Development of cell-sorting microscope

研制了一种新的微生物筛选设备--细胞分选显微镜(CSM)。它由光学设备、激光扫描细胞仪(LSC)和显微制作平台组成，LSC配备了细胞仪来召回载玻片上的细胞，激光光学观察到的图像和整个载玻片分析后的细胞学数据。对于玻片上有特殊内容的细胞或酶反应产物为荧光的细胞，该功能似乎是很好的。筛选出的细胞可以通过显微操作从其中挑选出来。第一个模型实验是用双荧光标记法进行酿酒酵母和腓骨酵母原生质体的细胞融合。将这两种酵母菌制成原生质体，分别用FITC和TRITC染色，按常规方法进行融合处理，在玻片上的普通琼脂表面显影，细胞计量学分析和…更重要的是，结果被绘制在基于FITC和TRITC荧光强度的细胞图中，这取决于父母。设置了一个窗口，这样就可以选择同时具有两种荧光的原生质体。第二个模型实验是以单细胞绿藻雨生红球藻为材料进行的，它在包囊形成过程中产生荧光类胡萝卜素虾青素(ASX)。正品ASX和雨生红藻细胞悬浮液在488 nm处激发荧光，与激光照射LSC的激发光相同，最大荧光强度在735 nm处，但强度很弱。因此，对整个绿色花期(&gt；570 nm)进行了分析，以进行筛选实验。用CSM监测藻类的生长，荧光面积的测量能很好地反映ASX的细胞含量。荧光面积宽的细胞在显微镜下观察时虾青素颜色很强，通过显微操作收集到的细胞。这些细胞再次生长并形成包囊，但LSM/CSM分析显示与亲本相似的细胞图，表明所选细胞对ASX的生产能力不稳定或不是遗传固定的特征。较少

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