(a) The localization of a stationary particle (190 nm multifluorescent bead, Spherotech) at a countrate of >1600 photons per orbit (i.e. 320 kHz) is shown as a function of time (acquisition rate 200 Hz) for x, y and z in black, dark gray and light gray, respectively. The localization precision, determined from the standard deviation of the position of the stationary particle, was <3 nm laterally and <21 nm axially. (b) The localization precision for a moving particle (with a maximum velocity of 6.2 μm/s). An immobilized particle was moved along a sinusoidal path using a 3-axis piezo stage and the position recorded as a function of time (acquisition rate, 200 Hz) in x, y or z shown in black, dark gray and light gray respectively. A sinusoidal fit was performed (green lines) and the standard deviation of the residuals was used to determine the precision. For a count rate of >1600 photons per orbit (i.e. 320 kHz), a localization precision of <3 nm laterally and 21 nm axially was measured. (c) Count-rate dependent localization precision (average values from stationary and dynamic particles, acquisition rate 200 Hz). The values for x, y and z are shown in black, dark gray and light gray, respectively. (d) Velocity-dependent localization precision for a particle moving along the x, y and z axes. The decreased accuracy of the x axis compared to the y axis at velocities above 5 μm/s is a result of a ~ 0.1 ms delay in updating the position of the particle at the starting point of the new orbit (ϕ = 0°). (e) To measure the in vivo localization precision of the orbital tracking approach, a stationary mitochondrion inside the zebrafish was tracked at a count rate of 500 photons per orbit (i.e. 100 kHz). The trajectory data along the x axis (minor axis of the mitochondria) shows a localization precision of ~21 nm. (f) To determine the dynamic localization precision, the stationary mitochondrion was externally moved along the minor axis using a piezo stage. Similar to the dynamic precision measurement using beads, the resulting trajectory was fit using a sine wave and the standard deviation of the residuals showed a localization precision of 42 nm. The localization precision for moving mitochondria, which are usually smaller than stationary ones, was estimated from the standard deviation of the y orbit displacement, which averages 4.6 nm after removing high frequency noise by smoothing the trajectory data by five points.