Page 306 - Vibrational Spectroscopic Imaging for Biomedical Applications

280 Cha pte r Ni ne

Many cell biological processes, such as division, differentiation,
apoptosis, necrosis, and phagocytosis, are accompanied by large spa-
tial reorganizations of the molecular components that constitute the
cell. The chemical composition of the cell too can strongly change
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over the time. In work of Uzunbajakava et al., nonresonant confocal
Raman point imaging has been used to map the DNA and the protein
distributions in individual apoptotic cells. Raman imaging of a single
cell took time about 15 to 20 minutes, with 0.5- to 2-second accumula-
tion per pixel, and a resolution of 250 nm. They investigated the dis-
tribution of cytoplasmic and nuclear materials of the cells in a late
stage of apoptosis using spectral band intensity images after PC fil-
tering of the spectra. In these experiments a high laser power (647 nm)
was used of 60 to 120 mW in a confocal spot (0.5 μm), but no damage
to the fixed cells was observed.
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In work by Yu et al. confocal Raman microspectroscopic map-
ping (with an excitation wavelength of 785 nm, and 10 mW laser
power on the sample) was used to investigate various HBE (human
breast epithelia) cell lines (one normal or untransformed and three
tumorigenic or transformed). Tumorigenesis is a genetic process
and it is expected that the early cancer effects will be found in the
nuclei. They extracted DNA, RNA, and proteins from the nuclei of
HBE cells and developed spectra-fitting models in order to identify
the compositional changes associated with tumorigenesis in cell
nuclei via in vivo investigation of single cells. Their results showed
that transformed and untransformed cells indeed show distinguish-
able differences in the Raman spectra of their nuclei.
High-spatial-resolution Raman mapping can reveal the struc-
ture and arrangement of subcellular organelles in detail and can
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provide insight into cellular biochemical dynamics. For example,
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in a study by van Manen et al. single-cell confocal Raman micros-
copy (with an excitation wavelength of 647 nm, and 100 mW laser
power on the sample) was used to analyze the chemical composi-
tion of lipid bodies, cytoplasmic organelles rich in triglycerides
and phospholipids that are associated with phagosomes in profes-
sional phagocytes. They found that incubation of phagocytes with
AA and latex beads leads to the accumulation of lipid bodies, rich
in esterified arachidonate, near the phagocytosed microspheres
(Fig. 9.7).
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Krafft et al. compared two different commercially available
Raman imaging instruments to investigate the chemical composi-
tion organelles and vesicles in single human lung fibroblast cells.
The setups were both dispersive point mapping instruments, but
had a different laser excitation wavelength and power, a different
spectral and spatial resolution, and a different signal integration
time per pixel. System 1 had 785 nm/100 mW excitation and 1 μm
spatial resolution, system 2 had 532 nm/20 mW excitation and 0.3 μm
resolution.