We introduce here multiplex nonlinear optical imaging as a powerful tool for studying the molecular organization and its transformation in cellular processes, with the specific example of apoptosis. Apoptosis is a process of self-initiated cell death, critically important for physiological regulation and elimination of genetic disorders. Nonlinear optical microscopy, combining the coherent anti-Stokes Raman scattering (CARS) microscopy and two-photon excited fluorescence (TPEF), has been used for analysis of spatial distribution of major types of biomolecules: proteins, lipids, and nucleic acids in the cells while monitoring their changes during apoptosis. CARS imaging revealed that in the nuclei of proliferating cells, the proteins are distributed nearly uniformly, with local accumulations in several nuclear structures. We have found that this distribution is abruptly disrupted at the onset of apoptosis and is transformed to a progressively irregular pattern. Fluorescence recovery after photobleaching (FRAP) studies indicate that pronounced aggregation of proteins in the nucleoplasm of apoptotic cells coincides with a gradual reduction in their mobility.
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