Yao Jie, Larson Daniel R, Vishwasrao Harshad D, Zipfel Warren R, Webb Watt W
School of Applied and Engineering Physics and Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14284-9. doi: 10.1073/pnas.0506523102. Epub 2005 Sep 16.
Water-soluble quantum dots (qdots) are now being used in life sciences research to take advantage of their bright, easily excited fluorescence and high photostability. Although the frequent erratic blinking and substantial dark (never radiant) fractions that occur in all available qdots may interfere with many applications, these properties of individual particles in biological environments had not been fully evaluated. By labeling Qdot-streptavidin with organic dyes, we were able to distinguish individual dark and bright qdots and to observe blinking events as qdots freely diffused in aqueous solution. Bright fractions were measured by confocal fluorescence coincidence analysis (CFCA) and two-photon cross-correlation fluorescence correlation spectroscopy (FCS). The observed bright fractions of various preparations were proportional to the ensemble quantum yields (QYs), but the intrinsic brightness of individual qdots was found to be constant across samples with different QYs but the same emission wavelengths. Increasing qdots' illuminated dwell time by 10-fold during FCS did not change the fraction of apparently dark qdots but did increase the detected fraction of blinking qdots, suggesting that the dark population does not arise from millisecond blinking. Combining CFCA with wide-field imaging of arrays of qdots localized in dilute agarose gel, the blinking of qdots was measured across five orders of magnitude in time: approximately 0.001-100 s. This research characterizes photophysical pathologies of qdots in biologically relevant environments rather than adhered on dielectric surfaces and describes methods that are useful for studying various bioapplicable nanoparticles.
水溶性量子点（量子点）目前正用于生命科学研究，以利用其明亮、易于激发的荧光和高光稳定性。尽管所有市售量子点都会频繁出现不稳定闪烁和大量暗态（无辐射）部分，这可能会干扰许多应用，但生物环境中单个粒子的这些特性尚未得到充分评估。通过用有机染料标记量子点-链霉亲和素，我们能够区分单个暗态和亮态量子点，并在量子点在水溶液中自由扩散时观察闪烁事件。通过共聚焦荧光符合分析（CFCA）和双光子交叉相关荧光相关光谱（FCS）测量亮态部分。观察到的各种制剂的亮态部分与总体量子产率（QYs）成正比，但发现单个量子点的固有亮度在具有不同QYs但发射波长相同的样品中是恒定的。在FCS期间将量子点的照射停留时间增加10倍，并没有改变明显暗态量子点的比例，但确实增加了检测到的闪烁量子点的比例，这表明暗态群体并非源于毫秒级闪烁。将CFCA与定位在稀琼脂糖凝胶中的量子点阵列的宽场成像相结合，在五个数量级的时间内测量了量子点的闪烁：约0.001 - 100秒。这项研究表征了生物相关环境中量子点的光物理病理，而非附着在介电表面上的量子点，并描述了对研究各种生物适用纳米颗粒有用的方法。
