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).