Fig. 8.

Dependence of the SMLM image quality on the duration of the image acquisition. (A) Reconstructed images of a Vero-B4 cell nucleus stained with 500 nM Vybrant® DyeCycle™ Violet. The images show that the total number of single molecule fluorescent bursts detected depends strongly on the total number of the image frames, i.e. on the length of the image acquisition. Average signal densities for the images: 1496 SM/µm² (5000 frames); 2901 SM/µm² (10,000 frames), 4285 SM/µm² (15,000 frames), 5716 SM/µm² (20,000 frames). SM signals per frame (in the following denoted by α): 5000 frames: α=1496/5000=0.299 SM/frame; 10,000 frames: α=0.290; 15,000 frames: α=0.286; 20,000 frames: α=0.286; ${\lambda }_{\mathit{exc}}$=491 nm (0.525 kW/cm²), ${\lambda }_{\mathit{em}}$: 585–675 nm. (B) A dependence of the number of the single molecule signals detected per 1000 acquired frames on acquisition time. A slight permanent bleaching (exponential decay) is observed. (C) A widefield image of fluorescence of VdcV in the same Vero-B4 cell nucleus; ${\lambda }_{\mathit{exc}}$= 405 nm, ${\lambda }_{\mathit{em}}$= 440–490 nm.