8 Super-Resolution Imaging in Raman Microscopy
203
Rieger et al. experimentally demonstrated the signal depletion by UV irradiation
at tris(bipyridine)ruthenium(ii). They also theoretically explained the Raman suppression effect by using rate equations and density matrix calculations [27, 28].
As introduced above, using the STED technique and higher-order nonlinearities
in material–light interaction can improve the spatial resolution beyond the diffraction
limit. However, these techniques often require a high intensity of laser light in order
to induce the higher-order nonlinearity and deleting Raman signal. In practice, this
is a significant limitation of the technique for being applied to organic and biological
samples. However, the techniques can be useful for imaging sample with a relatively higher damaging threshold such as inorganic materials, which still can claim
a benefit of super-resolution imaging since Raman microscopy can provide material
information and analysis in addition to their spatial resolutions without labeling.
8.2.2 Linear Techniques
There are also a few demonstrations for improvement of the spatial resolution of
Raman microscopy without inducing nonlinear effects in Raman scattering. One
simple approach is using a solid immersion lens in order to increase the numerical
aperture for sample illumination and signal detection [29]. It is also possible to use
the 4pi configuration to increase the axial resolution, which has also been developed
for fluorescence microscopy [30]. Tormo et al. demonstrated the use of two objective
lenses to irradiate a sample by two coherent laser spots. The interference fringe along
the optical axis gives narrower excitation PSF in the axial direction. Separation of
thin layers of PMMA, TiO 2 , and ARP of thicknesses 43, 23, and 65 nm, respectively,
has been demonstrated [31].
Image scanning microscopy (ISM) is also a technique to improve the spatial
resolution in linear imaging. The technique was first described by Sheppard in 1988 as
super-resolution technique for confocal microscopy using a pinhole placed at off-axis
positions [32]. Later, Müller et al. introduced this technique as ISM in laser scanning
fluorescence microscopy by using a 2D detector [33]. This approach can be used
for spontaneous Raman microscopy since it shares the same imaging property with
fluorescence microscopy. Roider et al. successfully implemented the ISM approach
using a fiber bundle as a 2D detector coupled with a spectrometer [34]. The spatially
segmented detection of Raman spectra improves the spatial resolution in confocal
Raman imaging without sacrificing the signal amount as shown in Fig. 8.6. ISM
has different implementations, such as pixel reassignment [35], instant SIM [36],
and rescan [37, 38], which can also be combined with different types of Raman
microscopy.
203
Rieger et al. experimentally demonstrated the signal depletion by UV irradiation
at tris(bipyridine)ruthenium(ii). They also theoretically explained the Raman suppression effect by using rate equations and density matrix calculations [27, 28].
As introduced above, using the STED technique and higher-order nonlinearities
in material–light interaction can improve the spatial resolution beyond the diffraction
limit. However, these techniques often require a high intensity of laser light in order
to induce the higher-order nonlinearity and deleting Raman signal. In practice, this
is a significant limitation of the technique for being applied to organic and biological
samples. However, the techniques can be useful for imaging sample with a relatively higher damaging threshold such as inorganic materials, which still can claim
a benefit of super-resolution imaging since Raman microscopy can provide material
information and analysis in addition to their spatial resolutions without labeling.
8.2.2 Linear Techniques
There are also a few demonstrations for improvement of the spatial resolution of
Raman microscopy without inducing nonlinear effects in Raman scattering. One
simple approach is using a solid immersion lens in order to increase the numerical
aperture for sample illumination and signal detection [29]. It is also possible to use
the 4pi configuration to increase the axial resolution, which has also been developed
for fluorescence microscopy [30]. Tormo et al. demonstrated the use of two objective
lenses to irradiate a sample by two coherent laser spots. The interference fringe along
the optical axis gives narrower excitation PSF in the axial direction. Separation of
thin layers of PMMA, TiO 2 , and ARP of thicknesses 43, 23, and 65 nm, respectively,
has been demonstrated [31].
Image scanning microscopy (ISM) is also a technique to improve the spatial
resolution in linear imaging. The technique was first described by Sheppard in 1988 as
super-resolution technique for confocal microscopy using a pinhole placed at off-axis
positions [32]. Later, Müller et al. introduced this technique as ISM in laser scanning
fluorescence microscopy by using a 2D detector [33]. This approach can be used
for spontaneous Raman microscopy since it shares the same imaging property with
fluorescence microscopy. Roider et al. successfully implemented the ISM approach
using a fiber bundle as a 2D detector coupled with a spectrometer [34]. The spatially
segmented detection of Raman spectra improves the spatial resolution in confocal
Raman imaging without sacrificing the signal amount as shown in Fig. 8.6. ISM
has different implementations, such as pixel reassignment [35], instant SIM [36],
and rescan [37, 38], which can also be combined with different types of Raman
microscopy.