NON-LINEAR OPTICAL FLUCTUATION STUDIES OF CARDIAC CELLS

动态激光光散射是一种强有力的研究方法 分子和颗粒系统的微观波动， 被广泛应用于生物物理学。 对复杂系统的适用性 例如完整细胞受到来自结构的非选择性散射的限制 这不是正在研究的。 基于我们广泛的理论研究，我们 提出发展一种新的非线性光学起伏技术， 准相干双共振荧光光子相关 这将允许选择性地测量平移、旋转 以及细胞内特定标记发色团的化学波动， 并将该技术应用于心脏兴奋-收缩的研究 偶合器. 我们将建造一种装置， 在与两个耦合光谱共振的波长处标记分子 过渡。 在激发频率的总和下发射的荧光 包含准相干分量，其边带直接与 的位置、方向和化学环境的波动， 标记分子。 该组分的光谱将通过 外差光子相关 为了验证理论原理，我们 将测量汞染料和其他大共轭染料的QDORF光谱， 分子，在各种粘度的自由溶液中， 固定在胶体上。 我们将从孤立的 单个心肌细胞，使用注射的标记物和天然发色团， 确定蜂窝网络中的干扰的灵敏度和来源 环境 在研究的后半部分，我们将与 合成染料化学家开发能够螯合钙的QDORF染料。 来自这种染料的化学动力学QDORF波动光谱是 预测产生细胞内钙浓度的线性估计 同时在几个隔间里 许多其他生物物理 可以预见QDORF的应用。

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.