Single-molecule imaging demands fluorophores with exceptional photostability and photon budget. This study presents an intramolecular energy transfer (IMET) strategy to enhance these critical properties. We developed xanthene-based IMET cassettes by covalently linking donor and acceptor, achieving significant improvements in single-molecule imaging performance. While extensive spectral overlap is generally beneficial in bulk systems, single-molecule imaging necessitates careful optimization to minimize direct acceptor excitation at the high excitation powers typically used. Our optimized cassettes, featuring rhodamine as donor and Si-rhodamine as acceptor, exhibit 94.8% energy transfer efficiency. This configuration effectively minimizes direct excitation-induced acceptor bleaching, as 95% of molecules exhibit donor photobleaching prior to acceptor photobleaching. This efficient energy management leads to a 670% enhancement in photostability, arising from the competition between IMET and photobleaching pathways, which effectively channels excitation energy away from photo-destructive processes. Time-resolved transient absorption spectroscopy revealed that IMET occurs on a picosecond timescale, significantly faster than both fluorescence relaxation (nanoseconds) and photobleaching (seconds). Notably, these IMET cassettes demonstrated superior performance in single-molecule tracking applications, including on supported lipid bilayers and in live-cell tracking of epidermal growth factor receptor (EGFR) dynamics, highlighting the broad potential of the IMET strategy for advancing single-molecule imaging.

Keywords: Intramolecular energy transfer; Single-molecule imaging; fluorescent probes; photostability.

© 2025 Wiley‐VCH GmbH.

单分子成像需要荧光团具有出色的光稳定性以及较大的光子预算。本研究提出了一种分子内能量转移（IMET）策略来增强这些关键特性。我们通过共价连接供体和受体开发了基于香豆素的IMET模块，从而在单分子成像性能方面取得了显著改进。虽然光谱重叠在整体系统中通常是有利的，但在通常使用的高激发功率下进行单分子成像时需要仔细优化以尽量减少直接受体激发。我们的优化模块采用罗丹明作为供体和Si-罗丹明作为受体，能量转移效率达到94.8%。这种配置有效地减少了直接激发引起的受体光漂白，因为有95%的分子在受体光漂白之前经历了供体光漂白。这种高效的能量管理使光稳定性提高了670%，这是因为IMET和光漂白途径之间的竞争有效将激发能从光破坏过程转移开。时间分辨瞬态吸收光谱显示，IMET发生在皮秒尺度上，远快于荧光松弛（纳秒）和光漂白（秒）。值得注意的是，这些IMET模块在单分子追踪应用中表现出色，包括支撑脂质双层上的追踪以及活细胞中表皮生长因子受体（EGFR）动力学的追踪，突显了IMET策略在推进单分子成像方面的广泛潜力。

关键词： 分子内能量转移；单分子成像；荧光探针；光稳定性。