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.