In single-molecule applications, the photostability of fluorescent molecules is a key parameter. We apply fluorescence correlation spectroscopy to compare the photostability of four fluorescein and four borondipyrromethene (BODIPY) dyes of similar structure but different triplet yields. The latter class of dyes are more stable. In the kinetic analysis the, diffusion and photobleaching are treated as competitive processes. Corrections, which account for saturation and for experimental artefacts, are achieved solely by using experimental data. Photobleaching is found to occur mainly through the first excited singlet state S(1), in contrast to previous findings.

We report the synthesis and characterization of the missing reference core compound 4,4-Difluoro-4-bora-(3a,4a)-diaza-s-indacene 1 of the BODIPY fluorescent dye class. The compound exhibits a fluorescence lifetime of 7.2 ns and has a high photostability.

The synthesis and properties of a ratiometric fluorescent dye system as a probe for the detection of oxidizing species are described. The dye consists of a strongly fluorescent Bodipy label attached to a styryl moiety. The oxidation of the exocyclic double bond by agents for syn-and anti-dihydroxylation leads to a visible change in fluorescence colour from orange to green. IR spectroscopy provides evidence for the generation of the epoxide in the reaction with peroxides, whereas a ketone and a product of unknown structure are produced in the reaction with osmiumtetroxide, each in contrast to the expected hydroxylations. Based on these first attempts, we discuss the significance of fluorescence spectroscopy to track chemical reactions at low concentrations and give a perspective of how present shortcomings can be overcome.

We highlight our recent achievements in the design of fluorescent dyes for the investigation of chemical reactions on the single-molecule level. These fluorophores are tailored to undergo changes in their photophysical properties upon chemical transformations. Three examples are presented: electrophilic aromatic substitution, phosphoester cleavage, and oxidation of double-bonds. Thin-layer chromatography and fluorescence correlation spectroscopy are used to separate and characterize the different reaction products, respectively. We are planning to develop more fluorescent synthons which enable us to perform single-molecule chemistry of various reactions.

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