Towards ultra-stable fluorescent dyes for single-molecule spectroscopy

The wide-spread use Of fluorescent dyes in molecular diagnostics and fluorescence microscopy together with new developments such as single-molecule fluorescence spectroscopy provide researchers from various disciplines with an ever expanding toolbox. Single-molecule fluorescence spectroscopy relies to a large extent on extraordinary bright and photostable organic fluorescent dyes such as rhodamine- or cyanine- derivatives. While in the last decade single-molecule equipment and methodology have significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields), instable emission ("blinking") and photobleaching become more and more the bottleneck of further development and spreading of single-molecule fluorescence studies. In recent years, agents and recipes have been developed to increase the photostability of conventional fluorescent dyes. Here, we investigate some of these strategies at the single-molecule level. In particular, we focus on the dye selection criteria for multi-color applications. We investigate fluorescent dyes from the rhodamine, carborhodamine, cyanine, and oxazine family and show that within one dye class the photophysical properties are very similar but that dyes from different classes show strikingly different properties. These findings facilitate dye selection and provide improved chemical environment for demanding fluorescence microscopic applications.