Analysis of biofunction by single molecule measurement

通过单分子测量分析生物功能

基本信息

批准号：
11794015
负责人：
TERAKAWA Susumu
金额：
$ 1.92万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for University and Society Collaboration
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11794015/
关键词：
evanescence microscope calcium transient ultra high NA lens neuronal cell death single molecule fluorescence single DNA total reflection illumination near field microscope CCDカメラ高速度カメラ光電子増倍管マルチアノード膜電位色素量子効率緑色蛍光タンパク膜蛋白

项目摘要

Using our newly developed CCD camera, we observed rhodamine molecules dissolved into water under an evanescent field microscope equipped with an objective lens of NA=1. 65. The quantal bleach was captured at time resolution of 5 ms. A similar response of Rhod-2molecule was also detected. In this molecule, the brightness depended on the concentration of calcium ions in the medium. This opened a possibility that microenvironments can be monitored by a probe consisting of a single molecule. Hippocampal neurons loaded with Rhod-2 were observed at the bottom through the evanescent field microscope equipped with the new camera. We could detect Ca images at a 5-ms frame rate. Lowering the Rhod-2 concentration to one hundredths of the commonly used one, images became punctuated, and some spots of an extremely high signal/DC ratio were observed. These spotty responses could be Ca transients detected by a single Rhod-2 molecule and possibly caused by a single Ca channel. We also succeeded in observing endocytotic responses by using GFP-conjugated dynamin expressed in PC12 cells. Upon activation of the cell, dynamin translocated near to the cell membrane, and moved around beneath there as if to capture the membrane pits for endocytosis. From this result, we proposed a hypothesis, "sweeping model of dynamin," for regulation of endocytosis. We observed a shape of DNA molecules obtained from a few hippocampal neurons in culture facing an excitotoxic cell death due to glutamate application. The evanescent field microscopy reveled for the first time that the DNAs were fragmented in such an acute process of neuronal cell death. In order to overcome the problems due to the diffraction limit, we developed a new imaging technique by which an evanescent shadow is fixed on a thin film made of a photosensitive resin. Images of small vesicles in the cell could be captured.

使用我们新开发的 CCD 相机，我们在配备 NA=1 物镜的倏逝场显微镜下观察了溶解在水中的罗丹明分子。 65. 量子漂白剂以 5 毫秒的时间分辨率捕获。 Rhod-2分子也检测到类似的反应。在该分子中，亮度取决于介质中钙离子的浓度。这开启了通过由单个分子组成的探针来监测微环境的可能性。通过配备新相机的渐逝场显微镜在底部观察装载有 Rhod-2 的海马神经元。我们可以以 5 毫秒的帧速率检测 Ca 图像。将 Rhod-2 浓度降低至常用浓度的百分之一时，图像变得断断续续，并且观察到一些具有极高信号/DC 比的斑点。这些斑点响应可能是由单个 Rhod-2 分子检测到的 Ca 瞬变，并且可能由单个 Ca 通道引起。我们还通过使用 PC12 细胞中表达的 GFP 缀合动力成功观察了内吞反应。细胞激活后，动力蛋白易位到细胞膜附近，并在细胞膜下方移动，仿佛要捕获膜凹坑进行内吞作用。根据这一结果，我们提出了一个用于调节内吞作用的假设，即“动力的清扫模型”。我们观察到从培养物中的一些海马神经元获得的 DNA 分子形状，这些神经元由于谷氨酸的应用而面临兴奋性毒性细胞死亡。倏逝场显微镜首次揭示了 DNA 在神经元细胞死亡的如此剧烈的过程中被片段化。为了克服衍射极限带来的问题，我们开发了一种新的成像技术，将消逝的阴影固定在由光敏树脂制成的薄膜上。可以捕获细胞中小囊泡的图像。