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

体内单分子荧光显微镜的研制及其在细胞信号研究中的应用

基本信息

批准号：
12558082
负责人：
SAKO Yasushi
金额：
$ 8.77万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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

本项目研制了一种“活体单分子荧光显微镜”，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体。在进行单分子观察的同时，可以对细胞进行各种光学操作。这台显微镜的规格如下。(1)荧光分子的单分子观察可以使用物镜型全内反射（TIR）光学器件来实现。(2)配备Kr-Ar离子激光器（488、568、647 nm）和Nd/YAG激光器（532 nm）作为光源。(3)在探测器前配备了双视角光学系统，用于同时进行双色观察。(4)具有卤素弧光灯的落射照明光学器件可以与使用TIR的照明同时使用。落射照明可用于各种细胞内荧光指示剂的测量和笼状化合物的光化学反应。(5)可以进行TIR光漂白实验 ...更多信息通过分析表皮生长因子（EGF）诱导的细胞内信号事件，该显微镜原型的性能得到了提高。首先，我们检查了足以诱导细胞内钙反应的细胞表面EGF结合的数量。将用荧光团Cy 3标记的EGF脉冲加入到加载有荧光钙指示剂Fluo-4的细胞中。在相同的细胞中，使用TIR显微镜计数EGF结合的数量，并使用由落射照明激发的Fluo-4测量细胞内钙浓度。我们已经发现，150个Cy 3-EGF分子的结合足以在每个细胞中诱导钙应答。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，少于1%的受体的活化足以诱导细胞应答。现在，我们正在研究细胞信号蛋白Ras和Raf 1在质膜下的横向运动。本研究将采用TIR光漂白技术。少