3 Label-Free, Ultrahigh-Speed, Direct Imaging and Tracking …
81
fluorescence labeling and imaging can identify nanoparticles of interest, and then
COBRI can be employed for long-term, high-resolution observation and tracking
of the targeted particles. This approach makes optimal use of the fluorescence photon budget and thus prolongs the observation time. When combining COBRI with
fluorescence superresolution microscopy [55], the nanoparticle trajectory can be
superimposed on a super-resolved molecular map, providing direct visualization of
the local interplay between the nanoparticle and molecules.
COBRI microscopy is more sensitive than conventional brightfield microscopy,
but what is the ultimate detection limit? As outlined in Sect. 3.2, the sensitivity of
COBRI is shot-noise limited, and thus the number of detected signal photons directly
determines whether the target can be perceived. For extremely small nanoparticles,
the scattering cross section is very small, and therefore it requires high illumination
intensity and long signal integration time to detect them. Conceptually, given sufficient illumination intensity or signal integration time, arbitrarily small nanoparticles
can be detected. In practice, illumination intensity is restricted by the damage threshold of the sample and optics, whereas the signal integration time is limited by the
signal and background stability that is required for proper background correction. It
is worth noting that detecting small signal under strong illumination is complicated
by the saturation of photodetector—the full well capacity of the camera sets the maximal light intensity it can detect, determining the strongest illumination intensity of
the sample in COBRI and other common-path interferometry. To further increase
the illumination intensity without saturation of the camera, pupil function engineering has been employed to selectively attenuate the reference beam and leave the
signal of nanoparticle nearly untouched [35, 52, 56–58]. This approach effectively
enhances the contrast of the signal by matching the amplitude of the reference and
the signal. Through pupil function engineering and image postprocessing, COBRI
microscopy detects very small nanoparticles (as small as 10 nm gold nanoparticles)
at 1000 fps [35]. By similar approaches of contrast enhancement, ultrahigh sensitivity is achieved by iSCAT microscopy where single protein macromolecules are
detected without any labels at an effective image acquisition rate of a few hertz [51,
52, 59, 60].
With enhanced sensitivity, live cells are expected to be highly dynamic and
heterogeneous under COBRI microscopy because cell molecules are in constant
motion. The spatial resolution of COBRI is diffraction limited, which means that
it cannot resolve individual molecules densely packed in live cells. Nevertheless,
the spatially resolved fluctuation of the COBRI signal contains information regarding local molecule dynamics that may be revealed by correlation spectroscopy [61,
62]. The high temporal resolution of COBRI microscopy enables investigation of cell
molecular dynamics in microsecond timescales. Further investigation could examine
whether diffusion, self-assembly, transportation, or even new dynamics are resolvable in a label-free manner.
Light doses are a concern for live cell imaging because of photo-toxicity [47, 63].
To minimize the light dose, ultrahigh-speed COBRI image recording at 100,000 fps
is only performed for a few seconds at an illumination intensity of 1 kW/cm
2 . Otherwise, continuous observation and sample exploration are conducted with a separate
81
fluorescence labeling and imaging can identify nanoparticles of interest, and then
COBRI can be employed for long-term, high-resolution observation and tracking
of the targeted particles. This approach makes optimal use of the fluorescence photon budget and thus prolongs the observation time. When combining COBRI with
fluorescence superresolution microscopy [55], the nanoparticle trajectory can be
superimposed on a super-resolved molecular map, providing direct visualization of
the local interplay between the nanoparticle and molecules.
COBRI microscopy is more sensitive than conventional brightfield microscopy,
but what is the ultimate detection limit? As outlined in Sect. 3.2, the sensitivity of
COBRI is shot-noise limited, and thus the number of detected signal photons directly
determines whether the target can be perceived. For extremely small nanoparticles,
the scattering cross section is very small, and therefore it requires high illumination
intensity and long signal integration time to detect them. Conceptually, given sufficient illumination intensity or signal integration time, arbitrarily small nanoparticles
can be detected. In practice, illumination intensity is restricted by the damage threshold of the sample and optics, whereas the signal integration time is limited by the
signal and background stability that is required for proper background correction. It
is worth noting that detecting small signal under strong illumination is complicated
by the saturation of photodetector—the full well capacity of the camera sets the maximal light intensity it can detect, determining the strongest illumination intensity of
the sample in COBRI and other common-path interferometry. To further increase
the illumination intensity without saturation of the camera, pupil function engineering has been employed to selectively attenuate the reference beam and leave the
signal of nanoparticle nearly untouched [35, 52, 56–58]. This approach effectively
enhances the contrast of the signal by matching the amplitude of the reference and
the signal. Through pupil function engineering and image postprocessing, COBRI
microscopy detects very small nanoparticles (as small as 10 nm gold nanoparticles)
at 1000 fps [35]. By similar approaches of contrast enhancement, ultrahigh sensitivity is achieved by iSCAT microscopy where single protein macromolecules are
detected without any labels at an effective image acquisition rate of a few hertz [51,
52, 59, 60].
With enhanced sensitivity, live cells are expected to be highly dynamic and
heterogeneous under COBRI microscopy because cell molecules are in constant
motion. The spatial resolution of COBRI is diffraction limited, which means that
it cannot resolve individual molecules densely packed in live cells. Nevertheless,
the spatially resolved fluctuation of the COBRI signal contains information regarding local molecule dynamics that may be revealed by correlation spectroscopy [61,
62]. The high temporal resolution of COBRI microscopy enables investigation of cell
molecular dynamics in microsecond timescales. Further investigation could examine
whether diffusion, self-assembly, transportation, or even new dynamics are resolvable in a label-free manner.
Light doses are a concern for live cell imaging because of photo-toxicity [47, 63].
To minimize the light dose, ultrahigh-speed COBRI image recording at 100,000 fps
is only performed for a few seconds at an illumination intensity of 1 kW/cm
2 . Otherwise, continuous observation and sample exploration are conducted with a separate