The photophysical properties of rhodamine molecules play a critical role in their performance across various applications. The spectroscopic single-molecule fluorescence (sSMF) technique overcomes the limitations of conventional SMF by distinguishing individual fluorophores based on their emission spectra. This enables precise measurement and direct comparison of photophysical properties among distinct molecules under identical conditions, without requiring separation of molecules. In this study, using a custom sSMF instrument, we successfully identified individual rhodamine B molecules and their various N-dealkylated intermediates, allowing for simultaneous investigation of their photophysical properties. Notably, we observed that rhodamine B undergoing a single dealkylation step exhibited a striking enhancement in photostability compared to its fully intact counterparts and those undergoing two dealkylation steps. This enhancement persisted across various buffer conditions, including different pH levels and the presence or absence of an oxygen scavenger system (OSS). Despite these differences in photostability, time-dependent density functional theory (TD-DFT) calculations revealed that all these rhodamine molecules examined shared a similar energy gap (∼0.6 eV) between their first excited singlet and triplet states.
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