Studying apoptosis, or programmed cell death, is imperative for understanding normal physiological functions as well as disorders such as cancer and neurodegenerative and autoimmune diseases. Being able to recognize the intracellular configurations of macromolecules that indicate apoptosis is essential for advancing biomedical science and applied medicine. Until now, however, it has not been possible to get an in-depth view of the apoptotic process. Only limited glimpses have been seen of the proteins, RNA, DNA and lipids that comprise the four major types of subcellular macromolecules. Imaging techniques have been unable to show all of these at the same time, so their spatial relationships and structural transformations during programmed cell death have been undetectable. To achieve real-time monitoring of apoptosis, a multidisciplinary team at the University at Buffalo developed a multiplex nonlinear optical imaging method that uses three types of complementary microscopies: coherent anti-Stokes Raman scattering (CARS), two-photon excitation fluorescence (TPEF) and fluorescence recovery after photobleaching (FRAP). The first two modes, operating in tandem, scan multiple pictures at almost exactly the same time, allowing all four types of macromolecules to be viewed in relation to each other. The third method, which quantifies two-dimensional lateral diffusion, characterizes the motion of nuclear proteins. The study, led by Paras N. Prasad, was published in the July 20, 2010, issue of Proceedings of the National Academy of Sciences. The four panels of this CARS/TPEF image demonstrate the redistribution of macromolecules during apoptosis over a 24-h period. Proteins are red, RNA is green, DNA is blue, and lipids are gray. Researchers at the Institute for Lasers, Photonics and Biophotonics at the University at Buffalo in New York have developed a multiplex biophotonic imaging platform that permits real-time imaging of dying cells. Courtesy of Paras N. Prasad. The CARS technique, a form of nonlinear optical microscopy that uses multiple photons to sense vibrational frequencies of specific chemical bonds, detected proteins and lipids at their characteristic vibrations of 2928 and 2890 cm—1, respectively. TPEF was required to sense the nucleic acids, selectively stained by acridine orange, in the green (DNA) and red (RNA) spectral channels. “This multiplex imaging is a step forward from traditional microscopy and provides direct information on the presence of nonlabeled molecules in the cell interior,” co-author Andrey N. Kuzmin said. Using this multiplex biophotonic platform permitted the researchers to see that, before apoptosis, while the cell is still proliferating, proteins in the nucleus are distributed nearly uniformly, although with accumulation in several subnuclear structures. However, at the onset of apoptosis, this pattern changes abruptly, with proteins forming progressively irregular clusters. The FRAP technique revealed that, during apoptosis, the diffusion of nuclear proteins gradually slows down. The most exciting potential applications of this multiplex imaging method include in vivo monitoring of infectious diseases and other cell function abnormalities, along with therapeutic drug administration, according to co-author Artem M. Pliss. “The ability to quantitatively characterize and monitor in real time biomolecular distribution and kinetics during drug-cell interactions would be in high demand for clinical administration and therapeutic drug development.” As for the future, the team’s plan is to concentrate on the “most essential and socially important biomedical challenges, such as disease diagnostics and monitoring and therapeutic drug interaction studies,” co-author Aliaksandr V. Kachynski said.