Doornbos R M, De Grooth B G, Kraan Y M, Van Der Poel C J, Greve J
Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands.
Cytometry. 1994 Mar 1;15(3):267-71. doi: 10.1002/cyto.990150312.
This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime.
本报告描述了一项关于在流式细胞仪中使用可见二极管激光器进行两项重要荧光应用的可行性研究。使用一台3 mW 635 nm二极管激光器，我们使用荧光团别藻蓝蛋白（APC）进行了免疫荧光测量。我们采用两步标记程序对CD8阳性淋巴细胞进行了测量，所得直方图显示阴性细胞与弱荧光和强荧光亚群之间有良好的分离。作为第二项荧光应用，我们选择了使用最近开发的DNA/RNA染料TOTO-3和TO-PRO-3进行DNA分析。在我们的设置中，TO-PRO-3产生了最佳结果，变异系数为3.4%。我们的结果表明，几毫瓦的635 nm可见二极管激光足以用别藻蓝蛋白进行单色免疫荧光测量，并用TO-PRO-3进行DNA分析。在流式细胞仪中使用二极管激光器的主要优点是体积小、价格低、效率高和寿命长。
