74
C.-L. Hsieh
intensity [35]. Different designs of the reference beam, however, lead to several distinctions between COBRI and iSCAT microscopy. First, COBRI and iSCAT have
different axial dependencies of particle contrast. In COBRI, when the particle moves
in the axial direction, its scattered field has a different phase evolution from the transmitted reference beam, leading to a full contrast inversion over the depth of field of
the microscope objective (typically around 1 μm for high NA microscope objective,
see Fig. 3.3a). In iSCAT, the reflection geometry makes the particle contrast more
sensitive to its axial position relative to the substrate from which the reflected reference beam is created. A full modulation of iSCAT contrast of a particle occurs over a
distance of 1/4 optical wavelength, and thus the iSCAT contrast inverts several times
over the depth of field [45]. Contrast inversion interrupts continuous imaging and
tracking of nanoparticle in the axial direction because the detection of nanoparticle
fails when the contrast is zero. As a result, COBRI has a larger working range in the
axial direction for continuous imaging and tracking, while iSCAT has a sharper axial
dependency of the particle contrast. Second, COBRI imaging is nearly insensitive to
the presence of semi-transparent interfaces in the sample because of its transmission
geometry. On the contrary, iSCAT detects weak reflections from those interfaces easily, and this property has been exploited to image the flatness of live cell membranes
at the nanoscale [34]. However, the sensitive interface detection of iSCAT could complicate the observation of small nanoparticles in live cells due to the highly dynamic
and random reflection from the cell membranes. Third, a beamsplitter is inevitably
needed in the reflection geometry of iSCAT, which not only increases the complexity
of optical alignment but also complicates signal collection at a high efficiency [46].
On the other hand, COBRI does not need a beamsplitter, offering higher signal collection efficiency in principle. Finally, in transmission geometry, COBRI can easily
be operated at low light intensity, typically 100-fold lower than the intensity at which
iSCAT can be operated under identical image acquisition conditions. For example,
COBRI works at an illumination intensity of 0.01 kW/cm
2 with an image acquisition
rate of 1000 fps, whereas iSCAT requires 1 kW/cm
2 correspondingly. The low operational intensity of COBRI makes it useful for live cell imaging where the light dose
is of a critical concern [47]. Increasing the illumination intensity in COBRI, and thus
its detection sensitivity, is possible through pupil function engineering. By inserting
a dot-shaped attenuator at the back focal plane of the microscope objective in the
detection path, the illumination intensity of COBRI can reach up to 150 kW/cm
2 at
1000 fps where single 10 nm gold nanoparticles are detected [35]. Therefore, COBRI
microscopy potentially covers a wider range of illumination intensities, providing a
large tunable range of sensitivity for different applications.
3.3 Scattering Background Estimation and Correction
Obtaining a high-quality micrograph where the object of interest (e.g., a nanoparticle) is clearly seen is the goal of microscope imaging. Scattering-based label-free
imaging is complicated by the random scattering background because any imperfect
C.-L. Hsieh
intensity [35]. Different designs of the reference beam, however, lead to several distinctions between COBRI and iSCAT microscopy. First, COBRI and iSCAT have
different axial dependencies of particle contrast. In COBRI, when the particle moves
in the axial direction, its scattered field has a different phase evolution from the transmitted reference beam, leading to a full contrast inversion over the depth of field of
the microscope objective (typically around 1 μm for high NA microscope objective,
see Fig. 3.3a). In iSCAT, the reflection geometry makes the particle contrast more
sensitive to its axial position relative to the substrate from which the reflected reference beam is created. A full modulation of iSCAT contrast of a particle occurs over a
distance of 1/4 optical wavelength, and thus the iSCAT contrast inverts several times
over the depth of field [45]. Contrast inversion interrupts continuous imaging and
tracking of nanoparticle in the axial direction because the detection of nanoparticle
fails when the contrast is zero. As a result, COBRI has a larger working range in the
axial direction for continuous imaging and tracking, while iSCAT has a sharper axial
dependency of the particle contrast. Second, COBRI imaging is nearly insensitive to
the presence of semi-transparent interfaces in the sample because of its transmission
geometry. On the contrary, iSCAT detects weak reflections from those interfaces easily, and this property has been exploited to image the flatness of live cell membranes
at the nanoscale [34]. However, the sensitive interface detection of iSCAT could complicate the observation of small nanoparticles in live cells due to the highly dynamic
and random reflection from the cell membranes. Third, a beamsplitter is inevitably
needed in the reflection geometry of iSCAT, which not only increases the complexity
of optical alignment but also complicates signal collection at a high efficiency [46].
On the other hand, COBRI does not need a beamsplitter, offering higher signal collection efficiency in principle. Finally, in transmission geometry, COBRI can easily
be operated at low light intensity, typically 100-fold lower than the intensity at which
iSCAT can be operated under identical image acquisition conditions. For example,
COBRI works at an illumination intensity of 0.01 kW/cm
2 with an image acquisition
rate of 1000 fps, whereas iSCAT requires 1 kW/cm
2 correspondingly. The low operational intensity of COBRI makes it useful for live cell imaging where the light dose
is of a critical concern [47]. Increasing the illumination intensity in COBRI, and thus
its detection sensitivity, is possible through pupil function engineering. By inserting
a dot-shaped attenuator at the back focal plane of the microscope objective in the
detection path, the illumination intensity of COBRI can reach up to 150 kW/cm
2 at
1000 fps where single 10 nm gold nanoparticles are detected [35]. Therefore, COBRI
microscopy potentially covers a wider range of illumination intensities, providing a
large tunable range of sensitivity for different applications.
3.3 Scattering Background Estimation and Correction
Obtaining a high-quality micrograph where the object of interest (e.g., a nanoparticle) is clearly seen is the goal of microscope imaging. Scattering-based label-free
imaging is complicated by the random scattering background because any imperfect