Ultraviolet laser-scanning confocal microscopy and application to the measurement of intracellular Ca^<2+>

研制了两种用于紫外激光扫描共焦显微镜（UV-CLSM）的光学系统。一种系统基于倒置显微镜（Nikon，TMD）并且由物镜（CF Fluor 40 X，N.A.，该系统包括一个新型的反射-折射混合物镜、一个新型的消色差中继镜和一个带载物台的普通镜筒，前者由石英玻璃和萤石制成，f=35 mm，用于紫外光和可见光，后者由平凸透镜（f=+500 mm）组成。这些光学系统中的任何一个与氩离子激光器的组合（Spectral Physics 2025-01，351 nm）和激光扫描头（MRC-600，Biorad）产生<0.4 μ m的横向分辨率（对于基于尼康的系统）和<0.7 μ m（对于混合物镜系统）和<1.5 μ m和2.5 μ m的光学分辨率，将这些UV-CLSM系统应用于培养的牛蛙（或大鼠）交感神经节细胞，所述交感神经节细胞装载有Ca^2+敏感的荧光探针indo-1，揭示了细胞内Ca^<2+>浓度动态变化的几个新发现[Ca^<2+>]_1对细胞膜兴奋和引起或阻断Ca^<2+>诱导的Ca^<2 +>释放的药物作用的反应（通过Ca ^2+释放通道）从储存Ca^2+的细胞器中释放。（1）Ca^<2 +>内流增加[Ca^<2 +>]_1的内向扩散速度加快（通过电压依赖性Ca^<2+>-通道）（40 mu/sec）与较深的细胞质相比（20 μ m/sec），（2）在咖啡因（6-10 mM）诱导的[Ca^<2+>]_1振荡中，膜下区的Ca^<2+>储存细胞器起着起搏作用;（3）[Ca^<2+]_1扩散较慢>]_1在核中上升。

Two types of optical system for ultraviolet laser-scanning confocal microscope (UV-CLSM) were developed. One system is based on an inverted microscope (Nikon, TMD) and composed of an objective (CF Fluor 40X, N.A., 0.85, used at a tube length of 230 mm), a newly-designed achromat relay lens (made of fused silica and fluorite, f=35 mm) for UV and visible rays, a planoconvex lens (f=+500 mm) in the laser path. Another consists of a newly-designed hybrid objective of reflective and refractive optics, and achromat relay lens and a conventional tube with a microscope stage. Combination of either of these optical systems with an argon ion laser (Spectral Physics 2025-01, 351 nm) and a laser scan head (MRC-600, Biorad) yielded the lateral resolution of <0.4 mum (for Nikon-based system) and <0.7 mum (for a hybrid objective system) and the optical resolution of <1.5 mum and 2.5 mum, respectively.Application of these UV-CLSM systems to cultured bullfrog (or rat) sympathetic ganglion cells which were loaded with a Ca^<2+>-sensitive fluorescent probe, indo-1, revealed several new findings on dynamic changes in the intracellular Ca^<2+> concentration ([Ca^<2+>]_1) in response to the cell membrane excitation and the action of drugs that causes or blocks Ca^<2+>-induced Ca^<2+> release (via Ca^<2+>-release channels) from Ca^<2+>-storing organelles. They are (1) the faster speed of the inward spread of an increased [Ca^<2+>]_1 by Ca^<2+> influx (through voltage-dependent Ca^<2+>-channels) at the submembrane region (40 mu/sec) compared to that at the deeper cytoplasm (20 mum/sec), (2) the pacemaking role of Ca^<2+>-storing organelles at the submembrane region in the [Ca^<2+>]_1 oscillation induced by caffeine (6-10 mM) and (3) a slower spread of [Ca^<2+>]_1 rise in the nucleus.