2001 Fiscal Year Final Research Report Summary
Analysis of biofunction by single molecule measurement
| Project/Area Number |
11794015
|
|---|
| Research Category |
Grant-in-Aid for University and Society Collaboration
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
General physiology
|
|---|
| Research Institution | Hamamatsu University School of Medicine |
|---|
Principal Investigator |
TERAKAWA Susumu Hamamatsu University School of Medicine, Photon Medical Research Center, Professor, 光量子医学研究センター, 教授 (50014246)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
IAWATA Futoshi Shizuoka University, Faculty of Engineering, Research Associate, 工学部, 助手 (30262794)
KAWATA Yoshimasa Shizuoka University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (70221900)
SAKURAI Takashi Hamamatsu University School of Medicine, Photon Medical Research Center, Research Associate, 光量子医学研究センター, 助手 (50283362)
|
|---|
| Project Period (FY) |
1999 – 2001
|
| Keywords | evanescence microscope / calcium transient / ultra high NA lens / neuronal cell death / single molecule fluorescence / single DNA / total reflection illumination / near field microscope |
|---|
| Research Abstract |
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.
|
