Development of Measurement System for Molecular Motion in Plasma Membrane using Pico-second Time-resolved Microscopic Photometry

时间分辨荧光退偏法是测量生物膜分子运动的有力工具。通常这种类型的测量已经应用于膜或细胞悬浮液，因此结果是膜或细胞中分子系综的平均值。然而，生理上重要的变化，分子运动的膜发生在局部地区。在这项工作中，我们使用显微镜来发展荧光退偏振方法，以微米分辨率检测分子在局部区域的运动。将脉冲Ar染料激光器引入位于显微镜载物台上的样品膜进行激发。入射角是可变的。该函数可用于检测膜中分子的取向角。通过单光子计数技术检测穿过物镜透镜的偏振荧光的衰减。利用该系统，我们可以在10平方微米的面积内测量含有1mol%的DPH-丙酸酯的硬脂酸单分子层的各向异性荧光衰减。这表明该系统可用于测量细胞膜局部区域的分子运动。

Time-resolved fluorescence depolarization method is a powerful tool to measure molecular motion of biological membranes. Usually this type of the measurement has been applied to membrane or cell suspension, and so the results were averaged values of the ensemble of molecules in the membranes or cells. However physiologically important changes of the molecular motions in the membranes occur at local areas. In this work, we use a microscope to develop the fluorescence depolarization method to detect the molecular motion at a local area with micrometer resolution. A pulsed Ar-dye laser was introduced to the sample membrane located on a microscope stage for excitation. The incident angle was variable. This function will be useful to detect the orientation angle of the molecules in membrane. Decay of the polarized fluorescent light passed through the objective lens was detected by a single photon counting technique. With this system, we could measure the anisotropic fluorescence decay from a single molecular layr of stearic acid containing 1 mol% of DPH-propionate in a 10 square micrometer area. This suggests that this system can be applied to measure molecular motion in a local area of cell membrane.