Diffusive, reactive and orientational dynamics of molecular systems using molecular Fourier imaging correlation spectroscopy

A novel spectroscopic technique for determining dynamical properties of small ensembles of isolated chromophores, termed Molecular Fourier Imaging Correlation Spectroscopy, has been developed. This technique is an extension of previous Fourier Imaging Correlation Spectroscopy methods which have been employed to study the dynamics of complex fluids including dilute and semi-dilute colloidal suspensions, as well as mitochondrial motion in living cells. In previous methods samples under investigation have contained high chromophore densities, a situation that is not practical in many systems as the introduction of such high densities of chromophore are likely to perturb the dynamics in question. This is especially true in biological systems where the dynamics of interest are often the result of complex interactions between individual proteins.; Molecular FICS expands on previous methods by significantly increasing the signal to noise ratio for low emission samples through improvements in stability, photon collection efficiency, and dynamic range. Through these modifications it is possible to achieve dynamic measurements on complex fluids containing small ensembles of isolated chromophores. Furthermore M-FICS measurements are polarization resolved and capable of resolving dynamics due to center of mass motion from those which are due to molecular reorientation and internal degrees of freedom. Current methods allow for determination of such dynamics across a wide range of spatial (0.75mum--5mum) and temporal (10-4s--10 2s) scales.; M-FICS measurements are presented here for small ensembles of two fluorophores of biological significance. The first system, the fluorescent protein DsRed, is a common fluorescent protein that can be readily transfected as a fluorescent probe for in-vivo cellular studies. DsRed has complex photophysical properties, and in these studies shows evidence of dynamic dispersion, possibly due to protein aggregation. The second system, molecular beacons, serves as a model chemically reactive system. These measurements represent the first time that the dynamics of small ensembles of isolated chromophores have been observed with this level of specificity and dynamic range. In addition, control measurements on a dilute colloidal system, for which the dynamics are well-characterized, are presented in order to demonstrate the effectiveness of M-FICS in reproducing the expected behavior.; This dissertation includes previously published and co-authored material.