2 Interferometric Scattering (iSCAT) Microscopy and Related Techniques
47
for extinction measurements. This is why quenched molecules can be detected in
extinction but not in fluorescence.
Gold nanoparticles, quantum dots and dye molecules all have resonances, with
their polarizability experiencing a maximum in a certain spectral range. While this
enhances the extinction cross section, and thus the iSCAT signal, it is not nearly as
decisive as the influence of the particle size. A consequence is that even a dielectric
nanoparticle can yield a large interferometric signal given it is of sufficient size. In
what follows, we show that all viruses and a vast range of proteins easily satisfy this
criterion, prompting efforts in their detection.
2.4.1.3 Viruses
Viruses play a crucial role in biology, whether causing harmful diseases or performing an integral symbiotic function within living systems [157] or even serving as
novel disease treatment vectors [158]. Detection of viruses, as with most other cell
biological entities, has traditionally been performed in fluorescence. However, it turns
out that viruses and virus-like particles such as X31 virus, H1N1, Zika, Ebola, and
SV40, which could range from dimensions of about 20 nm to beyond 200 nm [159],
can be easily detected via iSCAT. This has been demonstrated in environments such
as microfluidic channels [160, 161], on synthetic lipid bilayers (see Fig. 2.11a) [115,
116], and on dielectric substrates [77, 83]. Simultaneous iSCAT and fluorescence
tracking of a virus and a quantum dot on its surface made it possible to visualize
not only the nanoscopic binding domains on the membrane but also the rocking and
tumbling motion of single viruses (see Fig. 2.11a) [116]. Moreover, iSCAT has been
used to uncover to nanometer precision the position and orientational configuration
of bacteriophages interacting with a surface as well as to resolve, to a precision of
4200 basepairs, the kinetics of DNA ejection following stimulation [162]—shown
in Fig. 2.11b. These activities have also motivated attempts to detect pure scattering
of single viruses in conventional dark-field [139] and through light scattering from
a nanofluidic channel [163].
The large iSCAT signal of most viruses opens the way to label-free high-speed,
high-resolution and long-term imaging of single viruses and quantitative study of
their interactions with cells and cellular environment [106]. iSCAT imaging of viral
interactions is still in its infancy and is somewhat slowed down by the difficulty of
integrating biosafe conditions into conventional optical laboratories, but it promises
to provide invaluable insight about the secrets of viruses, which contain strong inherent inhomogeneities in their structure and function.
2.4.1.4 Proteins
Proteins are omnipresent in our body, taking on critical roles in essentially every step
of our physiology. They are responsible for the function, structure, and regulation of
our organs and tissue, as well as performing many of the functions of the cell. For
47
for extinction measurements. This is why quenched molecules can be detected in
extinction but not in fluorescence.
Gold nanoparticles, quantum dots and dye molecules all have resonances, with
their polarizability experiencing a maximum in a certain spectral range. While this
enhances the extinction cross section, and thus the iSCAT signal, it is not nearly as
decisive as the influence of the particle size. A consequence is that even a dielectric
nanoparticle can yield a large interferometric signal given it is of sufficient size. In
what follows, we show that all viruses and a vast range of proteins easily satisfy this
criterion, prompting efforts in their detection.
2.4.1.3 Viruses
Viruses play a crucial role in biology, whether causing harmful diseases or performing an integral symbiotic function within living systems [157] or even serving as
novel disease treatment vectors [158]. Detection of viruses, as with most other cell
biological entities, has traditionally been performed in fluorescence. However, it turns
out that viruses and virus-like particles such as X31 virus, H1N1, Zika, Ebola, and
SV40, which could range from dimensions of about 20 nm to beyond 200 nm [159],
can be easily detected via iSCAT. This has been demonstrated in environments such
as microfluidic channels [160, 161], on synthetic lipid bilayers (see Fig. 2.11a) [115,
116], and on dielectric substrates [77, 83]. Simultaneous iSCAT and fluorescence
tracking of a virus and a quantum dot on its surface made it possible to visualize
not only the nanoscopic binding domains on the membrane but also the rocking and
tumbling motion of single viruses (see Fig. 2.11a) [116]. Moreover, iSCAT has been
used to uncover to nanometer precision the position and orientational configuration
of bacteriophages interacting with a surface as well as to resolve, to a precision of
4200 basepairs, the kinetics of DNA ejection following stimulation [162]—shown
in Fig. 2.11b. These activities have also motivated attempts to detect pure scattering
of single viruses in conventional dark-field [139] and through light scattering from
a nanofluidic channel [163].
The large iSCAT signal of most viruses opens the way to label-free high-speed,
high-resolution and long-term imaging of single viruses and quantitative study of
their interactions with cells and cellular environment [106]. iSCAT imaging of viral
interactions is still in its infancy and is somewhat slowed down by the difficulty of
integrating biosafe conditions into conventional optical laboratories, but it promises
to provide invaluable insight about the secrets of viruses, which contain strong inherent inhomogeneities in their structure and function.
2.4.1.4 Proteins
Proteins are omnipresent in our body, taking on critical roles in essentially every step
of our physiology. They are responsible for the function, structure, and regulation of
our organs and tissue, as well as performing many of the functions of the cell. For