Taylor J G, Haigler C H, Kilburn D G, Blanton R L
Department of Biological Sciences, Texas Tech University, Lubbock 79409, USA.
Biotech Histochem. 1996 Sep;71(5):215-23. doi: 10.3109/10520299609117163.
Specific detection of cellulose has not been possible using laser based instruments such as laser scanning confocal microscopes (LSCM) and fluorescently activated cell sorters (FACS). Common cellulose dyes are nonspecific and/or nonexcitable with common lasers. Furthermore, many lasers emit wavelengths that overlap with autofluorescence from chlorophyll and other plant molecules. We demonstrate that a cellulase and an isolated bacterial cellulose binding domain (CBD) conjugated to fluorescent dyes can be used for laser detection of cellulose with improved specificity. Cell walls of differentiating tracheary elements and spores of Dictyostelium discoideum were tested in this study. For double labeling, autofluorescence interfering with the rhodamine signal was eliminated by collecting each excitation channel separately followed by computer recombination or by using a narrow band pass barrier filter allowing simultaneous channel collection. Using these methods, cellulose and microtubules tagged with a monoclonal antibody to alpha-tubulin were effectively colocalized in chlorophyll-containing tracheary elements using a LSCM. Also, Dictyostelium discoideum spores labeled or unlabeled with CBD-FITC were separated into two populations by FACS indicating that this tag should be useful in future mutagenesis experiments. Therefore, the presence or absence of cellulose can now be analyzed using common lasers.
使用基于激光的仪器，如激光扫描共聚焦显微镜（LSCM）和荧光激活细胞分选仪（FACS），无法实现对纤维素的特异性检测。常见的纤维素染料是非特异性的，并且/或者不能被常见的激光激发。此外，许多激光发射的波长与叶绿素和其他植物分子的自发荧光重叠。我们证明，与荧光染料偶联的纤维素酶和分离的细菌纤维素结合结构域（CBD）可用于以更高的特异性对纤维素进行激光检测。本研究对正在分化的管状分子的细胞壁和盘基网柄菌的孢子进行了测试。对于双重标记，通过分别收集每个激发通道然后进行计算机重组，或者使用允许同时收集通道的窄带通阻挡滤光片，消除了干扰罗丹明信号的自发荧光。使用这些方法，使用LSCM有效地将用抗α-微管蛋白单克隆抗体标记的纤维素和微管共定位在含有叶绿素的管状分子中。此外，通过FACS将用CBD-FITC标记或未标记的盘基网柄菌孢子分成两个群体，这表明该标签在未来的诱变实验中应该是有用的。因此，现在可以使用常见的激光分析纤维素的存在与否。
