Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection:  Evidence of Two-Step Photolysis.

The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, p(b), is in the order of 10(-)(6)-10(-)(7) for irradiances below 10(3) W/cm(2). However, a considerable increase of p(b) for irradiances above this level was observed which can only be described by photobleaching reactions from higher excited states (two-step photolysis). In view of these observations, the probability of photobleaching, p(b), as well as a closed expression of its dependence on the CW excitation irradiance considering a five-level molecular electronic state model with the possibility of photobleaching from higher excited electronic states, is derived. From this model, optimal conditions for SMD with respect to the number of emitted fluorescence photons and to the signal-to-background ratio are discussed, taking into account both saturation and photobleaching. The additional photobleaching due to two-step photolysis limits the applicable irradiance.