Analysis of biofunction by single molecule measurement

使用我们最新开发的CCD相机，我们在装有NA=1.65物镜的消逝视场显微镜下观察了罗丹明分子溶解在水中的情况。以5ms的时间分辨率捕获了量子漂白剂。Rhod-2分子也有类似的反应。在这种分子中，亮度取决于介质中钙离子的浓度。这为由单个分子组成的探测器监测微环境提供了可能性。在装有新相机的消逝视野显微镜下观察加载了Rhod-2的海马神经元。我们可以以5毫秒的帧速率检测钙图像。将Rod-2浓度降低到通常使用浓度的百分之一，图像变得点状，并观察到一些极高信号/直流比的斑点。这些点状反应可能是由单个Rhod-2分子检测到的钙瞬变，可能是由单个钙通道引起的。我们还成功地利用在PC12细胞中表达的GFP偶联动力蛋白观察到了内吞反应。当细胞被激活时，动力素转移到细胞膜附近，并在细胞膜下四处移动，就像捕获膜凹坑进行内吞一样。根据这一结果，我们提出了一个假说，即“动力素的扫荡模型”，用于调节内吞作用。我们观察到从培养的几个海马神经元中获得的DNA分子的形状，面临着由于谷氨酸应用而导致的兴奋毒性细胞死亡。瞬逝的视野显微镜第一次发现DNA在神经细胞死亡的如此剧烈的过程中是支离破碎的。为了克服衍射极限带来的问题，我们发展了一种新的成像技术，即在由光敏树脂制成的薄膜上固定一个逝去的阴影。可以捕捉到细胞内小泡的图像。

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.