report described the classification of 42 candida strains comprising
five species by confocal Raman microscopy to demonstrate the feasi-
53
bility of the technique for the rapid identification. With Raman
microspectroscopy it is furthermore possible to focus the excitation
laser down to about 1 μm in diameter, which is on the order of mag-
nitude of the size of bacteria. Therefore, even single cells can be
probed, which makes the time-consuming cultivation unnecessary
and reduces the amount of potentially hazardous biomaterial.
Excitation in the Visible Wavelength Range
Sample preparation for the Raman measurements is straight forward
and easy. To create a database with known bacterial strains, the bac-
teria are grown on agar plates at varying environmental conditions
(temperature, nutrition) which mimics the unknown history of indi-
vidual bacterial cells obtained from patients or found in hospitals,
clean room environments or food production lines. After varying
growth time, the bacteria are harvested and smeared on a fused silica
plate. Raman spectra of single bacterial cells are collected after excita-
tion with 532 nm from a frequency doubled Nd:YAG laser focused
down with a 100× objective and resulting in 10 mW laser power at the
sample. Typical exposure times are 60 seconds per spectrum.
Raman spectra of single bacterial cells from nine different species
are shown in Fig. 8.10a. The most intense Raman band is centered
−1
around 2940 cm which is assigned to symmetric and antisymmetric
C—H stretching vibrations of the CH and CH groups from lipids,
2 3
proteins and carbohydrates. The scissoring and deformation vibra-
−1
−1
tions of the C—H bond are found around 1450 cm and 1337 cm ,
respectively. Vibrations of the peptide linkage of proteins are located
−1
around 1660 cm (amide I) and with less intensity around 1242 cm −1
(amide III). The most prominent spectral contribution of the aromatic
−1
amino acid phenylalanine is found at 1003 cm . The band at 1575 cm −1
is assigned to the nucleotides guanine and adenine. The band at
−1
1128 cm is due to C—N and C—C stretching vibrations. Colored
bacteria, such as Micrococcus luteus, exhibit additional sharp signals