Registration protocol. (a) Schematics of the optical setup obtained to achieve axial stabilization. A near-infrared laser line from a pigtailed diode (Thorlabs, Newton, New Jersey) is collimated and focused off-axis in the back-focal plane of the high NA TIRF objective used for imaging. The totally internally reflected laser-line (blue) is then steered on a quadrant photodiode QP50-6SD2 (Pacific Silicon, West Lake Village, California). (b) The net output of this setup is to maintain the relative distance between the objective and the sample fixed (i.e. the focal position), avoiding blurring of the PSF over time. (c) A trace measured from the QPD over time, showing the feed-back signal (in red) and the z-displacement of the nano-positioning stage to counter the axial offset. The system is accurate over extended periods of time within 5 nm peak-to-peak.