In situ monitoring of cell death using Raman microspectroscopy.
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.
Verrier
S
S
Department of Materials, South Kensington Campus, London SW7 2BP, United Kingdom.
Notingher
I
I
Polak
J M
JM
Hench
L L
LL
D002241:Carbohydrates / Q000737:chemistry; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D008055:Lipids / Q000737:chemistry; D017830:Octoxynol / Q000494:pharmacology; D010710:Phosphates / Q000737:chemistry; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013997:Time Factors
D002241:Carbohydrates; D003902:Detergents; D008055:Lipids; D010710:Phosphates; D017830:Octoxynol; D004247:DNA; D020169:Caspases