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

Abstract 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.