SMLM microtubule images of sub-optimally fixed cells reveal sub-diffraction artifacts not observable in epifluorescence images.(A–C) Epifluorescence images of COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with PFA (A) for a shorter period than optimal, (B) at a lower concentration than optimal, and (C) for a longer period than optimal These images are not readily identified as having fixation artifacts. (D–F) Corresponding SMLM images of sub-areas from A–C show clear sub-diffraction scale damage to the microtubule architecture with some areas well preserved and continuously stained in D–E (white arrows) and other long stretches of filament missing or damaged beyond antibody-epitope recognition. Scale bars A–C: 5 μm, D: 1 μm, E–F: 500 nm.