| The spectroscopic detection of cell death has widespread applications in cancer diagnosis, treatment monitoring and drug development. In this dissertation, the use of Raman spectroscopy to identify the biochemical changes due to cell death has been demonstrated. Two models of cell death have been developed using a human melanoma cell line (MEL-28) - multicellular tumor spheroids to model cell death in tumors and cell death in monolayer cultures by combined oxygen and glucose deprivation. The mode of cell death in both models was necrosis. Raman spectroscopy measurements were made using 785 nm excitation in the 450--3130 cm-1 region. Raman spectra were fit to a sum of biochemical components - lipid, protein, RNA, DNA and glycogen - to estimate changes in their relative amounts due to cell death.; Raman spectroscopy measurements showed that spheroids with >45% necrosis had a smaller protein to lipid ratio and lower amounts of RNA compared to spheroids with no necrosis. Spectroscopic changes corresponding to protein, lipid and nucleic acids were observed in spheroids with necrosis. Flow cytometry analysis revealed that large spheroids had a greater fraction of G2 arrested cells. The spectroscopic changes observed in large spheroids were due to necrosis and G2 arrested cells. To the best of our knowledge, this is the first Raman study of cell death in 3D tumor models.; Raman spectroscopic measurements of cell death due to combined oxy-glucose deprivation showed that dead cells had relatively higher amounts of protein and DNA and lower amounts of lipid and RNA compared to live cells. Changes due to protein, lipid and nucleic acids were observed in the spectra of dead cells. To the best of our knowledge, this is the first Raman study of cell-death in monolayer cultures due to combined oxy-glucose deprivation.; Monte Carlo codes for light propagation in tissue were modified to incorporate Raman scattering. The Raman signal collected using our experimental setup was shown to originate from the top 1 mm of the sample. An increase in the beam diameter of the illumination source correlated positively with the fraction of photons collected from greater depths. |
