6 Absorption-Based Far-Field Label-Free Super-Resolution …
143
in possibilities of beating the diffraction limit of the pump beam by manipulating
the probe beam. Based on the preceding argument, super-resolution PTM imagings
have been achieved mainly in three different ways.
The first method is to choose shorter wavelength laser source as the probe beam.
This approach is the simplest way to realize sub-diffraction imaging and most used
in vibrational spectromicroscopy such as the detection of mid-IR absorptions. The
wavelength of mid-IR pump beam usually falls in the range of 2.5–10 µm and most IR
objectives have relatively small NA values (≤0.8) given the fact that IR source cannot
be used in water or immersion oil. Thus, the best spatial resolution of conventional
absorption-based IR microscopes is still limited to ~3 µm even with the synchrotronbased diffraction-limited system [31]. Besides, since the IR region covers such a
broad spectral window, the chromatic aberration will significantly degrade the image
quality. However, with the deployment of a visible probe beam (785 nm), Zhang and
colleagues achieved sub-micrometer (~0.6 µm) chemical imaging of living cells or
microorganisms via confocal mid-IR PTM with a Cassegrain objective (0.65 NA)
[59]. As the schematic shows in Fig. 6.3a, b, the probe focus is ~1/10 of the size of the
pump mid-IR beam such that objects outside the probe beam focus are not detected,
resulting in the ninefold improvement of the spatial resolution shown in Fig. 6.3c.
Since the mid-IR pump beam can fully exploit the rich chemical information encoded
in intrinsic chemical bond vibrations, mid-IR PTM emerges as a promising LFSRM
approach for biomedical and pharmaceutical applications [59, 84]. We will discuss
the recent advances and applications of mid-IR PTM in the following sections.
The second approach is to separate the beam paths and deploy a high NA objective
for the counter-propagating probe beam. As shown in Fig. 6.4, Li et al. reached an
ultimate resolution of 300 nm using a 532 nm probe beam and a 0.9 NA objective in
their counter-propagating mid-IR PT microscope [60]. The main advantage of this
approach is that the visible probe beam can go through a refractive objective instead
of low NA reflective objectives that are typically used for IR source. Since there
are numerous high NA refractive objectives available for visible probe beams, this
approach could potentially reach the spatial resolution on the scale of 100 nm. In addition, this approach is often combined with the first approach, which results in higher
resolution than those coaxial PT microscopes. However, the counter-propagating
configuration does increase the complexity for system alignment in order to overlay
the pump and probe beams in the sample plane and maximize the PT signal. Another
drawback is that this approach requires the epi-detection design in which the probe
photon collection efficiency is lower than that of the transmission mode, resulting
in slower data acquisition. Thus, there is a trade-off between spatial resolution and
imaging frame rate in this approach.
Last but not the least, super-resolution PTM can be achieved by probing the
nonlinear PT lens effect whose signal profile is much narrower than the linear PT
signal [61, 85–90]. Zharov first demonstrated the nonlinear dependence of PT signal
to the pump beam intensity when the laser energy reaches certain thresholds using
nanoparticles, through which the detected nonlinear PT signal arises only from the
nonlinear center of the samples [61].
143
in possibilities of beating the diffraction limit of the pump beam by manipulating
the probe beam. Based on the preceding argument, super-resolution PTM imagings
have been achieved mainly in three different ways.
The first method is to choose shorter wavelength laser source as the probe beam.
This approach is the simplest way to realize sub-diffraction imaging and most used
in vibrational spectromicroscopy such as the detection of mid-IR absorptions. The
wavelength of mid-IR pump beam usually falls in the range of 2.5–10 µm and most IR
objectives have relatively small NA values (≤0.8) given the fact that IR source cannot
be used in water or immersion oil. Thus, the best spatial resolution of conventional
absorption-based IR microscopes is still limited to ~3 µm even with the synchrotronbased diffraction-limited system [31]. Besides, since the IR region covers such a
broad spectral window, the chromatic aberration will significantly degrade the image
quality. However, with the deployment of a visible probe beam (785 nm), Zhang and
colleagues achieved sub-micrometer (~0.6 µm) chemical imaging of living cells or
microorganisms via confocal mid-IR PTM with a Cassegrain objective (0.65 NA)
[59]. As the schematic shows in Fig. 6.3a, b, the probe focus is ~1/10 of the size of the
pump mid-IR beam such that objects outside the probe beam focus are not detected,
resulting in the ninefold improvement of the spatial resolution shown in Fig. 6.3c.
Since the mid-IR pump beam can fully exploit the rich chemical information encoded
in intrinsic chemical bond vibrations, mid-IR PTM emerges as a promising LFSRM
approach for biomedical and pharmaceutical applications [59, 84]. We will discuss
the recent advances and applications of mid-IR PTM in the following sections.
The second approach is to separate the beam paths and deploy a high NA objective
for the counter-propagating probe beam. As shown in Fig. 6.4, Li et al. reached an
ultimate resolution of 300 nm using a 532 nm probe beam and a 0.9 NA objective in
their counter-propagating mid-IR PT microscope [60]. The main advantage of this
approach is that the visible probe beam can go through a refractive objective instead
of low NA reflective objectives that are typically used for IR source. Since there
are numerous high NA refractive objectives available for visible probe beams, this
approach could potentially reach the spatial resolution on the scale of 100 nm. In addition, this approach is often combined with the first approach, which results in higher
resolution than those coaxial PT microscopes. However, the counter-propagating
configuration does increase the complexity for system alignment in order to overlay
the pump and probe beams in the sample plane and maximize the PT signal. Another
drawback is that this approach requires the epi-detection design in which the probe
photon collection efficiency is lower than that of the transmission mode, resulting
in slower data acquisition. Thus, there is a trade-off between spatial resolution and
imaging frame rate in this approach.
Last but not the least, super-resolution PTM can be achieved by probing the
nonlinear PT lens effect whose signal profile is much narrower than the linear PT
signal [61, 85–90]. Zharov first demonstrated the nonlinear dependence of PT signal
to the pump beam intensity when the laser energy reaches certain thresholds using
nanoparticles, through which the detected nonlinear PT signal arises only from the
nonlinear center of the samples [61].