Axonal transport plays a significant role in the establishment of
neuronal polarity, axon growth, and synapse formation during neuronal
development. The axon of a naturally growing neuron is a highly complex
and multifurcated structure with a large number of bends and branches.
Nowadays, the study of dynamic axonal transport in morphologically
complex neurons is greatly limited by the technological barrier. Here, a
sparse gene transfection strategy was developed to locate fluorescent
mCherry in the lysosome of primary neurons, thus enabling us to track
the lysosome-based axonal transport with a single-particle resolution.
Thereby, several axonal transport models were observed, including
forward or backward transport model, stop-and-go model, repeated
back-and-forth transport model, and cross-branch transport model. Then,
the accurate single-particle velocity quantification by TrackMate
revealed a highly heterogeneous and discontinuous transportation process
of lysosome-based axonal transport in freely orientated axons. And,
multiple physical factors, such as the axonal structure and the size of
particles, were disclosed to affect the velocity of particle
transporting in freely orientated axons. The combined single-particle
fluorescence tracking and TrackMate assay can be served as a facile tool
for evaluating axonal transport in neuronal development and axonal
transport-related diseases.