Development of an in vivo single-molecule fluorescence microscope and its application to the studies of cell signaling

在这个项目中，我们开发了一种“体内单分子荧光显微镜”，可以观察活细胞中生物分子的单个颗粒和生物分子的复合物。细胞的各种光学操作可以在单分子观察的同时进行。这台显微镜的规格如下。(1)利用物镜型全内反射（TIR）光学可以实现对荧光分子的单分子观察。(2)采用488、568、647 nm的Kr-Ar离子激光器和532 nm的Nd/YAG激光器作为光源。(3)探测器前配置双视光学器件，可同时进行双色观测。(4)带卤素弧光灯的外延照明光学元件可与TIR照明同时使用。外伸照明可用于各种细胞内荧光指示剂和笼化化合物的光化学反应的测量。(5)在非常有限的激发体积（1 μm^2 x 150 nm）下，可进行TIR光漂白实验。通过对表皮生长因子（EGF）诱导的细胞内信号事件的分析，该显微镜原型的性能得到了改善。首先，我们检测了细胞表面上足以诱导细胞内钙反应的egf结合的数量。用荧光团Cy3标记的EGF脉冲加入到携带荧光钙指示剂Fluo-4的细胞中。在相同的细胞中，使用TIR显微镜计算egf结合的数量，并使用外照射激发的Fluo-4测量细胞内钙浓度。我们发现150个Cy3-EGF分子的结合足以在每个细胞中诱导钙反应。反应的大小与egf结合的数量无关。细胞表面有数万个egfr。因此，激活不到1%的受体就足以诱导细胞反应。目前，我们正在研究细胞信号蛋白Ras和Raf1在质膜下的横向运动。本研究将采用红外光漂白技术。少

In this project, we developed an "in vivo single-molecule fluorescence microscope", which can observe single particles of bio-molecules and complexes of bio-molecules in living cells. Various kinds of optical manipulation of cells can be executable simultaneously of the single molecule observation. The specifications of this microscope are as follows.(1) Single molecule observation of fluorescent molecules can be achieved using the objective-type total internal reflection (TIR) optics.(2) An Kr-Ar ion laser (488, 568, 647 nm) and a Nd/YAG laser (532 nm) are equipped as the light sources.(3) Dual-view optics are equipped before the detector for the simultaneous dual color observation.(4) Epi-illumination optics with a halogen arc lamp can be used simultaneously with the illumination using TIR. The epi-illumination can be used for the measurements with various intracellular fluorescence indicators and photochemical reactions of caged compounds.(5) TIR photobleaching experiment can be don … More e in a very limited excitation volume (1 μm^2 x 150 nm).Performance of the prototype of this microscope was improved through an application that is an analysis of intracellular signaling events induced by epidermal growth factor (EGF). First, we examined the number of EGF-binding on the cell surface that is enough to induce intracellular calcium response. A pulse of EGF labeled with a fluorophore Cy3 was added to cells loaded with a fluorescence calcium indicator Fluo-4. In the same cells, the number of EGF-binding was counted using TIR microscopy and the intracellular calcium concentrations were measured using Fluo-4 excited by epi-illumination. We have found that the binding of 150 molecules of Cy3-EGF is enough to induce calcium response in every cell. Magnitude of the response was not related to the number of EGF-binding. Cells has several tens thousands of EGFRs on the cell surface. Therefore, activation of less than 1 % of receptors was enough to induce cellular response. Now, we are studying the lateral movement of cell signaling proteins Ras and Raf1 under the plasma membrane. In this study, TIR photobleaching technique will be used. Less