68
C.-L. Hsieh
2]. In neuronal cells, active transport is especially critical for appropriate neuron
activities [3, 4]. For active transportation, cargos are enclosed in small lipid vesicles
or micelles, and their translocation is accomplished by motor proteins and cytoskeletons. The fundamental interaction between individual molecules underlies the regulatory mechanisms of intracellular transport, determining when and where the cargo
is delivered [5, 6]. However, this sophisticated cell machinery developed through
evolution is also utilized by viruses, which hijack the cell machinery by encapsulating their genomes in small particles with specific surface functionalities [7]. Thus,
examining how viruses infect cells is not only useful in the battle against disease but
also in understanding the cell machinery.
Through biochemical and molecular biology approaches, many proteins have
been identified that play crucial roles in cell uptake and transportation [8]. However,
these studies have tended to provide static results that lack spatial and temporal resolutions. Optical microscopy is a powerful tool that provides high spatiotemporal
resolution. Through labeling with fluorescent proteins and dyes, cell dynamics—
including intracellular transport and virus uptake—have been visualized in real time
[9–11]. Using this technique, individual cell vesicles and virus particles can be seen
under the microscope and their trajectories can be reconstructed by single-particle
tracking (SPT) [11–13]. The main challenge in fluorescence-based optical observation is the limited photon budget that strictly restricts the observation time and data
acquisition rate. Fluorescence-based SPT typically provides a spatial precision of
1–10 nm and a temporal resolution of a few to tens of milliseconds [14–16]. This
spatiotemporal resolution has enabled many studies of interactions between particles and their local environment. However, probing molecular interactions at the
nanometer scale requires higher spatiotemporal resolutions that fluorescence-based
approaches struggle to provide.
For high-precision and high-speed measurements, a robust signal is required. Linear scattering meets these requirements—unlike fluorescence, which suffers from
photobleaching, blinking and saturation, linear scattering from small particles is stable and indefinite, making it a promising contrast mechanism. Using scattering-based
optical microscopy, including darkfield and brightfield microscopy, many high-speed
and high-precision localization measurements have been reported that uncovered
important biophysical and biochemical processes [17–20]. For a biological nanoparticle with a diameter of 100 nm in an aqueous solution, its scattering cross section
in visible light is on the order of 10
−13 cm
2 (assuming a refractive index of 1.45),
which is three orders of magnitude larger than the fluorescence cross section of an
organic fluorophore. Therefore, intrinsic scattering from a bio-nanoparticle should be
sufficient for direct observation and dynamic study. Indeed, endogenous biological
nanoparticles (virus particles and extracellular vesicles) have been detected in cellfree in vitro environments by using several scattering-based imaging techniques,
including darkfield [21–23], holographic, and other interferometric microscopies
[24–28]. Among these imaging techniques, interferometric detection has the advantage of achieving shot-noise-limited sensitivity, independent of the detector noise
[29].
C.-L. Hsieh
2]. In neuronal cells, active transport is especially critical for appropriate neuron
activities [3, 4]. For active transportation, cargos are enclosed in small lipid vesicles
or micelles, and their translocation is accomplished by motor proteins and cytoskeletons. The fundamental interaction between individual molecules underlies the regulatory mechanisms of intracellular transport, determining when and where the cargo
is delivered [5, 6]. However, this sophisticated cell machinery developed through
evolution is also utilized by viruses, which hijack the cell machinery by encapsulating their genomes in small particles with specific surface functionalities [7]. Thus,
examining how viruses infect cells is not only useful in the battle against disease but
also in understanding the cell machinery.
Through biochemical and molecular biology approaches, many proteins have
been identified that play crucial roles in cell uptake and transportation [8]. However,
these studies have tended to provide static results that lack spatial and temporal resolutions. Optical microscopy is a powerful tool that provides high spatiotemporal
resolution. Through labeling with fluorescent proteins and dyes, cell dynamics—
including intracellular transport and virus uptake—have been visualized in real time
[9–11]. Using this technique, individual cell vesicles and virus particles can be seen
under the microscope and their trajectories can be reconstructed by single-particle
tracking (SPT) [11–13]. The main challenge in fluorescence-based optical observation is the limited photon budget that strictly restricts the observation time and data
acquisition rate. Fluorescence-based SPT typically provides a spatial precision of
1–10 nm and a temporal resolution of a few to tens of milliseconds [14–16]. This
spatiotemporal resolution has enabled many studies of interactions between particles and their local environment. However, probing molecular interactions at the
nanometer scale requires higher spatiotemporal resolutions that fluorescence-based
approaches struggle to provide.
For high-precision and high-speed measurements, a robust signal is required. Linear scattering meets these requirements—unlike fluorescence, which suffers from
photobleaching, blinking and saturation, linear scattering from small particles is stable and indefinite, making it a promising contrast mechanism. Using scattering-based
optical microscopy, including darkfield and brightfield microscopy, many high-speed
and high-precision localization measurements have been reported that uncovered
important biophysical and biochemical processes [17–20]. For a biological nanoparticle with a diameter of 100 nm in an aqueous solution, its scattering cross section
in visible light is on the order of 10
−13 cm
2 (assuming a refractive index of 1.45),
which is three orders of magnitude larger than the fluorescence cross section of an
organic fluorophore. Therefore, intrinsic scattering from a bio-nanoparticle should be
sufficient for direct observation and dynamic study. Indeed, endogenous biological
nanoparticles (virus particles and extracellular vesicles) have been detected in cellfree in vitro environments by using several scattering-based imaging techniques,
including darkfield [21–23], holographic, and other interferometric microscopies
[24–28]. Among these imaging techniques, interferometric detection has the advantage of achieving shot-noise-limited sensitivity, independent of the detector noise
[29].