Fig. 3: Combining STED with on/off switching and labeling by DNA hybridization.

a, Fluorophores (pentagons in gray and highlighted in green when able to fluoresce) attached to single-stranded DNA diffusing in solution, sporadically binding to molecular targets having complementary DNA strands; here the target is a DNA origami represented by gray spheres and sticks. The region in which fluorescence is possible (that is, E-PSF region) is shown in orange for the confocal case (top panel) and the STED case (lower panel). Suppression of the fluorescence of the quickly diffusing fluorophores by STED increases the ratio between the fluorescence signal of bound (on) and diffusing (off) fluorophores. The increased on/off ratio enhances the detection of single bound fluorophores. Conversely, it can be used to increase the concentration of diffusing fluorophores so to increase the imaging speed. b, Peak fluorescence from single DNA-bound Cy3B fluorophores (blue), fluorescence from diffusing fluorophores with excitation and STED, subtracted the STED-only signal. This is considered as the signal produced from the center peak of the E-PSF by the diffusing fluorophores (red), and STED beam induced fluorescence of the diffusing fluorophores (orange) as a function of the STED pulse energy E. The SBR increases by a factor >10 over that of confocal microscopy due to application of E = 1-nJ STED pulses.