Page 283 - Vibrational Spectroscopic Imaging for Biomedical Applications

258 Cha pte r Ei g h t

biofilm matrix is also responsible for reduced susceptibility of the bacte-
ria for antibiotics. Therefore, information about the bacterial surface
might lead to a more profound understanding of biofilm formation and
contribute to an improved treatment of S. epidermidis infections.
For the TERS investigations single bacterial cells of S. epidermidis
were drop casted on a glass slide and placed under the AFM. A silver-
coated AFM tip was approached from the top and operated in the
intermittent contact mode. A 568-nm laser beam from a krypton ion
laser was focused from below onto the AFM tip via an oil immersion
objective to excite the surface plasmons of the silver at the point of
the tip apex. The sample was moved in xy-direction by a piezo-driven
stage, while the enhancing tip was maintained in the laser focus, so
that changing areas of the sample experienced the high evanescent
field and TERS spectra could be recorded. 65,66 An example of a pseudo
three-dimensional topographic image of single S. epidermidis cells on
a glass surface is shown in Fig. 8.11b. The scanned area was 7 × 7 μm 2
and the bacterial cells were visible as round features on the glass sur-
face with a diameter of about 1 μm. When the silver-coated tip
approached on the cells, high signal-to-noise-ratio TERS spectra
could be recorded in only 1 second [Fig. 8.11(c), traces (A), (C), (D)],
while a flat baseline was observed when the tip was on the glass
substrate without cell [Fig. 8.11(c), trace (B)]. The enhanced Raman
signal originates just from a very small area in the vicinity of the tip,
corresponding roughly to the tip apex size. Taking this nominal tip
radius into account, chemical information from the vibrational spec-
tra of different spots on the cell can be resolved with a spatial reso-
lution down to a few 50 nm. Due to the much higher spatial
resolution, the chemical information contained in the TERS spectra
results from much less chemical species and is confined to the outer
most surface layer. In standard (confocal) Raman microspectros-
3
copy a much larger volume element (circa 0.8 × 0.8 × 3 μm ) is
probed.
Most of the TERS bands of the staphylococcus cells (Fig. 8.11(c),
traces (A), (C), (D)) can tentatively be assigned to contributions from
peptides and polysaccharides. Protein contributions are evident for
−1
the amide I band around 1660 cm , and N-acetyl-related bands
−1
−1
(amide II) around 1533 to 1567 cm and 1519 cm . For the amide III
band a wide spectral range is given in the literature, so that the bands
−1
between 1348 and 1187 cm might have contributions of the amide
III band from different amino acids. Bands at 1206 and 1198 are fur-
thermore reported to contain major contributions from tyrosine and
phenylalanine. Further peptide Raman bands are present with the
−1
C⎯C mode of phenylalanine at 1002 cm , the C—C skeletal modes
−1
in proteins between 930 and 938 cm , and the NCO deformation of
−1
tyrosine at 643 cm . Raman bands of different carbohydrate moieties
−1
are found with the CH bending of the CH OH group around 1200 cm ,
2
−1
and the OH deformation vibration around 1340 cm up to 1360 cm −1