Spatial two-photon fluorescence cross-correlation Spectroscopy for controlling molecular transport in microfluidic structures
Spatial two-photon fluorescence cross-correlation Spectroscopy for controlling molecular transport in microfluidic structures
复制标题
空间双光子荧光交叉相关光谱学用于控制微流体结构中的分子传输
DOI：
10.1021/ac025625p
复制
发表时间：
2002-09-01
影响因子：
7.4
通讯作者：
Schwille, P
中科院分区：
文献类型：
--
作者：
Dittrich, PS;Schwille, P
越来越多的各种几何形状和材料的微流体系统用于缩小化学或生化过程的规模，这提出了对精确确定流动参数和控制流体和颗粒操作的适当技术的强烈需求。在所有读出参数中，液体或悬浮颗粒的荧光分析特别有吸引力，因为它可以在没有机械干扰的情况下使用，并且灵敏度足够高，可以检测水环境中的单个分子。在这项研究中，我们提出了用荧光相关光谱（FCS）测定微观结构通道中的流动参数，如速度和方向，这是一种在共聚焦光学装置中进行的基于单分子光谱的方法。讨论了FCS的不同模式，如单光子激发和双光子激发的自动和双光束互相关技术。已知的双光子激发的优点，如高度受限的探测体积和低散射背景，被证明是特别有价值的测量在微小的通道系统。虽然传统的自相关足以描述单个分子的速度，但双光束相互关联允许各向同性和各向异性动力学的分离，例如，监测流动方向或区分可能被误认为迁移率参数的光物理效应。结果表明，双光束模式下的时间门控双光子激发显著降低了两个测量体积之间的不良串扰。最后，介绍了该方法在微流控分选装置标定和流型分析等方面的应用。
The increasing availability of microfluidic systems of various geometries and materials for the downscaling of chemical or biochemical processes raises a strong demand for adequate techniques to precisely determine flow parameters and to control fluid and particle manipulation. Of all readout parameters, fluorescence analysis of the fluid or suspended particles is particularly attractive, as it can be employed without mechanical interference and with a sensitivity high enough to detect single molecules in aqueous environments. In this study, we present the determination of flow parameters, such as velocity and direction, in microstructured channels by fluorescence correlation spectroscopy (FCS), a method based on single molecule spectroscopy carried out in confocal optical setups. Different modes of FCS, such as auto- and dual-beam cross-correlation techniques by one- and two-photon excitation, are discussed. Known advantages of two-photon excitation, such as highly restricted detection volumes and low scattering background, are shown to be particularly valuable for measurements in tiny channel systems. Although conventional autocorrelation is sufficient for describing the velocity of single molecules, dual-beam cross-correlation allows the separation of isotropic and anisotropic dynamics, for example, to monitor flow directions or to discriminate against photophysical effects that could be mistaken for mobility parameters. It can be shown that time-gated two-photon excitation in the dual-beam mode significantly lowers the undesired cross-talk between the two measurement volumes. Finally, some applications, such as the calibration of microfluidic sorting units and flow profiling, are demonstrated.