Figure 4. Photophysical characterization of photostabilizer–dye conjugates and their parent fluorophores with single-molecule TIRF microscopy.

Data was recorded in aqueous PBS buffer at pH 7.4 in the absence of oxygen under continuous 640 nm excitation with ≈50 W cm⁻² or 532 nm excitation with ≈20 W cm⁻². (a) Representative image frame (left panel) showing single fluorescent molecules (10 × 10 μm, exemplarily for Cy5). Subsequent images recorded over a period of 500 s showed an exponential decrease in the number of fluorescing molecules with a photobleaching lifetime τ_bleach, as shown for Cy5 (black), Cy5 with 2 mM TX (blue) and NPA–Cy5 (red) (middle panel). The curves shown were obtained by averaging over >5 TIRF movies. (b,c,d) Right panel: chemical structures (left panels) and respective photophysical parameters obtained from background-corrected fluorescence traces for Alexa555 (b, rhodamines), ATTO647N (c, carbopyronines) and Cy5 (d, cyanines). Values and error bars (s.d.) in bar graphs obtained from N>500 molecules. For further details of the experimental techniques, data acquisition and analysis, see the Methods section.