11 Label-Free Super-Resolution Microscopy by Nonlinear …
273
11.5 Modality Variations to Improve and Simplify NPMR
The use of NPMR to achieve SR converges as the nonlinearity order
√
n, while
the signal of high orders decays exponentially. Therefore, it is useful to seek for
additional effects that provide improvement in resolution and sensitivity, on top of
NPMR. Here we present two such approaches.
11.5.1 Spatial Modulation
Spatial overlap modulation (SPOM) technique improves three-dimensional sectioning by suppressing out-of-focus signals, as the pump and probe spatial overlap is
temporally modulated while scanning. In the focal region the modulation depth of
the spatial overlap peaks, and decays fast toward the out-of-focus regions [34]. In
SPOM nonlinear optical microscopy (SPOM-NOM), the spatial overlap between
two color pulses is temporally modulated by means of beam pointing modulation or
wavefront modulation, and nonlinear optical processes excited by a combination of
two color pulses are monitored. This technique was used for imaging with enhanced
spatial resolution in modalities such as sum frequency imaging [35] or stimulated
Raman scattering [34]. However, the PSF of SPOM, PSF SPOM , suffers from negative
lobes, since it practically represents the second derivative of the image, and generates
artifacts in the image.
The combination of NPMR with SPOM results in improved resolution and major
reduction of the negative lobes. The effective PSF of this combined modality amounts
to the product of PSF
n
pump and PSF SPOM , and the negative lobes of PSF SPOM overlap with low values of PSF
n
pump . In the experiments, the probe beam was spatially
modulated at ω SPAT using a galvanometer-mounted mirror (see Fig. 11.3), while the
intensity of the spatially fixed pump beam was modulated at ω m using the AOM.
The demodulated signal at frequency ω ref = nω m + 2ω SPAT provides the integrated
response of SPOM (second order) and NPMR. Figure 11.8a depicts a simulation
of the method using a pump beam at 392 nm and a probe beam at 785 nm, as in
the experiments. The simulated improvement in resolution, and the decrease of the
negative lobes of SPOM, is clearly demonstrated.
The experiments were performed on 100 nm thick silicon layers patterned on
sapphire. Figure 11.8b depicts a scan of a single silicon 125 nm wide stripe in
different modalities. The results are in good correspondence to our simulations. The
introduction of SPOM improved the resolution of the second harmonics NPMR
by ~15%, down to 85 ± 5 nm, consistent with the simulation. Theoretically, the
incorporation of SPOM is equivalent to additional 1–2 higher harmonics in NPMR,
yet with about tenfold higher signal levels. Indeed, the typical negative lobes of
SPOM are significantly reduced due to the narrow PSF of NPMR.
273
11.5 Modality Variations to Improve and Simplify NPMR
The use of NPMR to achieve SR converges as the nonlinearity order
√
n, while
the signal of high orders decays exponentially. Therefore, it is useful to seek for
additional effects that provide improvement in resolution and sensitivity, on top of
NPMR. Here we present two such approaches.
11.5.1 Spatial Modulation
Spatial overlap modulation (SPOM) technique improves three-dimensional sectioning by suppressing out-of-focus signals, as the pump and probe spatial overlap is
temporally modulated while scanning. In the focal region the modulation depth of
the spatial overlap peaks, and decays fast toward the out-of-focus regions [34]. In
SPOM nonlinear optical microscopy (SPOM-NOM), the spatial overlap between
two color pulses is temporally modulated by means of beam pointing modulation or
wavefront modulation, and nonlinear optical processes excited by a combination of
two color pulses are monitored. This technique was used for imaging with enhanced
spatial resolution in modalities such as sum frequency imaging [35] or stimulated
Raman scattering [34]. However, the PSF of SPOM, PSF SPOM , suffers from negative
lobes, since it practically represents the second derivative of the image, and generates
artifacts in the image.
The combination of NPMR with SPOM results in improved resolution and major
reduction of the negative lobes. The effective PSF of this combined modality amounts
to the product of PSF
n
pump and PSF SPOM , and the negative lobes of PSF SPOM overlap with low values of PSF
n
pump . In the experiments, the probe beam was spatially
modulated at ω SPAT using a galvanometer-mounted mirror (see Fig. 11.3), while the
intensity of the spatially fixed pump beam was modulated at ω m using the AOM.
The demodulated signal at frequency ω ref = nω m + 2ω SPAT provides the integrated
response of SPOM (second order) and NPMR. Figure 11.8a depicts a simulation
of the method using a pump beam at 392 nm and a probe beam at 785 nm, as in
the experiments. The simulated improvement in resolution, and the decrease of the
negative lobes of SPOM, is clearly demonstrated.
The experiments were performed on 100 nm thick silicon layers patterned on
sapphire. Figure 11.8b depicts a scan of a single silicon 125 nm wide stripe in
different modalities. The results are in good correspondence to our simulations. The
introduction of SPOM improved the resolution of the second harmonics NPMR
by ~15%, down to 85 ± 5 nm, consistent with the simulation. Theoretically, the
incorporation of SPOM is equivalent to additional 1–2 higher harmonics in NPMR,
yet with about tenfold higher signal levels. Indeed, the typical negative lobes of
SPOM are significantly reduced due to the narrow PSF of NPMR.