Fig. 1.

The influence of the enzymatic oxygen scavenging system (abbreviated as GOX/CAT), diluted in PBS and added to glycerol, on VdcV SMLM image quality. Under the conditions used in Fig. 1, glycerol alone provided an average density of about 300 single molecule (SM) signals per µm², or 17.3×17.3 SM/µm², i.e. one signal per 1000/17 nm=59 nm, corresponding to an average estimated structural resolution of ~2×59 nm. However, by adding the specifically designed enzymatic oxygen scavenging medium, the average density of SM signals was increased several times, resulting in an SMLM image with further enhanced structural resolution (based on the same number of image frames). For a quantitative comparison we used only VdcV at 10 nM, i.e. at a concentration where saturation of the detector does not occur at the beginning of the illumination procedure. This protocol made it possible to run a measurement without prior bleaching. Using an excitation wavelength of ${\lambda }_{\mathit{exc}}$=491 nm (illumination intensity 0.3 kW/cm²) and fluorescence emission registration in the range of 585–675 nm, 20,000 frames were collected. Signal densities obtained in the images of the cell nuclei: 207 SM/µm² in 100% glycerol (n=7) and 679 SM/µm² in the imaging buffer (n=9; n=number of cells analyzed). 207 SM/µm² (=14.4×14.4 SM µm²) corresponds to the next neighbor distance of one signal per 1000/14.4 nm=70 nm, resulting in an average estimated structural resolution of ~2×70 nm; 679 SM/µm² (=26×26 SM µm²) corresponds to the next neighbor distance of one signal per 1000/26 nm=38 nm, resulting in an average estimated structural resolution of ~2×38 nm. Note that the optical (two point) resolution depends on the precision of localization, whereas the structural resolution depends also on the density of signals, thus it may be different for different SMLM images, even if the localization precision remains the same.