Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens 1–5 . However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters 6 . Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Förster resonance energy transfer (FRET) to quantify molecular interactions 7 . In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.