Development of cell-sorting microscope

研制了一种用于微生物筛选的新型设备-细胞分选显微镜（CSM）。该系统由光学系统、激光扫描细胞仪（LSC）和显微操作系统组成，LSC配备有细胞分析仪，用于记录载玻片上的细胞、激光光学系统观察到的细胞图像以及整个载玻片上的细胞分析数据。此功能似乎很好地指定具有特殊内容物或酶反应产物的细胞，这些细胞在载玻片上是荧光的。第一个模型实验采用双荧光标记法进行了酿酒酵母（Saccharomycescerevisiae）和扣囊拟青霉（Acidomycopsis fibuligera）原生质体的细胞融合。从两种酵母制备原生质体，分别用FITC和TRITC染色，以常规方式进行融合处理，在载玻片上的普通琼脂表面上显影，细胞计数分析， ...更多信息 根据FITC和TRITC荧光的强度，将结果绘制在细胞图中，这取决于亲本。设置一个窗口，以便选择具有两种荧光的原生质体。第二个模型实验是用单细胞绿色雨生红球藻（Haematococcus pluvialis）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）。真实的ASX和雨生红球藻的细胞悬浮液在488 nm的激发光下发荧光，这与LSC配备的激光器的激发光相同，最大荧光在735 nm，尽管强度非常弱。因此，分析整个绿色荧光（> 570 nm）用于筛选实验。荧光面积的测定结果与细胞内ASX含量的测定结果吻合较好，荧光面积较大的细胞在显微镜下观察时具有较强的虾青素颜色，通过显微操作收集细胞。经LSM/CSM分析，其细胞图与亲本相似，说明所选细胞的ASX产率不稳定，或不是遗传固定的。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