The development of a two photon laser-scanning confocal microscope and its application to intracellular Ca^<2+> measurement

双光子激光扫描共聚焦显微镜的研制及其在细胞内Ca^2测量中的应用

• 批准号: 06557003
• 负责人: KUBA Kenji
• 金额: $ 11.65万
• 依托单位: Nagoya University (1996)佐賀医科大学 (1994-1995)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557003/
• 关键词: 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; C

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.

用Ar激光(488/512 nm，8-12W)激活的钛宝石激光器发射的脉冲激光(700-800 nm，80-150 fs，80 MHz，0.2-1W)被送入激光扫描荧光测量装置(Biorad MRC-600)扫描倒置显微镜工作台上的荧光团，产生两个光子激发的图像。(1)用荧光微珠(0.3um)测量的空间分辨率为0.3-0.4um(Nikon CFI Plan Fluor 40X，N.A.0.75)，低于单光子共聚焦激光扫描显微镜(CLSM)的空间分辨率。(2)轴向分辨率为0.3um，远远好于单光子CLSM。(3)荧光团的漂白速度比单光子CLSM慢得多。(4)INDO-1和Fura-2的激发光谱向短波长移动。Ca^lt；(6)长波脉冲激光扫描有不可忽视的加热效应。用这种双光子激光扫描显微镜观察了培养的海马神经元细胞内钙离子浓度([Ca^&lt；2+&gt；]i)对高钾诱导的去极化反应的变化。树突内[Ca~(2+)]_i的增加比胞体内[Ca~(2+)]_i的衰减更快。在用单光子激光共聚焦扫描显微镜测量破伤风引起的青蛙运动神经末梢[Ca^&lt；2+&gt；]_i的REISES时，与双光子成像的图像相比较，发现了钙诱导的钙释放的激活。分析了其活化和失活的具体机理。

项目成果

Kuba, K.: "Ca^<2+>-induced Ca^<2+> release in neurones." Jpn.J.Physiol.44. 613-650 (1994)

Kuba, K.：“Ca^<2> 诱导神经元中的 Ca^<2> 释放。”

Yoshizaki, K., Hoshino, T., Sato, M., Koyano, H., Nohmi, M., Hau, S.-Y.& Kuba, K.: "Ca^<2+>-induced Ca^<2+> release and its activation in response to a single action potential in rabbit otic ganglion cells." J.Physiol.lond.486. 177-187 (1995)

吉崎，K.，星野，T.，佐藤，M.，小矢野，H.，野美，M.，Hau，S.-Y。

Kuba,K.: "A UV laser-scanning confocal microscope for the measurement of intracellular Ca^<2+>" Cell Caleium.16. 205-218 (1994)

Kuba,K.：“用于测量细胞内 Ca^<2> 的紫外激光扫描共聚焦显微镜”Cell Caleium.16。

Yoshizaki,K.: "Ca^<2+>-induced Ca^<2+> release and its activation in response to a single action potential in rabbit otic ganglion cells." J.Physiol.(Lond.). 486. 177-187 (1995)

Yoshizaki,K.：“兔耳神经节细胞中 Ca^2 诱导的 Ca^2 释放及其对单一动作电位的激活。”

Hua,S.-Y.: "Cyclic ADP-ribose Modulates Ca^<2+> release channels for activation by physiological Ca^<2+>entry in bullfrog sympathetic neurones." Neuron. 12. 1073-1079 (1994)

Hua,S.-Y.：“环状 ADP-核糖调节 Ca^2 释放通道，以通过生理 Ca^2 进入牛蛙交感神经元进行激活。”