Figure 6. Axonal transport analysis with TrackMate algorithm.
(A) Single-particle velocity analysis during four phases in cross-branch
transport. Scale bar: 5 μm. (B) The velocity-time diagram about the
particle showed in (A). (C) Target particles selected from ring-like
axonal transport. Scale bar: 20 μm. (D) The correlation analysis between
velocity and diameter of particles marked in (C).
4. Conclusions
We successfully combine sparse neuron labelling, single-particle
fluorescence imaging with a concise and accurate quantitative algorithm
of single-particle velocity (TrackMate) for precisely tracking and
quantifying various lysosomal transport in freely orientated axons.
Thus, several axonal transport models were found, including forward or
backward transport model, stop-and-go model, repeated back-and-forth
transport model, and cross-branch transport model. Furtherly, this study
also revealed the velocity of single-particle transporting in freely
orientated axons was a highly heterogeneous and discontinuous
transportation process. Moreover, the axonal structure and particle size
were all found to affect the velocity of particle transporting in freely
orientated axons. We believe that the facile axonal transport assay may
be furtherly served as a kind of physiological steady-state parameter
assay to investigate neuronal development and axonal transport-related
diseases.
Though we have introduced TrackMate to accurately analyze single or
multiple particle velocity at the same time, we can’t get a better
understanding of the phenomenon that the velocity change during or after
the particle passing by the axon branching point due to the insufficient
temporal-spatial resolution to identify a single molecule in the axon.
Thus, the TrackMate together with an advanced single-molecule imaging
microscope may offer a more accurate velocity analysis to disclose the
molecular mechanisms involved in axonal transport during neuronal
development or diseases. On the other hand, this study only studied the
freely orientated neurons cultured on a coverslip due to the limited
tissue penetration depth of mCherry-based fluorescence imaging. We
believe that TrackMate in combination with a near-infrared fluorescence
imaging-based axonal transport tracking
technique[39,40] may offer a possibility for
exploring axonal transport in tissues or living animals.
Acknowledgements
This work was supported by the National Key Research and Development
Program (2017YFA0205503), the National Natural Science Foundation of
China (Grant No. 21778070, 22177128, 21934007), Chinese Academy of
Sciences (Grant No. XDB32030200, 121E32KYSB20180021, ZDBS-LY-SLH021),
the Youth Innovation Promotion Association of Chinese Academy of
Sciences, the Science and Technology Project of Suzhou (Grant No.
SZS201904). The authors thank Suzhou NIR-Optics Technology Co., Ltd. for
its instrumental and technique support on the fluorescence imaging.
Conflict of interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Author Contributions
Y.L., Y.L.: Conceptualization, investigation, methodology, validation,
visualization, software, writing the original draft and editing. Z.T.,
Y.C., D.H., W.F.: Formal analysis, investigation and editing. Y.Z. C.L.:
Funding acquisition and draft editing. G.C.: Conceptualization, formal
analysis, funding acquisition, supervision, writing the original draft
and editing; Q.W.: Conceptualization, project administration,
supervision, funding acquisition.
Data availability statement
All data that support the findings of this study are included within the
article (and any supplementary files).