Development of cell-sorting microscope

Research Project

Project/Area Number	08556014
Research Category	Grant-in-Aid for Scientific Research (A)
Allocation Type	Single-year Grants
Section	展開研究
Research Field	応用微生物学・応用生物化学
Research Institution	Nara Institute of Science and Technology
Principal Investigator	KATSURAGI Tohoru Nara Institute of Science and Technology, Graduate School of Biological Sciences, Associate Professor, バイオサイエンス研究科, 助教授 (80081579)
Co-Investigator(Kenkyū-buntansha)	YOSHIDA Nobuyuki Nara Institute of Science and Technology, Graduate School of Biological Sciences, バイオサイエンス研究科, 助手 (10273848) ONODERA Keiko Yamada Nara Institute of Science and Technology, Graduate School of Biological Sciences, バイオサイエンス研究科, 助手 (70202380) KOKUBO Tetsuro Nara Institute of Science and Technology, Graduate School of Biological Sciences, バイオサイエンス研究科, 助教授 (10271587) KITAGAWA Hisao Olympus Optical Company Limited, Optical Equipments Development Department, Mana, 光学機器開発部開発三グループ, (研究職)係長
Project Period (FY)	1996 – 1997
Project Status	Completed (Fiscal Year 1997)
Budget Amount *help	¥800,000 (Direct Cost: ¥800,000) Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Keywords	Screening / Cytometry / Laser-scanning cytometer / Micromanipulation / Cell fusion / Astaxanthin
Research Abstract	A novel equipment, cell-sorting microscope (CSM), was developed for screening of microorganisms. It was constructed of an optic equipment, laser-scanning cytometer (LSC), and a micromanuplator. LSC is equiped with a cytometric analyzer to recall a cell on the slide stage, its image as was observed by the laser optics, and the cytometric data after total analysis throughout the slide glass. This function seemed good for specifying cells with special contents or products of enzymatic reaction which are fluorescent on the slide glass. Selected cells may be picked up from there by micromanipulation. The first model experiment was done with a cell fusion between protoplasts of Saccharomyces cerevisiae and Saccharomycopsis fibuligera with double fluorescence labelling method. Protoplasts were made from the two kinds of yeast, stained separately with FITC and TRITC,treated for fusion in a conventional manner, developed on the surface of plain agar on a slide glass, cytometrically analyzed, and … More the results were plotted in a cytogram based on the intensity of FITC and TRITC fluorescence, which depends on the parents. A window was set so that protoplasts with both kinds of fluorescence would be selected. Desired cells, which were in the window, were recalled and acconfirmed for the morphology on the computer display, or directly under the microscope. The second model experiment was done with a unicellular green alga Haematococcus pluvialis, which produces a fluorescent carotenoid astaxanthin (ASX) during cyst formation. Authentic ASX and the cell suspension of H.pluvialis fluoresced with excitation light at 488 nm, which is the same as that of the laser equipped to LSC,with a maximum fluorescence at 735 nm, although the intensity was very weak. Therefore the whole green florescence (>570nm) was analyzed for the screening experiments. The algal growth was monitored with the CSM,and the measure of fluorescence area was found to well respond the cellular content of ASX.The cells with wide fluorescence area had strong color of astaxanthin when observed under microscope, and were collected by micromanipulation. These cells were grown again and made into cysts, which however gave the similar cytogram as tha parents when analyzed with LSM/CSM.It suggests that the productivity for ASX of the selected cells was unstable or was not genetically fixed as their characteristic. Less