The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.

We report the first Raman spectra of individual living and dead cells (MLE-12 line) cultured on bioinert standard poly-L-lysine coated fused silica and on bioactive 45S5 Bioglass®measured at 785 nm laser excitation. At this excitation wavelength no damage was induced to the cells even after 40 minutes irradiation at 115 mW power, as indicated by cell morphology observation and trypan blue viability test. We show that shorter wavelength lasers, 488 nm and 514 nm, cannot be used because they induce damage to the cells at very low laser powers (5 mW) and short irradiation times (5–20 minutes). The most important differences between the spectra of living and dead cells are in the 1530–1700 cm−1range, where the dead cells have strong peaks at 1578 cm−1and 1607 cm−1. Other differences occur around the DNA peak at 1094 cm−1. Our study establishes the feasibility of using the 785 nm laser for anin situreal-time non-invasive method to follow biological events (proliferation, differentiation, cell death, etc.) within individual cells cultured on bioactive scaffolds in their physiologic environment over long periods of time.

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We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.