Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-induc... More
Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.
