Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Gunma University|
ISHIKAWA Harunori Gunma University School of Medicine, 医学部, 教授 (90010058)
PEACHEY Lee d. University of Pennsylvania, Department of Biology, 生物学科, 教授
L D Peachey ペンシルバニア大学, 生物学教室, 教授
藤巻 昇 群馬大学, 医学部, 講師 (10008261)
|Project Period (FY)
1992 – 1994
Completed(Fiscal Year 1994)
|Budget Amount *help
¥7,200,000 (Direct Cost : ¥7,200,000)
Fiscal Year 1994 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1993 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1992 : ¥3,400,000 (Direct Cost : ¥3,400,000)
|Keywords||Three-dimensional image analysis / Striated muscle / T-system / Electron Microscopy / Confocal laser microscopy / LM-EM correlation / Myofibril / Membranous system / 横細管系 / 選択的染色|
1.Three-Dimensional Image Analysis of Membranous Systems in Skeletal Muscle
The T-system in frog skeletal muscle fibers was selectively stained with the Golgi silver impregnation method followed by specimen preparation for electron microscopy. We found that the muscle fibers in which T-system was selectively stained appeared transparent, while those with sarcoplasmic reticulum staining were pink and those with no membranous staining were in black under reflected light. Thus, the muscle fibers with T-system staining were easily identified under the light microscope.
Sections of 1-3mum thick were cut from the Golgi-stained muscle tissue and examined with the intermediate voltage electron microscope. Such stereo-pair micrographs were taken to be used for development of computer programs for three-dimensional image analysis.
When Golgi-stained muscle fibers were examined under the confocal laser microscope with reflection mode, the silver-stained T-system was clearly visualized in three dimen
sions. Such confocal images were found to be useful for studies of the overall distribution of the T-system.
2.Correlated Light and Electron Microscopy of Muscle Cells.
We developed the method to correlate the fluorescence and immunofluorescence findings to ultrastructures observed by electron microscopy. For cultured cells, cardiac muscle cells from chick embyros were cultured on formval-filmed gold EM grids, stained fluorescently, and examined by confocal laser microscopy. The same samples were then fixed, dehydrated, and critical-point-dried for whole-mount cell specimen preparations, which were then examined three-dimensionally with the intermediate voltage electron microscope. In this method, we could successfully correlate the fluorescence images with ultrastructures in the same cells. The similar correlation was also made for tissue sections. Cryo-stat sections were stained for immunofluorescence using anti-dystrophin antibody and examined by confocal laser microscopy. Serial optical section images of about 0.5 mum thick were reconstructed three-dimensionally to corelate in a point-to-point manner with stereo-pair images obtained from electron microscopy. Less