The (un)known issue with using rose bengal as a standard of singlet oxygen photoproduction

Rose bengal (RB) is a widely used photosensitizer for determining quantum yields of singlet oxygen generation. While it is known to aggregate in polar environments at concentrations above 2 μM, the relationship between RB concentration and singlet oxygen photogeneration remains unclear. This study investigates the shift from monomeric to dimeric RB with increasing concentration and its impact on singlet oxygen generation in D₂O-based solutions and DMPC liposomes. Absorbance maxima for RB were observed at 514 nm (dimer) and 549 nm (monomer), with ionic environments influencing aggregation rates. Singlet oxygen phosphorescence showed non-linear dependency above 2 μM, indicating the effects of aggregation. Results suggest that RB concentrations should be kept at 1 μM or lower in photochemical studies to avoid aggregation-related discrepancies in singlet oxygen yield determination. These findings highlight the importance of considering RB aggregation in photochemical research and medical applications.