Retrograde transport by dynein is essential for neuronal growth and function. However, quantitative knowledge of dyneins on axonal endosomes during this long-range transport requires bright, photostable and non-blinking single-molecule probes. Here we report long-term single-particle tracking using upconversion nanoparticles (UCNPs). The probes enabled imaging cargo transport in live neurons over tens of minutes. Using the fluctuation theorem, the number of active dynein is shown to switch between one to five pairs during the transport of a single cargo. The high brightness of these UCNPs allowed 8-nm dynein steps to be clearly resolved with one millisecond/video frame resolution. Data taken at 22, 30 and 37 °C reveal that the dwell-time between each molecular step is described by two sequential and equally thermal-activated rate constants. Anew model is proposed for dynein operation in neurons. Finally, the data indicates that cargo-motor operates out of thermal equilibrium with its cellular environment.