TRIAL FOR A MULTI-MODE MICROSCOPE WITH EVANECSENT OPTICS

带有 EVANECENT 光学器件的多模式显微镜的试用

• 批准号: 12557002
• 负责人: KATAYAMA Yoshifumi
• 金额: $ 3.9万
• 依托单位: TOKYO MEDICAL AND DENTAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557002/
• 关键词: laser light beam; total internal reflection fluorescence microscopy; evanescent light field; cooled CCD camera; cultured neurons; growth cones; cell adhesion; fluorescence staining; 近接陽光; 脊髄後根神経節; 三叉神経節細胞; 免疫染色法; 微分干渉顕微鏡; 接着; 脊髄後根神経節細胞; レーザ

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

我们构建了一个全内反射荧光显微镜（TIRFM），配备了两个不同的系统来产生倏逝光。当激光束通过附在盖玻片上的三角玻璃棱镜引入时，在沿着光路的有限区域的斑点上产生倏逝光场，有时远离培养的神经元；也就是说，可观测区域相当有限。另一方面，当激光束通过物镜在视场中心产生渐逝场时，神经元及其部分可以通过移动显微镜载物台自由定位在中心。我们通过冷却 CCD 相机观察散射在表面的荧光珠图像，确认激光束通过三角棱镜和物镜在盖玻片的上表面产生倏逝光场。使用从新生 Wister 大鼠（出生后 1 至 6 天）的背神经节、三叉神经节和颈上神经节分离的培养神经元进行了多项实验来测试该显微镜。使用荧光钙指示剂 Fluo-3 处理的神经元，TIRFM 图像显示出斑点的明亮区域，而传统的落射荧光显微镜图像显示出均匀的明亮区域。响应于电刺激，荧光强度随着不同的时间进程而增加，TIRFM 的增加更快，表明TIRFM 可以测量膜下区域的[Ca^2+]_i-变化。此外，通过 TIRFM 观察到通过免疫荧光染色可视化的粘附分子、整合素 α1β1 (VLA-1) 和整合素 α6β1 (VLA-6)。前者的荧光是从体细胞和生长锥附着到盖玻片上的广阔区域观察到的，而后者的荧光是从它们附着到盖玻片的区域中的斑点位点观察到的。将进行进一步的研究，使用 TIRFM 和两个产生倏逝光的激光束路径来定位生长锥上的钙通道。较少的

项目成果

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