This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Fluorescence correlation spectroscopy (FCS) based on statistical analysis of photons emitted by fluorescent particles as they move through a femtoliter observation volume provides quantitative information about mobility of molecules, concentration, composition of molecular complexes, dissociation constants, and reaction kinetics. The established theory of FCS predicts the statistics of photon arrival times based on free diffusion of particles, and particles involved in certain kinds of reactions. However explicit solutions do not exist for more complex particle systems found in live cells and involving membrane structures, cytoskeleton, interactions with multiple partners, and molecular crowdedness. To interpret the FCS data obtained from live cells, the in vivo environment will be modeled by spatial Monte Carlo method. Currently we are developing software that models individual particles randomly diffusing inside a reflective reaction volume. Particles can have different diffusion coefficient and fluorescence quantum yield and can engage in different first and second order reactions. Various reflective surfaces can be defined inside the reaction volume. The observation volumes is defined as a spatial brightness function. Photon emission probability depends on particle position relative to the center of the observation volume. Simulation results are used to validate data analysis methods for in vivo FCS data.
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