| Co-Investigator(Kenkyū-buntansha) |
TATSUMI Hitoshi NAGOYA UNIVERSITY, GRADUATE SCHOOL, ASSOCIATE PROFESSOR, 大学院・医学研究科, 助教授 (20171720)
HOMMA Tommo TOKYO MEDICAL AND DENRAL UNIVERSITY, MEDICAL RESEARCH INSTITUTE, ASSISTANT PROFESSOR, 難治疾患研究所, 助手 (80242246)
HIRAI Kenji TOKYO MEDICAL AND DENRAL UNIVERSITY, MEDICAL RESEARCH INSTITUTE, ASSOCIATE PROFESSOR, 難治疾患研究所, 助教授 (70156628)
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| Research Abstract |
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
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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
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