10 Super-Resolution Imaging Based on Nonlinear Plasmonic Scattering
241
microscopy. It features an imaging modality in which a sample is illuminated from
the side perpendicular to the observation optic axis, by a beam engineered into a wide
and relatively thin sheet. SPIM offers an axial resolution better than confocal (2×),
wide-field (2.5×), and two-photon microscopy (3×), while the lateral resolution is
the same as wide-field [11].
All these far-field approaches such as laser scanning confocal, multi-photon
microscopy, SPIM, and so on have enhanced spatial resolution along lateral or
axial axes, but are still considered as diffraction limited. Generally speaking, superresolution microscopy techniques are defined as those capable of resolving structures
beyond the classical diffraction limit and can be mainly divided into two categories,
that is near-field and far-field. The examples of the former are near-field scanning
optical microscopy (NSOM) and total internal reflection fluorescence microscopy
(TIRFM). NSOM [12] uses fibre optic probe to funnel light to the nano-dimensions.
This is accomplished by focussing the excitation laser through an aperture having a
much smaller diameter than the excitation wavelength. This results in the formation
of evanescent waves on the other side of the aperture. NSOM achieves a resolution
of 20 nm in lateral and about 5 nm in axial, but suffers by the limitations such as
zero working distance, small field depth, long scan times, and so on. On the other
hand, TIRFM uses evanescent waves to excite fluorophores in a very small volume
of the specimen immediately adjacent to the glass-medium interface. These evanescent waves are generated only when the incident light is totally internally reflected
at the glass-medium interface. The evanescent EM field decays exponentially from
the interface, and thus limits axial imaging depth to about 100 nm, that is improving
the axial sectioning beyond diffraction [13].
For far-field super-resolution microscopy, Gustafsson [14] had explained the following three assumptions that limit light microscope resolution: (i) light for imaging
is collected by a single objective; (ii) the excitation light is uniform throughout the
sample; and (iii) fluorescence takes place through normal, linear absorption and emission of a single photon. The resolution limit can be challenged if somehow these
assumptions are wisely tackled either by using multiple objectives, non-uniform
illumination , or nonlinear absorption and emission. Below we introduce different
techniques that are explored in the last two decades towards advancement of superresolution microscopy based on the three limiting assumptions.
(a) Objectives modifications: In the early 1990s, Stefan Hell proposed that by
two opposing objectives, the collection aperture angle could be significantly
improved, up to maximally 4Pi, and thus could enhance axial resolution. The
technique is called 4Pi microscopy [15], wherein two face-to-face objectives,
above and below the sample, were used. Both objectives illuminate and collect
the emission from the same focal plane of the sample. The image is reconstructed
by superposition of signals collected from both excitation and emission optical
paths. This method achieves four times better axial resolution than CLFM. Later,
Gustafsson developed image interference microscopy (I
n M) [16], which used
incoherent light source and also, two objectives for illumination and emission,
wherein constructive interference between the excitation and emission light
241
microscopy. It features an imaging modality in which a sample is illuminated from
the side perpendicular to the observation optic axis, by a beam engineered into a wide
and relatively thin sheet. SPIM offers an axial resolution better than confocal (2×),
wide-field (2.5×), and two-photon microscopy (3×), while the lateral resolution is
the same as wide-field [11].
All these far-field approaches such as laser scanning confocal, multi-photon
microscopy, SPIM, and so on have enhanced spatial resolution along lateral or
axial axes, but are still considered as diffraction limited. Generally speaking, superresolution microscopy techniques are defined as those capable of resolving structures
beyond the classical diffraction limit and can be mainly divided into two categories,
that is near-field and far-field. The examples of the former are near-field scanning
optical microscopy (NSOM) and total internal reflection fluorescence microscopy
(TIRFM). NSOM [12] uses fibre optic probe to funnel light to the nano-dimensions.
This is accomplished by focussing the excitation laser through an aperture having a
much smaller diameter than the excitation wavelength. This results in the formation
of evanescent waves on the other side of the aperture. NSOM achieves a resolution
of 20 nm in lateral and about 5 nm in axial, but suffers by the limitations such as
zero working distance, small field depth, long scan times, and so on. On the other
hand, TIRFM uses evanescent waves to excite fluorophores in a very small volume
of the specimen immediately adjacent to the glass-medium interface. These evanescent waves are generated only when the incident light is totally internally reflected
at the glass-medium interface. The evanescent EM field decays exponentially from
the interface, and thus limits axial imaging depth to about 100 nm, that is improving
the axial sectioning beyond diffraction [13].
For far-field super-resolution microscopy, Gustafsson [14] had explained the following three assumptions that limit light microscope resolution: (i) light for imaging
is collected by a single objective; (ii) the excitation light is uniform throughout the
sample; and (iii) fluorescence takes place through normal, linear absorption and emission of a single photon. The resolution limit can be challenged if somehow these
assumptions are wisely tackled either by using multiple objectives, non-uniform
illumination , or nonlinear absorption and emission. Below we introduce different
techniques that are explored in the last two decades towards advancement of superresolution microscopy based on the three limiting assumptions.
(a) Objectives modifications: In the early 1990s, Stefan Hell proposed that by
two opposing objectives, the collection aperture angle could be significantly
improved, up to maximally 4Pi, and thus could enhance axial resolution. The
technique is called 4Pi microscopy [15], wherein two face-to-face objectives,
above and below the sample, were used. Both objectives illuminate and collect
the emission from the same focal plane of the sample. The image is reconstructed
by superposition of signals collected from both excitation and emission optical
paths. This method achieves four times better axial resolution than CLFM. Later,
Gustafsson developed image interference microscopy (I
n M) [16], which used
incoherent light source and also, two objectives for illumination and emission,
wherein constructive interference between the excitation and emission light