3 Label-Free, Ultrahigh-Speed, Direct Imaging and Tracking …
79
erally on the plasma membrane with a very high diffusion coefficient (~1 μm
2 /s),
which is as high as its free diffusion in water. This observation indicates that the
virus weakly adhered to the membrane and was free to explore the cell surface.
Strikingly, from the trajectory, numerous zones of 10–20 nm were present where the
virus was transiently confined for sub-milliseconds. The observed viral dynamics
are hypothesized to reflect the interaction between the virus and the cell membrane
molecules (e.g., glycosaminoglycan). The transient nano-confinements may be evidence of virus interplay with immobile membrane receptors. Notably, the trajectory
was 3D, which indicates the 3D cell membrane morphology.
3.4.2 Nanoscopic Dynamics of a Cell Vesicle During Active
Transportation
COBRI microscopy also makes it possible to visualize the intracellular dynamics
of individual cell vesicles and organelles. With improved sensitivity and speed, a
native fibroblast cell appears highly dynamic under COBRI microscopy. Many cell
vesicles are locally confined and restricted to diffuse within small volumes created
by cytoskeleton networks inside the cytosol. Rapid and directional transportations of
cell vesicles are also occasionally observed. Through lipophilic dye labeling, most
vesicles were found to be lipid-rich, presumably lipid droplets. Estimated from their
COBRI contrast, most vesicles under observation were approximately 300 nm in
diameter. The sensitivity of COBRI microscopy is sufficient for tracking single vesicles with nanometer spatial precision in 3D. By capturing vesicle motions at 30,000
fps, their nanoscopic motion is unveiled. Figure 3.6 shows typical transportation of
a vesicle with clearly resolved stepwise motion by motor proteins. The bidirectional
motion of the vesicle resulting from a tug-of-war between oppositely directed motor
proteins attached to the same cargo can clearly be seen. Under ultrahigh-resolution
observation, the transient pausing at the moment the vesicle switches directions is
clearly resolved where an oscillating behavior over 10-nm is disclosed. In addition
to the ultrahigh spatiotemporal resolution, label-free COBRI microscopy offers the
opportunity to probe intracellular dynamics in their native condition.
3.5 Conclusions and Future Perspectives
In this chapter, COBRI microscopy was shown to enable ultrahigh-speed imaging with a frame time of microseconds. The high sensitivity of COBRI facilitates
nanometer-precise 3D tracking of endogenous biological nanoparticles with diameters as small as 100–200 nm. The cell scattering background can be selectively
removed through proper background estimation and correction. The current strategy
of background correction is favorable when tracking mobile nanoparticles in a rela-
79
erally on the plasma membrane with a very high diffusion coefficient (~1 μm
2 /s),
which is as high as its free diffusion in water. This observation indicates that the
virus weakly adhered to the membrane and was free to explore the cell surface.
Strikingly, from the trajectory, numerous zones of 10–20 nm were present where the
virus was transiently confined for sub-milliseconds. The observed viral dynamics
are hypothesized to reflect the interaction between the virus and the cell membrane
molecules (e.g., glycosaminoglycan). The transient nano-confinements may be evidence of virus interplay with immobile membrane receptors. Notably, the trajectory
was 3D, which indicates the 3D cell membrane morphology.
3.4.2 Nanoscopic Dynamics of a Cell Vesicle During Active
Transportation
COBRI microscopy also makes it possible to visualize the intracellular dynamics
of individual cell vesicles and organelles. With improved sensitivity and speed, a
native fibroblast cell appears highly dynamic under COBRI microscopy. Many cell
vesicles are locally confined and restricted to diffuse within small volumes created
by cytoskeleton networks inside the cytosol. Rapid and directional transportations of
cell vesicles are also occasionally observed. Through lipophilic dye labeling, most
vesicles were found to be lipid-rich, presumably lipid droplets. Estimated from their
COBRI contrast, most vesicles under observation were approximately 300 nm in
diameter. The sensitivity of COBRI microscopy is sufficient for tracking single vesicles with nanometer spatial precision in 3D. By capturing vesicle motions at 30,000
fps, their nanoscopic motion is unveiled. Figure 3.6 shows typical transportation of
a vesicle with clearly resolved stepwise motion by motor proteins. The bidirectional
motion of the vesicle resulting from a tug-of-war between oppositely directed motor
proteins attached to the same cargo can clearly be seen. Under ultrahigh-resolution
observation, the transient pausing at the moment the vesicle switches directions is
clearly resolved where an oscillating behavior over 10-nm is disclosed. In addition
to the ultrahigh spatiotemporal resolution, label-free COBRI microscopy offers the
opportunity to probe intracellular dynamics in their native condition.
3.5 Conclusions and Future Perspectives
In this chapter, COBRI microscopy was shown to enable ultrahigh-speed imaging with a frame time of microseconds. The high sensitivity of COBRI facilitates
nanometer-precise 3D tracking of endogenous biological nanoparticles with diameters as small as 100–200 nm. The cell scattering background can be selectively
removed through proper background estimation and correction. The current strategy
of background correction is favorable when tracking mobile nanoparticles in a rela-