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

由氩激光（488/512nm, 8-12W）激活的ti -蓝宝石激光器发出脉冲激光（700- 800nm, 80- 150fsec, 80MHz, 0.2-1W），送入激光扫描荧光测量单元（Biorad MRC-600），扫描转换显微镜显微镜台上的荧光团，得到两幅光子激发图像。获得了成像技术的以下特征。(1)使用荧光珠（0.3mum）测量的空间分辨率为0.3 ~ 0.4mum（尼康CFI Plan Fluor 40X，N.A.0.75），低于单光子共聚焦激光扫描显微镜（CLSM）。(2)轴向分辨率为0.3mum，远优于单光子CLSM。(3)荧光团的漂白速率比单光子CLSM慢得多。(4) indo-1和fura-2， Ca^<2+>-敏感探针的激发光谱有向短波长的偏移。(5)成像能力优于单光子CLSM。(6)长波长脉冲激光扫描加热的影响不可忽略。利用双光子CLSM，观察了培养海马神经元细胞内Ca^<2+>浓度（[Ca^<2+>]_i）对高K^<+->诱导去极化的反应。树突中[Ca^<2+>]_i的增加比胞体中的衰减更快。在用单光子CLSM测量破伤风诱导的青蛙运动神经末梢[Ca^<2+>]_i的升高，并与双光子成像图像进行比较时，发现Ca^<2+>-诱导的Ca^<2+>释放被激活。详细分析了其活化和失活的机理。

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.