Applications of Raman Spectroscopy to Virology and Microbial Analysis - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 446

tion referring to size or shape. Endospores, for example, exhibit characteristic
birefringence clearly visible under phase contrast conditions. Also fluorescence
characteristics among single cells are exploited, either by utilizing the intrin-
sic autofluorescence to differentiate between biotic/abiotic particles [69] or by
implementing active fluorescence staining methods to perform a live/dead as-
sessment [70, 71] or to find bacteria co-localized within amorphous background
[72].
A clever way to minimize the unwanted background from the cover plate or
the environment is combining laser tweezers and confocal Raman spectroscopy
(LTRS) [73, 74]. While the single cells are levitated well off the surface and
held in the focus of the laser beam the Raman spectral patterns of these cells
are recorded with high sensitivity. Another appealing fact is the usage of one
laser for both Raman excitation and optical trapping to keep the instrumental
efforts as low as possible. Additionally the trapped cells can also be micro-
manipulated and moved from one place to another, e.g., from the native matrix
to a clean collection chamber.
With this technique the CaDPA content inside single Bacillus endospores
could be determined [75] to > 800 mM per single endospore, way above the
CaDPA solubility. Using LTRS also bacteria in spoiled milk samples were
localized, among other food particles and thereafter manipulated to a new
position [76]. Also the discrimination of bacteria according to their species,
various growing conditions or growth phases [74] and preliminary heat treat-
ments [77] were studied with LTRS.
19.3.3 UV-Resonance Raman Spectroscopy of Bacteria
Non-resonant Raman spectroscopy has been applied with success to bacte-
rial characterization and identification. However, the drawback of this tech-
nique is the low probability of Raman scattering. A solution for this problem
is to use a Raman signal-enhancing method, e.g., SERS, TERS, or UVRR.
As already mentioned above an additional advantage of using deep UVRR
spectroscopy is the avoidance of a strong fluorescence background of most
biological samples. Furthermore resonance Raman spectroscopy allows one to
selectively monitor certain chromophoric groups present in macromolecules
and depends greatly on the Raman excitation wavelength applied [78]. Raman
spectra excited at 218-231 nm reflect aromatic acid contributions, while spec-
tra excited at 242-257 nm are dominated by bands from nucleic acids [79].
Figure 19.4 compares bacterial Raman spectra recorded for different excita-
tion wavelengths in the UV and visible regions. The non-resonant Raman
spectrum (532 nm) displays Raman bands originating from all cell compo-
nents while the UVRR spectrum (257 nm) is dominated by bands from nucleic
acids. Here, the signals at 1330 cm − 1 can be assigned to adenine, whereas the
bands at 1480 and 1575 cm − 1 are due to adenine and guanine. Although these
bands can also be seen in the non-resonant Raman spectrum, their intensities
are much lower. In the UVRR spectrum, the proteins are represented only