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

研制了两种用于紫外光激光扫描共焦显微镜的光学系统。其中一个系统基于倒置显微镜(尼康，TMD)，由一个物镜(CF Fluor 40X，N.A.，0.85，管长230 mm)，一个新设计的用于紫外光和可见光的消色差中继透镜(由熔融石英和萤石制成，f=35 mm)，一个激光路径中的平凸透镜(f=+500 mm)组成。另一种是由新设计的反射和折射光学混合物镜、消色差继电器透镜和带有显微镜工作台的传统管组成。将这两种光学系统与Ar离子激光器(光谱物理2025-01,351 nm)和激光扫描头(MRC-600，Biorad)相结合，分别获得了-lt；0.4um(对于尼康系统)和&lt；0.7um(对于混合物镜系统)的横向分辨率和&lt；1.5um和2.5um的光学分辨率。敏感的荧光探针INDO-1揭示了细胞内钙离子浓度([Ca^&lt；2+&gt；]_1)的动态变化的几个新发现，这些变化响应于细胞膜的兴奋和药物的作用，这些药物导致或阻断了Ca^&lt；2+&gt；诱导的Ca^&lt；2+&gt；释放通道。它们是：(1)[Ca^&lt；2+&gt；]_1通过电压依赖的[Ca^&lt；2+&gt；-通道]在细胞膜下区增加的内流速度(40亩/秒)比在细胞质更深处(20微米/秒)快，(2)[Ca^&lt；2+&gt；]1亚膜区储存细胞器的起搏作用；咖啡因(6-10 mM)诱发的[Ca~(2+)]_1振荡；(3)[Ca~(2+)]_1在核内扩散较慢。

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.