Leif Robert C, Yang Sean, Jin Dayong, Piper James, Vallarino Lidia M, Williams John W, Zucker Robert M
Newport Instruments, 5648 Toyon Road, San Diego, California 92115-1022, USA.
J Biomed Opt. 2009 Mar-Apr;14(2):024022. doi: 10.1117/1.3103646.
The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, would be enhanced by the availability of narrowband emitting, UV excited lanthanide calibration beads. 0.5-, 3-, and 5-microm beads containing a luminescent europium-complex are manufactured. The luminescence distribution of the 5-microm beads is measured with a time-delayed light-scatter-gated luminescence flow cytometer to have a 7.0% coefficient of variation (CV) The spacial distribution of the europium-complex in individual beads is determined to be homogeneous by confocal microscopy. Emission peaks are found at 592, 616 (width 9.9 nm), and 685 nm with a PARISS spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation of the 0.5- and 5-microm beads measured under LED excitation with a fluorescence microscope indicate that bleaching does not interfere with their imaging. The luminescence lifetimes in water and air were 340 and 460 micros, respectively. Thus, these 5-microm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.
通过流式细胞术和数字显微镜进行镧系元素发光测量的可靠性，将因窄带发射、紫外激发的镧系元素校准微珠的可用性而得到提高。制造了含有发光铕配合物的0.5微米、3微米和5微米微珠。用延时光散射门控发光流式细胞仪测量5微米微珠的发光分布，变异系数（CV）为7.0%。通过共聚焦显微镜确定铕配合物在单个微珠中的空间分布是均匀的。用PARISS分光光度计在592、616（宽度9.9纳米）和685纳米处发现发射峰。在LED激发下用荧光显微镜测量0.5微米和5微米微珠的紫外照射引起的发光漂白动力学表明，漂白不影响它们的成像。在水和空气中的发光寿命分别为340和460微秒。因此，这些5微米微珠可用于配备光谱仪的显微镜的光谱校准，作为延时发光流式细胞仪的测试颗粒，并可能作为大分子和细胞的标记物。
