2 Interferometric Scattering (iSCAT) Microscopy and Related Techniques
39
We remark that the background subtraction methods discussed above could also be
equally well applied to dark-field microscopy [139]. Thus, one might wonder whether
dark-field microscopy might not match the performance of iSCAT microscopy.
Indeed, one can show that the shot-noise-limited advantage of interferometric (homodyne) over dark-field (scattering intensity) detection is only a factor of two. The
practical implementation of ultrasensitive dark-field, however, is nontrivial because
the very small dark-field signal proportional to the sixth power of the particle size
(see (2.1)) puts higher demands on the detector technology.
2.3.4 Long Measurements: Indefinite Photostability
One of the key advantages of detecting scattering instead of fluorescence is that the
former does not suffer from photobleaching. Whether one uses the inherent scattering
of a bioparticle such as a virus or if one detects a GNP label, the scattering signal
does not degrade over time. Thus, very long—in principle indefinite—measurements
become possible. Such experiments might encounter technical difficulties such as the
particle moving out of the field of view, but these can be easily overcome with more
sophisticated instrumentation.
2.3.5 Fast Measurements: No Saturation
Another favorable feature of scattering contrast as compared to fluorescence is lack
of saturation. A fluorophore is a quantum mechanical system with an inherent anharmonicity, implying that only one photon at a time can be absorbed. The fluorescence
lifetime of the excited-state places a limit on how fast the photon can be emitted,
imposing a bottleneck for the rate at which the fluorophore can radiate, and thus a
limit to how fast one can image. A nanoparticle behaves like a classical oscillating
dipole which does not suffer from saturation: the stronger the illumination, the higher
the rate of scattering. Indeed, iSCAT imaging speeds up to about 1 MHz has been
demonstrated [118]. The immediate limitation is currently a technological matter of
availability of suitable cameras. A more fundamental limit is introduced when concerned over photodamage of the sample when using very strong illuminations since
every realistic substance also absorbs light at every wavelength, even if very weakly.
When performing iSCAT on biological samples, illumination intensities in the order
of 0.001–0.1 mWµm
−2 have been reported for membranes [118, 138], with powers
as high as 5 mWµm
−2 [118] for the fastest MHz imaging rates.
39
We remark that the background subtraction methods discussed above could also be
equally well applied to dark-field microscopy [139]. Thus, one might wonder whether
dark-field microscopy might not match the performance of iSCAT microscopy.
Indeed, one can show that the shot-noise-limited advantage of interferometric (homodyne) over dark-field (scattering intensity) detection is only a factor of two. The
practical implementation of ultrasensitive dark-field, however, is nontrivial because
the very small dark-field signal proportional to the sixth power of the particle size
(see (2.1)) puts higher demands on the detector technology.
2.3.4 Long Measurements: Indefinite Photostability
One of the key advantages of detecting scattering instead of fluorescence is that the
former does not suffer from photobleaching. Whether one uses the inherent scattering
of a bioparticle such as a virus or if one detects a GNP label, the scattering signal
does not degrade over time. Thus, very long—in principle indefinite—measurements
become possible. Such experiments might encounter technical difficulties such as the
particle moving out of the field of view, but these can be easily overcome with more
sophisticated instrumentation.
2.3.5 Fast Measurements: No Saturation
Another favorable feature of scattering contrast as compared to fluorescence is lack
of saturation. A fluorophore is a quantum mechanical system with an inherent anharmonicity, implying that only one photon at a time can be absorbed. The fluorescence
lifetime of the excited-state places a limit on how fast the photon can be emitted,
imposing a bottleneck for the rate at which the fluorophore can radiate, and thus a
limit to how fast one can image. A nanoparticle behaves like a classical oscillating
dipole which does not suffer from saturation: the stronger the illumination, the higher
the rate of scattering. Indeed, iSCAT imaging speeds up to about 1 MHz has been
demonstrated [118]. The immediate limitation is currently a technological matter of
availability of suitable cameras. A more fundamental limit is introduced when concerned over photodamage of the sample when using very strong illuminations since
every realistic substance also absorbs light at every wavelength, even if very weakly.
When performing iSCAT on biological samples, illumination intensities in the order
of 0.001–0.1 mWµm
−2 have been reported for membranes [118, 138], with powers
as high as 5 mWµm
−2 [118] for the fastest MHz imaging rates.