2000 Fiscal Year Final Research Report Summary
Electron Microscopic Recording of Dynamic Structural Changes in Living Biomolecule
| Project/Area Number |
11357001
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (A).
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
General physiology
|
|---|
| Research Institution | Teikyo University Medical School |
|---|
Principal Investigator |
SUGI Haruo Teikyo Univ, Med.Sch.Professor, 医学部, 教授 (20082076)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SHIRAKAWA Ibuki Teikyo Univ, Med.Sch.Instructor, 医学部, 助手 (80236190)
KOBAYASHI Takakazu Teikyo Univ, Med.Sch.Assistant Professor, 医学部, 講師 (00112756)
SUGIWURA Seiryo Univ.Tokyo, Med.Sch.Instructor, 医学部, 助手 (10272551)
福島 球琳男 日本電子株式会社, 研究員
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Keywords | bilmolecules / gas environmental chamber / electron microscope / myofilament / image analysis |
|---|
| Research Abstract |
The results of the present research work are summarized as follows :
1. Preparation of bipolar myosin filaments : We succeeded in preparing long and thick bipolar myosin filaments by mixing myosin and myosin rod at a ratio of 1 : 10, and slowly polymerizing them into filaments. The bipolar filaments thus prepared are 2-3um long and 100-200nm thick, and are suitable for electron microscopic recording under unstained conditions. The myosin head on the filament is marked with a gold particle (diameter, 15nm), and the position of each gold position marker does not change appreciably with time. When F-actin is added to the filaments in the absence of ATP, F-actin filaments are attached to the myosin filaments in parallel with each other due to multiple rigor linkage formation between the myosin head and F-actin.
2. We constructed software programs to determine the centroid position of each gold particle. By the use of the new imaging plate system, we can now reduce EM magnification from 10,000X to 7,000X without reducing special resolution attained with the former imaging plate system. As the result, the image of the same filament can be recorded 8 times without any serious electron beam damage to the spacimen.
3. The carbon sealing film, which we have been using in our gas environmental chamber, is readily damaged by laser or xenon lamp flash. We attempted to prepare a new sealing film with SiO, which is resistant to laser or xenon lamp flash, to make it possible to use caged compounds which rapidly release active compounds on photolysis with light flash. The SiO film is, however, readily damaged with electron beam. After a number of trials and errors we have succeeded in preparing a SiO film suitable for our use in the gas environmental chamber.
|
