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Raman Micr oscopy for Biomedical Applications 245
of Raman bands. This hypochromic effect was already reported in
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isolated DNA more than 30 years ago. Similarly, a reduced ratio of
RNA to protein bands was detected in the cytoplasm as a function of
cell stress. Finally, more intense nucleic acid bands were detected in
blisters at the cell surface. This result is consistent with apoptotic par-
ticles by which cells export material out of the cell. In summary, all
observations agree with the key processes of apoptosis, the term for
programmed cell death. Among them are shrinkage of the cell, reor-
ganization of cell organelles, condensation of chromatin, decomposi-
38
tion of cell nucleoli and fragmentation of the nucleus. The cell stress
process was selected for the first application of Raman imaging
because the morphology and molecular composition change in a
well-known way. Such experiments are important before new tech-
niques can be applied to unknown processes.
8.3.2 Red Blood Cells
Resonance Raman (RR) spectroscopy is a variant of Raman spectros-
copy that can be used to enhance its specificity and sensitivity. Here,
the excitation wavelength is matched to an electronic transition of the
molecule of interest so that vibrational modes associated with the
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excited state are enhanced (by as much as a factor of 10 ). Electronic
transitions of proteins with prosthetic groups are found in the visible
spectral region. As the enhancement is restricted to vibrational modes
of the chromophore, the complexity of Raman spectra from cells is
reduced. Functional red blood cells (also known as erythrocytes)
are an excellent subject for RR microspectroscopy and imaging
because of the high concentration of hemoglobin (22 mM) and its
unique spectral properties. The resonance-enhanced Raman sig-
nals of the chromophore heme are rich in information that enabled a
deeper understanding of the structure and function of red blood cells.
RR spectroscopy of red blood cells has recently been reviewed. 39
Unlike traditional histopathological methods (e.g., Gimsa staining),
this approach allows studying unlabeled red blood cells.
Malaria research is an interesting diagnostic application of RR
spectroscopy which has been reviewed by Wood and McNaughton. 40
Malaria is one of the most common infectious diseases and an enor-
mous public health problem. The disease is caused by protozoan
parasites of the genus Plasmodium that are transmitted by mosqui-
toes. The parasites multiply within red blood cells, where they digest
a major proportion of red blood cell hemoglobin. As free heme, a
product of hemoglobin digestion, is toxic for the parasite it detoxifies
free heme by conversion into an insoluble crystal called hemozoin or
“malaria pigment.” The spatial distribution of heme species in red
blood cells was probed by RR imaging. 41–43 An example is shown in
Fig. 8.9. Typical RR spectra of hemozoin and hemoglobin inside a
parasitized red blood cell are compared with RR spectra of β-hematin
and hemoglobin as reference compounds. The synthetic compound
β-hematin is a structural analogue of hemozoin with a strong marker