Analysis of biofunction by single molecule measurement

使用我们新开发的CCD摄像机，我们观察了溶解在水中的罗丹明分子的倏逝场显微镜配备了一个物镜透镜NA=1。65.量子漂白被捕获的时间分辨率为5 ms。一个类似的响应Rhod-2分子也被检测到。在这种分子中，亮度取决于介质中钙离子的浓度。这开启了一种可能性，即微环境可以通过由单个分子组成的探针来监测。通过装备有新相机的渐逝场显微镜在底部观察负载Rhod-2的海马神经元。我们可以在5 ms的帧速率下检测Ca图像。将Rhod-2浓度降低至常用浓度的百分之一，图像变得间断，并且观察到极高信号/DC比的一些斑点。这些斑点反应可能是由单个Rhod-2分子检测到的Ca瞬变，并且可能由单个Ca通道引起。我们还成功地观察到内吞反应，通过使用在PC 12细胞中表达的GFP缀合的发动蛋白。当细胞被激活时，发动蛋白移位到细胞膜附近，并在细胞膜下移动，似乎是为了捕获细胞膜上的小凹进行内吞作用。根据这一结果，我们提出了一个假说，“发动蛋白清扫模型”，内吞作用的调节。我们观察到一种形状的DNA分子从几个海马神经元在文化面临兴奋性细胞死亡，由于谷氨酸盐的应用。消逝场显微镜首次揭示了神经元细胞死亡的急性过程中DNA的断裂。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，通过该技术，将渐逝阴影固定在由光敏树脂制成的薄膜上。细胞中的小泡的图像可以被捕获。

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.