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

点击翻译按钮获取中文摘要

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.