STED simulations of AFM ribosome clusters display aspect ratios overlapping with objects observed by STED in cells. (a) Example of a typical AFM image. (b and c) STED and confocal images of MCF-7 cells, respectively, where RPL26 has been immunolabeled with ATTO-488. (d) Applying a threshold to the raw AFM image, we generated a binary image that we convolved with the simulated STED point spread function (PSF) after noise addition. The amount of noise was calculated measuring the mean standard deviation of counts in the background of the STED images. (e and f) Zoom of the squared regions of interest in b and c, respectively. The zoom has been chosen to match the size of the AFM image. (g–i) Zoom of the squared regions of interest in d–f, respectively. Fluorescence spots in STED (e and h) are comparable with simulated images of HMW polysomes obtained by convolution of AFM scans with STED PSF (d and g). (j and k) Shown are the result of the object localization procedure (see Materials and methods) applied to the g and h images, respectively. The blue lines show the perimeter of the objects identified. (l) Distribution of the aspect ratio of the objects that arise from the analysis of 10 STED images and 10 simulated STED images from AFM originals. The two graphs are normalized by the whole number of counted objects, respectively, and show a good matching. All images were acquired at 1024 × 1024 pixels with 14-nm pixel size. Bars, 1 µm.