Due to its high lateral resolution, Raman microspectrsocopy is rapidly becoming an accepted technique for
the subcellular imaging of single cells. Although the potential of the technique has frequently been
demonstrated, many improvements have still to be realised to enhance the relevancy of the data collected.
Although often employed, chemical fixation of cells can cause modifications to the molecular composition
and therefore influence the observations made. However, the weak contribution of water to Raman spectra
offers the potential to study live cells cultured in vitro using an immersion lens, giving the possibility to
record highly specific spectra from cells in their original state. Unfortunately, in common 2-D culture
models, the contribution of the substrates to the spectra recorded requires significant data pre-processing
causing difficulties in developing automated methods for the correction of the spectra. Moreover, the 2-D
in vitro cell model is not ideal and dissimilarities between different optical substrates within in vitro cell
cultures results in morphological and functional changes to the cells. The interaction between the cells and
their microenvironment is crucial to their behavior but also their response to different external agents such
as radiation or anticancer drugs. In order to create an experimental model closer to the real conditions
encountered by the cell in vivo, 3-D collagen gels have been evaluated as a substrate for the spectroscopic
study of live cells. It is demonstrated that neither the medium used for cell culture nor the collagen gels
themselves contribute to the spectra collected. Thus the background contributions are reduced to that of the
water. Spectral measurements can be made in full cell culture medium, allowing prolonged measurement
times. Optimizations made in the use of collagen gels for live cells analysis by Raman spectroscopy are
encouraging and studying live cells within a collagenous microenvironment seems perfectly accessible.
Raman spectroscopy, as an evaluation of the products of ionising radiation exposure in biological systems, has been utilised mainly in the evaluation of the impact of exposure in tissue, cellular constituents and live animals. It has also been recently demonstrated that Raman spectroscopy can demonstrate key spectroscopic changes in the live cell associated with significant apoptotic and necrotic chemical damage. The present preliminary work utilises Raman spectroscopy at 514.5 nm to evaluate the results of exposure to γ-rays in HaCaT cells from a Co-60 therapy source, in tandem with other biological assays. The results demonstrate that Raman spectral changes may be correlated with changes in the cell also identified in parallel biochemical assays.