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首页> 外文期刊>Applied optics >Confined diffusion in tubular structures analyzed by fluorescence correlation spectroscopy on a mirror
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Confined diffusion in tubular structures analyzed by fluorescence correlation spectroscopy on a mirror

机译：通过镜面荧光相关光谱分析的管状结构中的受限扩散

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摘要

In fluorescence correlation spectroscopy (FCS) analysis it is generally assumed that molecular species diffuse freely in volumes much larger than the three-dimensional FCS observation volume. However, this standard assumption is not valid in many measurement conditions, particularly in tubular structures with diameters in the micrometer range, such as those found in living cells (organelles, dendrites) and microfluidic devices (capillaries, reaction chambers). As a result the measured autocorrelation functions (ACFs) deviate from those predicted for free diffusion, and this can shift the measured diffusion coefficient by as much as approx50percent when the tube diameter is comparable with the axial extension of the FCS observation volume. We show that the range of validity of the FCS measurements can be drastically improved if the tubular structures are located in the close vicinity of a mirror on which FCS is performed. In this case a new fluctuation time in the ACF, arising from the diffusion of fluorescent probes in optical fringes, permits measurement of the real diffusion coefficient within the tubular structure without assumptions about either the confined geometry or the FCS observation volume geometry. We show that such a measurement can be done when the tubular structure contains at least one pair of dark and bright fringes resulting from interference between the incoming and the reflected excitation beams on the mirror surface. Measurement of the diffusion coefficient of the enhanced green fluorescent protein (EGFP) and IscS-EGFP in the cytoplasm of living Escherichia coli illustrates the capabilities of the technique.
机译：在荧光相关光谱法（FCS）分析中，通常假定分子种类以比三维FCS观测体积大得多的体积自由扩散。但是，此标准假设在许多测量条件下均无效，尤其是在直径在微米范围内的管状结构中，例如在活细胞（细胞器，树突）和微流控设备（毛细管，反应室）中发现的那些。结果，测得的自相关函数（ACF）偏离了预测的自由扩散函数，当管径与FCS观测体积的轴向扩展相当时，这会使测得的扩散系数偏移多达约50％。我们显示，如果管状结构位于执行FCS的镜子的附近，则可以大大改善FCS测量的有效范围。在这种情况下，由于荧光探针在光学条纹中的扩散而在ACF中产生了新的波动时间，从而可以测量管状结构内的实际扩散系数，而无需假设受限的几何形状或FCS观测体积的几何形状。我们表明，当管状结构包含至少一对暗条纹和亮条纹时，可以进行这种测量，该暗条纹和亮条纹是由于镜面上入射光束和反射光束之间的干涉而产生的。增强的绿色荧光蛋白（EGFP）和IscS-EGFP在活的大肠杆菌细胞质中的扩散系数的测量说明了该技术的功能。

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