Simple method to enhance the photostability of the fluorescence reporter R6G for prolonged single-molecule studies

For fluorescence-based single-molecule studies, photobleaching of the dye reporter often limits the time window over which individual molecules can be followed. As such, many strategies, for example, using a cocktail of chemical reagents, have been developed to decrease the rate of photobleaching. Herein, we introduce a new and highly effective method to enhance the photostability of one of the commonly used fluorescent dyes, rhodamine 6G (R6G). We show that micrometer-sized polydimethylsiloxane (PDMS) wells, when the PDMS surface is properly treated, not only provide a confined environment for single-molecule detection but can also significantly increase the survival time of individual R6G molecules before photobleaching. Moreover, our results suggest, consistent with several previous studies, that R6G photobleaching involves a radical state.