| Project/Area Number |
06557003
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (A)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 試験 |
|---|
| Research Field |
General physiology
|
|---|
| Research Institution | Nagoya University (1996)
佐賀医科大学 (1994-1995) |
|---|
Principal Investigator |
KUBA Kenji Nagoya University, School of Medicine, Professor, 医学部, 教授 (60080561)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
能見 光雄 佐賀医科大学, 医学部, 助教授 (80117209)
林 尚久 大日本スクリーン製造(株), 技術研究所, 係長補佐
|
|---|
| Project Period (FY) |
1994 – 1996
|
| Project Status |
Completed (Fiscal Year 1996)
|
| Budget Amount *help |
¥18,200,000 (Direct Cost: ¥18,200,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1994: ¥11,000,000 (Direct Cost: ¥11,000,000)
|
|---|
| Keywords | Two photon excitation / Pulsed laser / Intracellular Ca^<2+> / Neurons / Indo-1 / Ti-Sapphire laser / Fluorescent measurement / Presynaptic terminals / 起短パルスレーザー / 共焦点走査装置 / 二光子励起 / Ca^<2+>感受性蛍光プローブ / ニューロン内Ca^<2+>イメージ / 細胞内Ca^<2+>遊離機構 / Ca^<2+>遊離チャネル / Ca^<2+>チャネル |
|---|
| Research Abstract |
Pulsed laser (700-800 nm, 80-150 fsec, 80MHz, 0.2-1W) emitted from Ti-sapphirelaser which was activated by Argon laser (488/512nm, 8-12W) was fed into a laser scanning fluorescence measurement unit (Biorad MRC-600) to scan fluorophores on the microscope stage of an onverted microscope, yielding two photon-excited images. The following characteristics of the imaging technique were obtained.
(1) The spatial resolution measured with a fluorescent bead (0.3mum) was 0.3-0.4mum (Nikon CFI Plan Fluor 40X,N.A.0.75) which was inferior to that of single photon confocal laser-scanning microscope (CLSM).
(2) The axial resolution was 0.3mum, which was far better than that of single photon CLSM.
(3) The rate of bleaching of fluorophores was much slower than that with single photon CLSM.
(4) There was a shift toward a short wavelength of the excitation spectra of indo-1 and fura-2, Ca^<2+>-sensitive probes.
(5) The ability to image deeper layrs was superior to that of single photon CLSM.
(6) There was non-negligible effects of heating by scanning with long-wavelength pulsed laser.
Using this two photon CLSM,changes in the intracellular Ca^<2+> concentration ( [ Ca^<2+> ]_i ) in cultured hippocampal neurons in response to high K^<+-> induced depolarization were observed. An increase in[ Ca^<2+> ]_i in dendrites had the faster decay than that in the cell soma. During the measurement of tetanus-induced reises in[ Ca^<2+> ]_i in the frog motor nerve terminals using single photon CLSM to compare with the images taken by two photon imaging, the activation of Ca^<2+>- induced Ca^<2+> release was found to occur. The detailed mechanisms of its activation and inactivation were analyzed.
|
