Hudson J C, Porcelli R T, Russell T R
Coulter Corp., Miami, Florida, USA.
Cytometry. 1995 Oct 1;21(2):211-7. doi: 10.1002/cyto.990210214.
We evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS Elite with a 76 microns Sortsense flow cell. The two applications chosen were immunofluorescence and DNA analysis. We measured the fluorescence threshhold of phycoerytherin calibration beads to be approximately 336 MESF. Cell analysis with a HeNe laser and Argon laser correlated well for the CD4PE, CD56PE, CD19PE conjugates, with correlation coefficients of 0.98, 0.99, 0.94, respectively. The % positive and mean channel fluorescence were comparable to the results obtained with a 15 mW Argon laser. In addition, a three-color configuration yielded excellent results. Cell analysis of CD4PE, CD3ECD and CD19Cy-Chrome with the HeNe laser and Argon laser correlated well with correlation coefficients of 0.96, 0.95, and 0.92, respectively. The histograms showed good separation between the negative cells, the dimly staining cells and the brightly staining cells. Propidium Iodide was chosen for DNA analysis. Full CV values for whole blood DNA fluorescence using the green laser were good at 2.6%. These data indicate the low power 544 nm laser is sufficient to do immunophenotyping and DNA analysis. Results may be explained by higher quantum efficiency and lower background fluorescence. The wavelength of the 544 nm laser is much closer to the excitation peaks of PI, PE, and the tandem dyes ECD and Cy-Chrome. Also, the Raman scattering of water for the 544 nm laser has a longer wavelength maximum than the emission peaks of PI, PE, and ECD. The major advantages of this laser for the research laboratory are small size, no cooling fan, low power requirements and low cost.
我们评估了一台1.5毫瓦的氦氖激光器（544纳米），用于配备76微米Sortsense流动池的EPICS Elite仪器上。选择的两个应用是免疫荧光和DNA分析。我们测得藻红蛋白校准微珠的荧光阈值约为336个MESF。使用氦氖激光器和氩离子激光器进行细胞分析时，对于CD4PE、CD56PE、CD19PE偶联物，相关性良好，相关系数分别为0.98、0.99、0.94。阳性百分比和平均通道荧光与使用15毫瓦氩离子激光器获得的结果相当。此外，三色配置产生了优异的结果。使用氦氖激光器和氩离子激光器对CD4PE、CD3ECD和CD19Cy-铬进行细胞分析，相关性良好，相关系数分别为0.96、0.95和0.92。直方图显示阴性细胞、弱阳性细胞和强阳性细胞之间有良好的区分。选择碘化丙啶用于DNA分析。使用绿色激光对全血DNA荧光的全CV值良好，为2.6%。这些数据表明低功率544纳米激光器足以进行免疫表型分析和DNA分析。结果可能是由于较高的量子效率和较低的背景荧光。544纳米激光器的波长更接近PI、PE以及串联染料ECD和Cy-铬的激发峰。此外，544纳米激光器的水拉曼散射的最大波长比PI、PE和ECD的发射峰更长。这种激光器对于研究实验室的主要优点是体积小、无冷却风扇、低功率要求和低成本。
