17 Microsphere-Assisted Interference Microscopy
459
point of view than DHM, PSM needs however several acquisition frames compared
with just one for DHM.
Other interference imaging techniques exist and also allow non-invasive and labelfree imaging to be performed with a high-axial sensitivity. Nevertheless, the theoretical lateral resolving power of interference microscopes is limited by the diffraction
of light [147] as in classical light microscopy. For example, the lateral resolution in
DHM cannot be better than the wavelength of the monochromatic light source λ or
λ/2 using oblique illumination [148]. In practice, a lateral resolution of λ is unfortunately never achieved. Indeed, the fabrication of interference objectives having an NA
superior to 0.6 is relatively complex. Furthermore, the use of high-numerical aperture
microscope objectives in an interferometer are not trivial due to the misalignments
of optical components which can occur, leading to wavefront errors. This restriction
yields a non-homogeneous spatial resolution along the transverse and axial dimensions, i.e. a non-homogeneous voxel size. While the remarkable nanometric axial
resolution of automated interference techniques has been recognized and appreciated by optical metrologists for over three decades, it has been frustrating to have to
put up with a lateral resolution that is over two orders of magnitude worse. The recent
idea of combining interference microscopy with super-resolution imaging aided by a
microsphere is thus perceived as a major breakthrough in providing a more uniform
3D resolution of samples.
17.3.2 Super-Resolution Interference Methods
The first paper on microsphere-assisted interference microscopy showing a potential increase in lateral resolution was reported using DHM, in 2015 [149]. It was
underlined that the super-resolution phenomenon is applicable not only to the amplitude image but also to the phase image. Moreover, a 90 µm-diameter polystyrene
microsphere acting as a near-field to far-field transforming optical element allows
the diffraction limit to be broken, passing from 1.38 to 1 µm. Here, the magnification
factor of the microsphere equals 2.3.
A few months later, F. Wang et al. demonstrated experimentally a concrete and
significant enhancement of the lateral resolution in 3D label-free super-resolution
imaging using microspheres [150]. Indeed, the lateral resolution was increased by
a factor of 2.3 (according to the Abbe criterion). Here, the interferometric method
used is CSI and the configuration consists of a Linnik interferometer using a white
light lamp and two 0.6-numerical-aperture objectives. Microspheres with different
diameters were placed in water (made of barium-titanate glass) and in air (made of
polystyrene). F. Wang et al. claim to discern features having a smallest size of 50 nm
(in reality, the highest lateral resolution achieved experimentally is around 200 nm)
while keeping 10 nm of axial sensitivity. The surface topography of complex-shaped
samples (e.g. gratings, nanodot array and structures of a CPU chip) was successfully
reconstructed as shown in Fig. 17.12. Furthermore, the influence of the illumination
conditions on the image contrast was reported.
459
point of view than DHM, PSM needs however several acquisition frames compared
with just one for DHM.
Other interference imaging techniques exist and also allow non-invasive and labelfree imaging to be performed with a high-axial sensitivity. Nevertheless, the theoretical lateral resolving power of interference microscopes is limited by the diffraction
of light [147] as in classical light microscopy. For example, the lateral resolution in
DHM cannot be better than the wavelength of the monochromatic light source λ or
λ/2 using oblique illumination [148]. In practice, a lateral resolution of λ is unfortunately never achieved. Indeed, the fabrication of interference objectives having an NA
superior to 0.6 is relatively complex. Furthermore, the use of high-numerical aperture
microscope objectives in an interferometer are not trivial due to the misalignments
of optical components which can occur, leading to wavefront errors. This restriction
yields a non-homogeneous spatial resolution along the transverse and axial dimensions, i.e. a non-homogeneous voxel size. While the remarkable nanometric axial
resolution of automated interference techniques has been recognized and appreciated by optical metrologists for over three decades, it has been frustrating to have to
put up with a lateral resolution that is over two orders of magnitude worse. The recent
idea of combining interference microscopy with super-resolution imaging aided by a
microsphere is thus perceived as a major breakthrough in providing a more uniform
3D resolution of samples.
17.3.2 Super-Resolution Interference Methods
The first paper on microsphere-assisted interference microscopy showing a potential increase in lateral resolution was reported using DHM, in 2015 [149]. It was
underlined that the super-resolution phenomenon is applicable not only to the amplitude image but also to the phase image. Moreover, a 90 µm-diameter polystyrene
microsphere acting as a near-field to far-field transforming optical element allows
the diffraction limit to be broken, passing from 1.38 to 1 µm. Here, the magnification
factor of the microsphere equals 2.3.
A few months later, F. Wang et al. demonstrated experimentally a concrete and
significant enhancement of the lateral resolution in 3D label-free super-resolution
imaging using microspheres [150]. Indeed, the lateral resolution was increased by
a factor of 2.3 (according to the Abbe criterion). Here, the interferometric method
used is CSI and the configuration consists of a Linnik interferometer using a white
light lamp and two 0.6-numerical-aperture objectives. Microspheres with different
diameters were placed in water (made of barium-titanate glass) and in air (made of
polystyrene). F. Wang et al. claim to discern features having a smallest size of 50 nm
(in reality, the highest lateral resolution achieved experimentally is around 200 nm)
while keeping 10 nm of axial sensitivity. The surface topography of complex-shaped
samples (e.g. gratings, nanodot array and structures of a CPU chip) was successfully
reconstructed as shown in Fig. 17.12. Furthermore, the influence of the illumination
conditions on the image contrast was reported.