NON-LINEAR OPTICAL FLUCTUATION STUDIES OF CARDIAC CELLS

点击翻译按钮获取中文摘要

Dynamic laser light scattering is a powerful method for studying microscopic fluctuations of molecular and particulate systems, which has been widely applied in biophysics. Its applicability to complex systems such as intact cells is limited by non-selective scattering from structures which are not under study. Based on our extensive theoretical studies, we propose to develop a novel non-linear optical fluctuation technique, quasi-coherent double resonance fluorescence photon correlation (QDORF) which will permit selective measurement of the translational, rotational and chemical fluctuations of specific labeling chromophores within cells, and apply the technique to studies of cardiac excitation-contraction coupling. We will construct apparatus which will simultaneously excite the labeling molecules at wavelengths resonant with two coupled spectral transitions. Fluorescence emitted at the sum of the exciting frequencies contains a quasi-coherent component whose sidebands are directly related to fluctuations in the position, orientation and chemical environment of the labeling molecules. The spectrum of this component will be analyzed by heterodyne photon correlation. To validate the theoretical principles, we will measure QDORF spectra from merocyanine dyes and other large conjugated molecules, in free solution of various viscosities, and partially immobilized on colloids. We will make QDORF measurements from isolated single cardiac myocytes, using injected labels and native chromophores, to determine the sensitivity and sources of interference in the cellular environment. In the latter part of the study we will collaborate with synthetic dye chemists to develop QDORF dyes capable of chelating calcium. The chemical kinetic QDORF fluctuation spectrum from such a dye is predicted to yield a linear estimate of intracellular calcium concentration in several compartments simultaneously. Numerous other biophysical applications of QDORF are foreseen.