Figure 1.

Stochastic optical reconstruction microscopy (STORM) with photo-switchable fluorophores. (a) The diagram shows a STORM imaging sequence using a hypothetical hexameric object labeled with red fluorophores that can be switched between a fluorescent and a dark state by a red and green laser, respectively. All fluorophores are first switched to the dark state by a strong red laser pulse. In each imaging cycle, a green laser pulse is used to switch on only a fraction of the fluorophores to give an optically resolvable set of active fluorophores. Next, under red illumination, these molecules emit fluorescence until they are switched off, allowing their positions (indicated by white crosses) to be determined with high accuracy. The overall image is then reconstructed from the fluorophore positions obtained from multiple imaging cycles. (b) A single Cy5 switch on DNA can be turned on and off for hundreds of cycles before being permanently photobleached. A red laser (633 nm, 30 W/cm²) is used to excite fluorescence (black line) from Cy5 and to switch Cy5 to the dark state. A green laser (532 nm, 1 W/cm²) is used to return Cy5 to the fluorescent state. The alternating red and green line indicates the laser excitation pattern. The recovery rate of Cy5 depends critically on the close proximity of Cy3¹⁴.