3.5. Single-particle tracking and velocity analysis
To achieve an accurate particle velocity analysis of lysosomal transport, we selected a recently published algorithm TrackMate[19] for single-particle tracking and compared it with the mainstream algorithm KymographClear[18] for the same goal. It is previously reported that the velocity analysis by KymographClear is not continuous because the software exclusively identifies moving particles and does not record the static velocity at the same time,[34] and the static velocity in the whole period needs to be uniformly set to zero to draw in a diagram. In contrast, the velocity analysis energized by the TrackMate algorithm can recognize the target particle and record its velocity in the whole period no matter which state of motion it is, moving or stationary. Unless the moving particle is exactly stationary, the recorded speed should always be greater than zero. In other words, KymographClear only obtains the velocity of moving particles (the static velocity is not recorded) while TrackMate can extract the velocity in a whole period (both moving and static velocities are recorded).
As shown in Figure 5A, the location of particle a was recorded at time t1, t2,…, t7respectively, and the location-time diagram was drawn in the red coordinate axis. By exerting the KymographClear algorithm, all the positions of particle a were marked at time t instantaneously, then its track would be depicted in Figure 5C. The kymograph record starting from the upper left corner is particle a’s track, another two lines represent the other two particles that can’t be excluded but analyzed at the same time. Then, we further obtained the velocity-time diagram drawn in Figure 5D (marked in red) after setting the speed of the unrecognized motion to zero. Figure 5B shows the initial analysis result of particle a with the TrackMate algorithm. The green line referred to the track of particle a, and the tracking length was measured as 25.67 μm. The purple mark meant several other moving particles were identified at the same time. Eventually, the velocity-time diagram of particle a was shown in Figure 5D (marked in green) in a whole period without data interrupt. We then compared the subtle difference of velocity data extracted by these two algorithms. One point to state is that the average velocity of particle a calculated by these two algorithms is similar, approximately 0.103 μm/s (KymographClear algorithm) and 0.094 μm/s (TrackMate algorithm) (Figure 5D), suggesting that these two algorithms work well for average velocity analysis. However, these two algorithms are significantly different in the analysis of instantaneous velocity. As revealed by the velocity-time diagram (Figure 5D), the lysosomal transport in the axon is a highly heterogeneous and discontinuous transportation process with numerous velocity fluctuations. TrackMate algorithm shows more concise and accurate than the KymographClear algorithm for single-particle velocity assay. For example, three velocity fluctuations around 50 s were recorded by TrackMate (green line), while only one velocity change was recorded by KymographClear (red line). Moreover, after 100 s, the red line tended to flatten out, while the green line still showed several significant changes (three spikes) with a significant unsmooth curve. It is suggested that there is a brief unbalance between motor proteins that respond to the dynamic concentration changes of kinesins and dyneins, or the frequency of spikes represent other axonal transport features. These phenomena can just be visualized by the TrackMate algorithm. Undoubtedly, we obtained the same results by analyzing the motion of particle b (Figure S2). It proves that, on the premise of ensuring the consistent average velocity of particles, the TrackMate algorithm can better retain the velocity fluctuations and provide better fidelity than the KymographClear algorithm in restoring the motion details.