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

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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.