BIOMOLECULE INTERACTIONS W/ FLUORESCENCE CORRELATION SPECTROSCOPY

分子聚集是一种重要的调节机制。 生物系统。然而，大多数实验技术都需要 相对较高的浓度(从毫米到毫米)。因为有很多人 有趣的生理事件发生在浓度低于 微摩尔，我们探索了检测分子的可能性 低浓度范围(从mm到PM)的聚集 双光子荧光显微镜。双光子荧光 显微镜具有固有的3-D分辨率，并使用高数值 光圈物镜的激发体积可以小到0.1 FL，在1 nM浓度下仅含有0.06个分子。一小块 激发体积是检测荧光波动的关键 在分子中。通过分析光子爆发的直方图，一个 可以从它们的分布(H。 Oian和E.Elson，1990，PNAS，87,5479)。我们为我们的光子建模 直方图与简单两种群系统的比较实验 结果与理论预测相符。这种分析方法是 本质上比涨落相关技术更简单 光谱学，这需要更高阶的计算 多物种系统的关联函数。

Molecular aggregation is a crucial regulatory mechanism in biological systems. However, most experimental techniques require relatively high concentrations (from mM to mM). Since many interesting physiological events happen at concentrations below micromolar, we have explored the possibility to detect molecular aggregation in the low concentration range (from mM to pM) using two-photon fluorescence microscopy. Two-photon fluorescence microscopy has an inherent 3-D resolution and using high numerical aperture objectives the excitation volume can be made as small as 0.1 fL, which only contains 0.06 molecules at 1 nM concentration. A small excitation volume is essential for detecting fluorescence fluctuations among molecules. By analyzing the histogram of photons bursts, one can obtain the degrees of aggregation from their distribution (H. Oian and E. Elson, 1990, PNAS, 87, 5479). We model our photon histograms with a simple two species system and compare experimental results to the theoretical predictions. This method of analysis is intrinsically simpler than the technique of fluctuation correlation spectroscopy, which requires the calculation of higher order correlation functions for the multiple species system.