16 Theoretical Foundations of Super-Resolution in Microspherical Nanoscopy
423
incidence or other conditions allow resolving much finer features, but this type of
imaging should not be confused with the classical resolution defined for incoherent
point sources.
It is important to note that although the enhancement of the classical resolution
offered by high-index (n = 2) microspheres can be viewed as modest, there are many
straightforward techniques to increase the resolution much further by combining MN
with a series of ideas and methods developed for increasing the resolution in conventional microscopy. As we discussed above, the MN drawback related to its limited
field of view can be solved by developing the surface scanning technologies. On the
other hand, for each fixed position of dielectric microsphere MN represents a simplest widefield and white light imaging technique similar to standard microscopy.
Another limitation in comparison with the standard microscopy is the fact that MN
works for imaging objects located at ∼λ/2 proximity to the surface of the spheres.
This condition can be realized by reducing the thickness of the samples (by pressing slightly the spheres or slabs with embedded spheres into the substrate). In the
case of thick samples, it can be achieved by depositing the investigated biomedical
samples directly on the surface of the microspheres. This can be viewed as a disadvantage or a “price to pay” in comparison with the convenience of conventional
microscopy. However, from the pure imaging point of view, MN has all other advantages of conventional microscopy including parallel acquisition with a low-intensity
level not damaging for the biomedical objects. Another advantage is related with its
millisecond dynamical range enabling real-time study of dynamical processes. Most
importantly, it opens a possibility to increase the MN resolution further by combining this technique with the whole series of label-free super-resolution imaging
techniques developed in standard microscopy.
The use of nanostructured plasmonic objects represent the first step on this path,
and from the very beginning MN was applied for imaging nanoholes fabricated in
metal sheets, plasmonic dimers, etc., that resulted in resolution values ∼λ/7 which
far exceeded the resolution limits determined by the classical resolution theory developed for incoherent point sources. The reasons for increased resolution of nanoplasmonic objects include stronger near fields with much larger k-vectors which are
intrinsic for plasmons compared to photons in air, especially in the case of resonant excitation of localized surface plasmon resonances. Another important factor is
connected with a coherent excitation of out-of-phase plasmonic oscillations in such
structures which can contribute to the observation of subdiffraction features.
The use of confocal microscopy represents the next step which was taken at the
initial stage of developing MN [33, 45]. Due to the effect of the confocal pinhole,
it allowed to increase the optical contrast and signal-to-noise ratio of the images
obtained through the microspheres which was an important factor in improving the
experimental quantification of resolution in this method. Initially, the resolution of
confocal microscopy was overestimated at ∼λ/17 level [45], however eventually
the resolution ∼λ/7 was demonstrated using convolution with PSF for imaging
nanoplasmonic structures. Another technique was based on nonlinear reduction of
PSF using MN integration with two-photon photoluminescence [85]. This method
was used for imaging dimer gap region where the resolution of ∼λ/7 was demon-
423
incidence or other conditions allow resolving much finer features, but this type of
imaging should not be confused with the classical resolution defined for incoherent
point sources.
It is important to note that although the enhancement of the classical resolution
offered by high-index (n = 2) microspheres can be viewed as modest, there are many
straightforward techniques to increase the resolution much further by combining MN
with a series of ideas and methods developed for increasing the resolution in conventional microscopy. As we discussed above, the MN drawback related to its limited
field of view can be solved by developing the surface scanning technologies. On the
other hand, for each fixed position of dielectric microsphere MN represents a simplest widefield and white light imaging technique similar to standard microscopy.
Another limitation in comparison with the standard microscopy is the fact that MN
works for imaging objects located at ∼λ/2 proximity to the surface of the spheres.
This condition can be realized by reducing the thickness of the samples (by pressing slightly the spheres or slabs with embedded spheres into the substrate). In the
case of thick samples, it can be achieved by depositing the investigated biomedical
samples directly on the surface of the microspheres. This can be viewed as a disadvantage or a “price to pay” in comparison with the convenience of conventional
microscopy. However, from the pure imaging point of view, MN has all other advantages of conventional microscopy including parallel acquisition with a low-intensity
level not damaging for the biomedical objects. Another advantage is related with its
millisecond dynamical range enabling real-time study of dynamical processes. Most
importantly, it opens a possibility to increase the MN resolution further by combining this technique with the whole series of label-free super-resolution imaging
techniques developed in standard microscopy.
The use of nanostructured plasmonic objects represent the first step on this path,
and from the very beginning MN was applied for imaging nanoholes fabricated in
metal sheets, plasmonic dimers, etc., that resulted in resolution values ∼λ/7 which
far exceeded the resolution limits determined by the classical resolution theory developed for incoherent point sources. The reasons for increased resolution of nanoplasmonic objects include stronger near fields with much larger k-vectors which are
intrinsic for plasmons compared to photons in air, especially in the case of resonant excitation of localized surface plasmon resonances. Another important factor is
connected with a coherent excitation of out-of-phase plasmonic oscillations in such
structures which can contribute to the observation of subdiffraction features.
The use of confocal microscopy represents the next step which was taken at the
initial stage of developing MN [33, 45]. Due to the effect of the confocal pinhole,
it allowed to increase the optical contrast and signal-to-noise ratio of the images
obtained through the microspheres which was an important factor in improving the
experimental quantification of resolution in this method. Initially, the resolution of
confocal microscopy was overestimated at ∼λ/17 level [45], however eventually
the resolution ∼λ/7 was demonstrated using convolution with PSF for imaging
nanoplasmonic structures. Another technique was based on nonlinear reduction of
PSF using MN integration with two-photon photoluminescence [85]. This method
was used for imaging dimer gap region where the resolution of ∼λ/7 was demon-