Fig. 6 |. Limitations of single-molecule localization microscopy techniques.

a | Single-molecule localization microscopy (SMLM) image of microtubules before and after drift correction. Arrows show a fluorescent bead used to estimate the drift. b,c | Artefacts caused by point spread function (PSF) overlaps in simulated images. Ground truth image without localization errors, shown as a scatter plot (part b, left). Corresponding SMLM image for a low density of activated fluorophores (10 localizations per square micrometre (locs μm⁻²), no PSF overlaps) (part b, middle). Corresponding SMLM image for a high activation density (50 locs μm⁻²); overlapping PSFs cause artefactual localizations near the intersection of filaments at the centre (part b, right). Simulated molecular clusters, with a 10-fold higher density for the top cluster (part c). Simulated ground truth shown as a scatter plot (part c, left). Corresponding SMLM image without filtering (part c, middle). Corresponding SMLM image after filtering out poor localizations caused by overlapping PSFs (part c, right). After filtering, the high-density cluster is barely visible. d | Artefacts in SMLM images of microtubules resulting from subpixel localization bias. Left: without bias. Right: with bias caused by using localization software with an incorrect PSF model. Because of the bias, the reconstructed image shows a grid pattern. Insets show the entire field of view. The localization bias is readily apparent in the histogram of x coordinates relative to the centre of camera pixels. Drift correction was not applied to these data to better highlight the effect of localization bias.