379
13 Vibrational Spectroscopy: Disease Diagnostics and Beyond
spatial resolution in the future, but at present such short pulse systems cannot be
considered benchtop techniques.
Cisplatin, a dNA major groove binder [121], was chosen as a model as its mechanism of action is well characterised. to fully validate the technique, the study
should be extended to other chemotherapeutic agents of known interaction mechanisms, such as tallimustine, a known dNA minor groove binder, and ethidium bromide, a known dNA intercalator. In this way the spectroscopic signatures of their
interaction can be established and subsequently can act as a database against which
novel chemotherapeutic agents can be evaluated. ultimately, the techniques should
be validated in tumour tissue.
13.5 Live Cell Imaging [122]
Raman microspectroscopy presents several advantages for the study of live cells,
combining molecular analysis with optical imaging. In comparison to infrared spectroscopic analysis, the weak contribution from water offers the possibility to study
the cells in an aqueous environment and thus to keep them alive for the duration
of the measurement [101]. the specific information contained in the Raman spectra provides a signature of the samples studied, which can be related to molecular
content or changes to the physiology as a result of external stimuli [123, 124]. the
spatial resolution is of the order of 0.5-2 µm, providing access to the subcellular
organisation of the cells at a molecular level [125, 126].
Previous studies have demonstrated the capacity of Raman spectroscopy to map
single cells and their subcellular structures. the identification of the sub-cellular
structures such as the nucleus, cytoplasm and mitochondria can be achieved [127]
and chemical maps can be constructed giving information about the distribution of
differentiating molecules such as proteins [128]. however, these have been performed on chemically fixed cells and it has since been demonstrated that chemical fixation induces significant changes to the biochemistry of the cell [129, 130].
ultimately, if subtle changes in metabolism due to for example chemotherapeutic
agents, toxicants, or radiation are to be discerned, live cells should be studied.
over the last decade, new models such as collagen gels and more complex multicomponent systems such as matrigel have emerged, providing the cells a matrix
which more accurately reproduces the extra cellular matrix (ECm) [131–133]. It
has been demonstrated in various studies that the behaviour of cells cultured in
such an environment differs considerably compared to those cultured on traditional
2d surfaces [134]. In terms of cellular viability and spectral quality, collagen gels
have been recently demonstrated to be a substantially improved substrate for live
cells analysis using Raman spectroscopy [122]. the gels provide a 3-d matrix for
cell growth, although it should be noted that the cells are cultured on the surface of
the matrix. As measured using the Alamar Blue cytotoxicity assay, in comparison
to uncoated quartz, a human lung cell line exhibited a 39 % increase in viability in
collagen gels whereas as dermal cell line seemed to be more affected by the differ-
13 Vibrational Spectroscopy: Disease Diagnostics and Beyond
spatial resolution in the future, but at present such short pulse systems cannot be
considered benchtop techniques.
Cisplatin, a dNA major groove binder [121], was chosen as a model as its mechanism of action is well characterised. to fully validate the technique, the study
should be extended to other chemotherapeutic agents of known interaction mechanisms, such as tallimustine, a known dNA minor groove binder, and ethidium bromide, a known dNA intercalator. In this way the spectroscopic signatures of their
interaction can be established and subsequently can act as a database against which
novel chemotherapeutic agents can be evaluated. ultimately, the techniques should
be validated in tumour tissue.
13.5 Live Cell Imaging [122]
Raman microspectroscopy presents several advantages for the study of live cells,
combining molecular analysis with optical imaging. In comparison to infrared spectroscopic analysis, the weak contribution from water offers the possibility to study
the cells in an aqueous environment and thus to keep them alive for the duration
of the measurement [101]. the specific information contained in the Raman spectra provides a signature of the samples studied, which can be related to molecular
content or changes to the physiology as a result of external stimuli [123, 124]. the
spatial resolution is of the order of 0.5-2 µm, providing access to the subcellular
organisation of the cells at a molecular level [125, 126].
Previous studies have demonstrated the capacity of Raman spectroscopy to map
single cells and their subcellular structures. the identification of the sub-cellular
structures such as the nucleus, cytoplasm and mitochondria can be achieved [127]
and chemical maps can be constructed giving information about the distribution of
differentiating molecules such as proteins [128]. however, these have been performed on chemically fixed cells and it has since been demonstrated that chemical fixation induces significant changes to the biochemistry of the cell [129, 130].
ultimately, if subtle changes in metabolism due to for example chemotherapeutic
agents, toxicants, or radiation are to be discerned, live cells should be studied.
over the last decade, new models such as collagen gels and more complex multicomponent systems such as matrigel have emerged, providing the cells a matrix
which more accurately reproduces the extra cellular matrix (ECm) [131–133]. It
has been demonstrated in various studies that the behaviour of cells cultured in
such an environment differs considerably compared to those cultured on traditional
2d surfaces [134]. In terms of cellular viability and spectral quality, collagen gels
have been recently demonstrated to be a substantially improved substrate for live
cells analysis using Raman spectroscopy [122]. the gels provide a 3-d matrix for
cell growth, although it should be noted that the cells are cultured on the surface of
the matrix. As measured using the Alamar Blue cytotoxicity assay, in comparison
to uncoated quartz, a human lung cell line exhibited a 39 % increase in viability in
collagen gels whereas as dermal cell line seemed to be more affected by the differ-