Institute of Physical Chemistry, University of Zurich, Switzerland.
Chemphyschem. 2012 Nov 12;13(16):3655-60. doi: 10.1002/cphc.201200395. Epub 2012 Sep 3.
The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non-membrane-bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non-ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample-related artifacts in the diffusion measurement.
细胞膜中分子扩散的荧光相关光谱（FCS）测量中，细胞的复杂形状和可塑性能是一个复杂的问题。准确扩散测量的一个重要前提条件是考虑区域内的细胞膜有足够的平整度，并且不存在非膜结合荧光扩散。本文提出了一种基于同时测量样品/玻璃界面的临界角以上和以下的荧光发射的方法，来识别由于细胞膜的非理想几何形状引起的轴向运动分量。由此，生成了两个横向重合但在样品轴向穿透深度上不同的检测体积。超临界角荧光（SAF）和次临界角荧光（UAF）的强度轨迹之间的相似性强烈依赖于细胞膜的平整度和细胞内荧光，并且可以帮助避免扩散测量中的与样品相关的伪影。
