为了表征上转换纳米荧光微粒的发光特性, 设计了一个可以对单个纳米微粒进行荧光寿命测量的系统。该系统首先使用基于检流计振镜的双光子显微镜系统对单分散状态的上转换纳米微粒样品进行扫描成像。然后, 通过单分子荧光纳米定位算法精确找出每个纳米微粒的准确位置, 再依次将激光聚焦到每个纳米微粒上, 在该点施加一个500 μs宽度的激光脉冲, 并通过光电倍增管探测随时间变化的荧光强度信号。最后对荧光衰减曲线进行拟合,计算得到该纳米微粒的荧光寿命。实验结果表明: 单个上转换纳米荧光微粒的荧光发射曲线符合单指数衰减规律, 其荧光寿命为195.3 μs。与之相比, 聚集状态的纳米微粒的荧光寿命为358.9 μs。这表明聚集状态对上转换纳米微粒的发光特性有显著影响。

In order to characterize the photon luminescence properties of up-conversion nanoparticles(UCNPs), a system to measure fluorescence life time of single nanoparticle was established based on a custom-built two-photon microscope scanned with a pair of galvo-mirrors. First, a sample of mono-disperse UCNPs was imaged by the two-photon microscope. Then, the exact location of each nanoparticle was precisely determined through the single molecule fluorescence localization algorithm. The laser was focused on the nanoparticles one by one with a laser pulse with 500 μs duration exerted on the nanoparticle. A photomultiplier was employed to measure the time-lapse fluorescence intensity which varied with time. Finally, the fluorescence decay curve was fitted to calculate the life time of single nanoparticle. The experiment results show that the fluorescence curves of single nanoparticle fit with single exponential decay, and the fluorescence life time is 195.3 μs. For contrast, the life time of the bulk sample is 358.9 μs. This indicates a dramatic effect of particle aggregation on the photon luminescence properties of UCNPs.