8 Super-Resolution Imaging in Raman Microscopy
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peak of the laser spot becomes sharper in return for a significant increase of side
lobes. However, in their CARS microscopy, the side lobes are not visible in resultant
CARS images, thanks to the multiplicative interaction of pump and Stokes beams to
generate CARS signal. Although the technique can provide a sharper main peak in
the image of a point object, the bandwidth for transferring spatial frequency is still
the same as CARS microscopy uses the Gaussian beams. The technique enhances
the efficiency of transferring components with relatively high spatial frequencies and
does not provide the expansion of the theoretical bandwidth. However, the technique
is effective since laser scanning microscopy using a Gaussian spot typically has
the low-pass characteristic, where the practical resolution is strongly affected by
the noise in the detected signal. The enhancement of the transfer efficiency of high
spatial frequency components effectively brings up the fine sample structures above
the noise level and makes them visible in the resultant image.
In order to achieve the resolving power beyond the bandwidth that is determined
by the Raman excitation volume, the strategies used for super-resolution fluorescence
microscopy are also available. In laser scanning fluorescence microscopy, the area of
fluorescence detection in a sample is reduced by using a highly nonlinear interaction
between laser intensity and fluorescence emission.
Introducing saturation in optical effect is one of the ways to introduce high-order
nonlinear relations between light intensity and the Raman effect. As shown in Fig. 8.1,
higher-order nonlinear responses can be localized within a volume smaller than that
of a laser spot and improve the spatial resolution in laser scanning microscopy. This
approach is originally demonstrated for fluorescence imaging [18, 19] and also for
plasmonic scattering [20]. Since coherent Raman effects can also be saturated due
to the saturation of the population at vibrational excitation, the use of optical effect
can also be utilized to improve the spatial resolution of CARS microscopy further.
Yonemaru et al. used the harmonic demodulation technique to extract the higherorder nonlinear CARS signal (Fig. 8.3) and demonstrated super-resolution imaging of
diamond nanoparticles [21]. This technique improves the spectral resolution in CARS
spectroscopy. This is because the saturation is seen prominently when the difference
between the frequencies of pump and Stokes beams matches with the vibrational
frequency of molecules to be excited. For the same reason, it is also possible to
reduce the effect of non-resonant background that gives image contrasts unrelated to
vibrational excitation in CARS imaging. This technique was also demonstrated for
improving the spatial resolution in SRS microscopy [22].
Another strategy is to use a laser beam for controlling the Raman effect, which
is similar to the technique used in STED microscopy [2]. In STED microscopy
for fluorescence imaging, a fluorescent sample is excited by a Gaussian laser spot.
In addition, in order to reduce the detection volume, another laser beam, called
a STED beam, is irradiated to the sample collinearly to the excitation beam. The
STED beam is in the shape of a donut and induces stimulated emission from the
sample, which depletes the fluorescence signal by spontaneous emission. Since the
efficiency of stimulated emission is saturated with a strong STED beam, the area
of stimulated emission becomes larger, while the spontaneous emission remains at
the center of excitation spot due to zero intensity of the STED beam. Therefore, the
199
peak of the laser spot becomes sharper in return for a significant increase of side
lobes. However, in their CARS microscopy, the side lobes are not visible in resultant
CARS images, thanks to the multiplicative interaction of pump and Stokes beams to
generate CARS signal. Although the technique can provide a sharper main peak in
the image of a point object, the bandwidth for transferring spatial frequency is still
the same as CARS microscopy uses the Gaussian beams. The technique enhances
the efficiency of transferring components with relatively high spatial frequencies and
does not provide the expansion of the theoretical bandwidth. However, the technique
is effective since laser scanning microscopy using a Gaussian spot typically has
the low-pass characteristic, where the practical resolution is strongly affected by
the noise in the detected signal. The enhancement of the transfer efficiency of high
spatial frequency components effectively brings up the fine sample structures above
the noise level and makes them visible in the resultant image.
In order to achieve the resolving power beyond the bandwidth that is determined
by the Raman excitation volume, the strategies used for super-resolution fluorescence
microscopy are also available. In laser scanning fluorescence microscopy, the area of
fluorescence detection in a sample is reduced by using a highly nonlinear interaction
between laser intensity and fluorescence emission.
Introducing saturation in optical effect is one of the ways to introduce high-order
nonlinear relations between light intensity and the Raman effect. As shown in Fig. 8.1,
higher-order nonlinear responses can be localized within a volume smaller than that
of a laser spot and improve the spatial resolution in laser scanning microscopy. This
approach is originally demonstrated for fluorescence imaging [18, 19] and also for
plasmonic scattering [20]. Since coherent Raman effects can also be saturated due
to the saturation of the population at vibrational excitation, the use of optical effect
can also be utilized to improve the spatial resolution of CARS microscopy further.
Yonemaru et al. used the harmonic demodulation technique to extract the higherorder nonlinear CARS signal (Fig. 8.3) and demonstrated super-resolution imaging of
diamond nanoparticles [21]. This technique improves the spectral resolution in CARS
spectroscopy. This is because the saturation is seen prominently when the difference
between the frequencies of pump and Stokes beams matches with the vibrational
frequency of molecules to be excited. For the same reason, it is also possible to
reduce the effect of non-resonant background that gives image contrasts unrelated to
vibrational excitation in CARS imaging. This technique was also demonstrated for
improving the spatial resolution in SRS microscopy [22].
Another strategy is to use a laser beam for controlling the Raman effect, which
is similar to the technique used in STED microscopy [2]. In STED microscopy
for fluorescence imaging, a fluorescent sample is excited by a Gaussian laser spot.
In addition, in order to reduce the detection volume, another laser beam, called
a STED beam, is irradiated to the sample collinearly to the excitation beam. The
STED beam is in the shape of a donut and induces stimulated emission from the
sample, which depletes the fluorescence signal by spontaneous emission. Since the
efficiency of stimulated emission is saturated with a strong STED beam, the area
of stimulated emission becomes larger, while the spontaneous emission remains at
the center of excitation spot due to zero intensity of the STED beam. Therefore, the