A General Strategy for Enhanced Single‐Molecule Imaging Through Intramolecular Energy Transfer

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 94.9% 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.