Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor single molecules beyond the range accessible to normal microscopes. However, several aspects of the ZMW influence on the photophysics of fluorophores remain inadequately documented and sometimes controversial. Here, we thoroughly investigate the ZMW influence on the fluorescence of single immobilized Cy3B and Alexa 647 molecules, detailing the interplays between brightness, lifetime, photobleaching time, the total number of emitted photons, and Förster resonance energy transfer (FRET). Despite the plasmonic-enhanced excitation intensity in the ZMW, we find that the photostability is preserved with similar photobleaching times as on the glass reference. Both the fluorescence brightness and the total number of photons detected before photobleaching are increased, with an impressive gain of nearly five times that found for Alexa 647 dyes. Finally, the single-molecule data importantly allow a loophole-free characterization of the ZMW influence on the FRET process. We show that the FRET rate constant is enhanced by 50%, demonstrating that nanophotonics can mediate the energy transfer. These results deepen our understanding of the fluorescence enhancement in ZMWs and are of immediate relevance for single-molecule biophysical applications.

中文翻译：

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零模式波导纳米孔径中固定单分子的荧光亮度、光稳定性和能量转移增强

零模式波导 (ZMW) 纳米孔径被广泛用于监测超出普通显微镜可访问范围的单个分子。然而，ZMW 对荧光团光物理影响的几个方面仍然没有得到充分记录，有时还存在争议。在这里，我们彻底研究了 ZMW 对单个固定 Cy3B 和 Alexa 647 分子荧光的影响，详细说明了亮度、寿命、光漂白时间、发射光子总数和 Förster 共振能量转移 (FRET) 之间的相互作用。尽管 ZMW 中的等离子体增强了激发强度，但我们发现光稳定性与玻璃参考相似的光漂白时间得以保持。荧光亮度和光漂白前检测到的光子总数都增加了，与 Alexa 647 染料相比，其效果显着提高了近五倍。最后，单分子数据重要地允许无漏洞表征 ZMW 对 FRET 过程的影响。我们表明 FRET 速率常数提高了 50%，表明纳米光子学可以介导能量转移。这些结果加深了我们对 ZMWs 荧光增强的理解，并与单分子生物物理应用直接相关。