Page 114 - Vibrational Spectroscopic Imaging for Biomedical Applications

90 Cha pte r T h ree

and spectral distinction between cytoplasmic and nuclear compartments
in living cells was not effected in Raman maps of two different cell
types (human osteogenic sarcoma cell and human embryonic lung
epithelial fibroblast) that required long collection times (up to 20
60
60
hours). In this study by Krafft et al., difference spectra of the
cytoplasm and nucleus identified the important discriminatory
variables that distinguished these compartments: nucleic acids and
lipids. Infact, analysis of living cells grown on quartz and analyzed
in media, provided Raman spectra containing features of subcellu-
lar components that were more pronounced than those obtained
from frozen-hydrated cells. This was attributed to conformational
changes and aggregation of biomolecular constituents caused by
the freeze-drying process and which are not present when the cells
are analyzed hydrated.
The acquisition time for a Raman map of a cell can be improved
by increasing the sensitivity of the technique. Kneipp et al. 61–62 have
demonstrated that enhanced Raman signals (10 to 14 orders of mag-
nitude) for the native constituents of a cell can be achieved by incor-
porating colloidal gold particles into the cell. The gold nanoparticles
give rise to surface-enhanced Raman scattering (SERS), where Raman
molecules close to the vicinity of the nanoparticles experience elec-
tronic interaction with enhanced optical fields due to resonances of
the applied optical fields with the surface plasmon oscillations of the
metallic nanostructures. This process results in an increase in the scat-
tering cross section of the Raman molecules, which enabled Raman
maps to be collected at 1-μm lateral resolution (1 second for one map-
ping point), where each spectrum in the map consisted of the spectral
−1 61
region 400 to 1800 cm .
Delivery of the nanoparticles into the cell interior can be carried
61
out in two ways, sonication or fluid-phase uptake. The fluid-phase
uptake method involves supplementing the culture medium with
colloidal gold suspensions (60 nm in size), 24 hours prior to experi-
ments. The cells internalize the nanoparticles through endocytosis
62
and without further induction (Fig. 3.14a). This can result in the
formation of colloidal aggregates inside the cell that may be 100 nm
61
to a few micrometers in size. The cells are washed in buffer to
remove nonincorporated nanoparticles and replaced in fresh buffer
for SERS analysis. The second method of delivering nanoparticles
into the cell is by sonication, where rupture of the cell membrane
enables an influx of nanoparticles before self-annealing within a
few seconds. However, in low-intensity ultrasound mediated gene
transfection, it has been found that sonication can induce stress
63
responses in the cell and so should be carried out 24 hours prior to
experiment to allow enough time for the cell to repair any damage.
The authors report that incorporation of the nanoparticles into
the cell using the fluid-phase uptake method did not yield any visible
changes in growth characteristics such as signs of apoptosis or cell