8 Super-Resolution Imaging in Raman Microscopy
205
8.3 Structure Illumination Microscopy
Structure illumination microscopy (SIM) uses patterned illumination in order to shift
the spatial frequency of the sample structure to lower frequency [7]. As a result, the
information of structures with sub-diffraction-limited size can be transferred to the
imaging optics, and post-processing of the obtained images provides images with a
spatial resolution twice higher than that of conventional wide-field microscopy. For
fluorescence microscopy, SIM can be realized by modifying the illumination optics
using a conventional wide-field microscopy. Therefore, similar strategies have been
proposed for improving the spatial resolution in various types of Raman microscopy.
Typical SIM requires the use of a 2D detector to record images under structured
illumination. Therefore, optical imaging systems that image monochrome signal light
can be easily combined with SIM. Since in typical CARS microscopy, the signal is
given with a wavelength determined by the wavelengths of pump and Stokes beams,
the structured illumination technique can be implemented in a wide-field CARS
microscopy [39]. Hajek et al. proposed the introduction of structured illumination into
a wide-field CARS microscope [40] and theoretically described that the structured
illumination can improve the spatial resolution about three times compared to that
with wide-field CARS microscopy using uniform illumination. CARS-SIM using
2D has also been proposed [41]. Although CARS microscopy is a coherent imaging
technique, the signal is given as a product of pump and Stokes beams. This nonlinear
process in signal generation can shift the spatial frequency of sample structured to
lower frequency, allowing the wide-field imaging optics to resolve the sub-diffraction
limit structures, which is different from SIM using linear coherent signals [42]. The
cut-off frequency of CARS-SIM can be equivalent to a typical spot-scanning CARS
microscopy. The wide-field configuration can have a higher contrast in imaging
fine structures due to the illumination pattern that only contains the high spatial
frequency. However, the wide-field CARS configuration requires high intensity to
produce CARS process in the entire region of view area, and therefore the practical
implementation of CARS-SIM is limited by available laser sources.
SIM can be combined easily with wide-field Raman microscopy using a narrowband filter for spectral separation. The structured illumination is produced by an
interference of two parallel beams similar to typical fluorescence SIM. The narrowband filter transmits Raman scattering light of interest to produce a Raman image on a
2D detector. Chen et al. demonstrated the resolution improvement by using surfaceenhanced Raman scattering to observe the distribution of SERS nanoparticles on
glass substrate and in a living cell with a lateral resolution of 109 nm.
The structured illumination technique can be combined with line illumination
Raman microscopy to obtain the benefits of both high spatial resolution and analytical capability [43]. In line illumination Raman microscopy, a sample is irradiated
by a lined-shaped focus with which Raman scattering under the illumination line is
detected simultaneously by using a 2D sensor [44, 45]. The scanning of line illuminated in the perpendicular direction produces hyper spectral Raman images of the
sample. The line illumination microscope has the same imaging property as wide-
205
8.3 Structure Illumination Microscopy
Structure illumination microscopy (SIM) uses patterned illumination in order to shift
the spatial frequency of the sample structure to lower frequency [7]. As a result, the
information of structures with sub-diffraction-limited size can be transferred to the
imaging optics, and post-processing of the obtained images provides images with a
spatial resolution twice higher than that of conventional wide-field microscopy. For
fluorescence microscopy, SIM can be realized by modifying the illumination optics
using a conventional wide-field microscopy. Therefore, similar strategies have been
proposed for improving the spatial resolution in various types of Raman microscopy.
Typical SIM requires the use of a 2D detector to record images under structured
illumination. Therefore, optical imaging systems that image monochrome signal light
can be easily combined with SIM. Since in typical CARS microscopy, the signal is
given with a wavelength determined by the wavelengths of pump and Stokes beams,
the structured illumination technique can be implemented in a wide-field CARS
microscopy [39]. Hajek et al. proposed the introduction of structured illumination into
a wide-field CARS microscope [40] and theoretically described that the structured
illumination can improve the spatial resolution about three times compared to that
with wide-field CARS microscopy using uniform illumination. CARS-SIM using
2D has also been proposed [41]. Although CARS microscopy is a coherent imaging
technique, the signal is given as a product of pump and Stokes beams. This nonlinear
process in signal generation can shift the spatial frequency of sample structured to
lower frequency, allowing the wide-field imaging optics to resolve the sub-diffraction
limit structures, which is different from SIM using linear coherent signals [42]. The
cut-off frequency of CARS-SIM can be equivalent to a typical spot-scanning CARS
microscopy. The wide-field configuration can have a higher contrast in imaging
fine structures due to the illumination pattern that only contains the high spatial
frequency. However, the wide-field CARS configuration requires high intensity to
produce CARS process in the entire region of view area, and therefore the practical
implementation of CARS-SIM is limited by available laser sources.
SIM can be combined easily with wide-field Raman microscopy using a narrowband filter for spectral separation. The structured illumination is produced by an
interference of two parallel beams similar to typical fluorescence SIM. The narrowband filter transmits Raman scattering light of interest to produce a Raman image on a
2D detector. Chen et al. demonstrated the resolution improvement by using surfaceenhanced Raman scattering to observe the distribution of SERS nanoparticles on
glass substrate and in a living cell with a lateral resolution of 109 nm.
The structured illumination technique can be combined with line illumination
Raman microscopy to obtain the benefits of both high spatial resolution and analytical capability [43]. In line illumination Raman microscopy, a sample is irradiated
by a lined-shaped focus with which Raman scattering under the illumination line is
detected simultaneously by using a 2D sensor [44, 45]. The scanning of line illuminated in the perpendicular direction produces hyper spectral Raman images of the
sample. The line illumination microscope has the same imaging property as wide-