TRIAL FOR A MULTI-MODE MICROSCOPE WITH EVANECSENT OPTICS

我们构建了一个全内反射荧光显微镜（TIRFM），配备了两种不同的系统来产生倏逝光。当激光束通过附着在覆盖玻璃上的三角形玻璃棱镜入射时，在光通路上的有限区域上产生消失的光场，偶尔远离培养的神经元；也就是说，可观测的区域相当有限。另一方面，当激光束通过物镜在光场中心产生一个消失场时，通过移动显微镜台，神经元及其组成部分可以自由地定位在光场中心。利用冷却CCD相机观察散射在盖板玻璃上表面的荧光珠图像，证实了激光束通过三角棱镜和物镜在盖板玻璃上表面都产生了倏逝光场。利用新生Wister大鼠（出生后1 ~ 6天）背房神经节、三叉神经节和颈上神经节分离的神经元进行了实验。使用荧光钙指示剂fluo-3处理的神经元，TIRFM图像显示斑点状明亮区域，而传统的表观荧光显微镜图像显示均匀明亮区域。在电刺激作用下，荧光强度随时间的增加而增加，其中TIRFM的荧光强度增加得更快，说明TIRFM可以测量膜下面积[Ca^<2+>]_i的变化。免疫荧光染色观察粘附分子、整联素α1β1 （vla1）和整联素α6β1 （vla6）。前者的荧光是从附着在覆盖玻璃上的躯体和生长锥的广阔区域观察到的，而后者的荧光是从附着在覆盖玻璃上的区域的斑点处观察到的。进一步的研究将使用TIRFM和两个产生倏逝光的激光束路径波来定位生长锥上的钙通道。少

We constructed a total internal reflection fluorescence microscope (TIRFM) equipped with two different systems for generating evanescent light. When laser beam was introduced through a trianglular glass prism attached to cover glasses, evanescent light fields were generated on limited area of spots along light pathway, occasionally away from cultured neurons ; that is, observable area was rather limited. On the other hand, when an evanescent field was generated in the center of the optic field by laser beam through objective lens, neurons and their parts could be freely positioned on the center by moving the microscope stage. We confirmed that laser beam through both trianglular prism and objective lens generated the evanescent light field(s) on the upper surface of cover glasses, by observing images of fluorescent beads scattered on the surface by means of a cooled CCD camera. Several experiments were performed to test this microscope using cultured neurons dissociated from dorsal roo … More t, trigeminal and superior cervical ganglia of newborn Wister rats (1 to 6 days after birth). Using neurons treated with fluorescent calcium indicator, fluo-3, TIRFM images showed spotted bright areas, whereas conventional epifluorescence microscopy images showed a uniformly bright area. In response to electrical stimulation, fluorescence intensity increased with different time course, more rapid with TIRFM, indicating [Ca^<2+>]_i-changes in the submembrane area could be measured by TIRFM. Furthermore, adhesion molecules, integrin α1β1 (VLA-1) and integrin α6β1 (VLA-6) visualized with immunofluorescence staining were observed with TIRFM. Fluorescence of the former was observed from the wide area on which somata and growth cones attached to the cover glasses, whereas fluorescence of the latter was from spotted sites in the area on which they attached to the cover glasses. Further investigations will be performed to localize calcium channels on growth cones using the TIRFM with two laser beam pathwavs generating evanescent light. Less