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C. Li and J.-X. Cheng
objects that are closer or smaller than about half the wavelength cannot be discerned
and those sub-cellular structures such as organelles can only be visualized down to
this scale [3]. To break the diffraction limit, the last two decades have seen an emerging interest of developing fluorescence-based far-field super-resolution microscopy,
or the so-called “far-field fluorescence nanoscopy”. Among them, photoactivated
localization microscopy and stochastic optical reconstruction microscopy use the
on/off blinking and switching behavior of fluorescent proteins to partially excite and
localize the fluorophores with ~10 nm precision [4–7]. Stimulated emission depletion microscopy [8–10], along with ground-state depletion (GSD) microscopy [11]
and saturated structured illumination microscopy [12, 13] are based on nonlinear
optical effects that reduce the point spread function (PSF) of the focused light by
controlling the excitation beam geometry.
Although the power of far-field fluorescence nanoscopy approaches enabled many
breakthrough and discoveries in cell biology and biomedical sciences, due to the
necessity of employing fluorescent labeling, many important intracellular processes
that could be perturbed by the fluorophores remain unexplored [14–18]. In addition, many intrinsic biomolecules and nanomaterials, such as hemes, nanotubes, and
graphene, have strong absorption yet low quantum efficiency [19–21], which makes
it hard to visualize these species using far-field fluorescence nanoscopy. Label-free
super-resolution microscopy (LFSRM), which uses other inherent chemical or physical properties from the samples to provide the contrast, thus shows advantages over
fluorescence-based approaches for in vivo imaging of cells and organisms and draws
increasing interests in the recent years.
One way to provide contrast mechanism in the label-free imaging manner is to
use the intrinsic molecular absorption that is prevalent from inorganic materials to
biomolecules. Linear absorption has been well studied since the early twentieth century and inspired various widely used measurement methods, including infrared (IR)
spectroscopy (vibrational state absorption) [22] and UV–Vis spectroscopy (electronic
state absorption) [23]. On comparing with scattering or multiphoton processes, we
found that the large cross-section of linear absorption allows researchers to push
the sensitivity of absorption spectroscopy to single-molecule detection [24, 25].
The application of Fourier transform in the interferometric spectroscopy enables
the extraction of absorption spectra with high spectral resolution from the measured
interferogram [26–29]. The development of bright coherent sources, like synchrotron
radiation or quantum cascade lasers [30, 31], along with the deployment of fast electronics or sensitive detectors [27, 32] has improved not only the signal-to-noise ratio
to an extreme but also the spectral resolution and has reduced the data acquisition
time. These advancements in linear absorption spectroscopy have all facilitated the
deployment of spectroscopic imaging in chemistry, materials science, pharmaceutics, and polymer sciences [33–36]. Meanwhile, the advancements in nonlinear optics
have also seen the growth of nonlinear absorption-based spectroscopy, such as transient absorption spectroscopy and stimulated Raman scattering spectroscopy [15, 19,
37–41]. Transient absorption microscopy, being able to provide the time-resolved
spectroscopic imaging of various ultra-fast processes, is deployed in the field of nanomaterials, semiconductors, catalysts, and so on [42–46]. Stimulated Raman scattering
C. Li and J.-X. Cheng
objects that are closer or smaller than about half the wavelength cannot be discerned
and those sub-cellular structures such as organelles can only be visualized down to
this scale [3]. To break the diffraction limit, the last two decades have seen an emerging interest of developing fluorescence-based far-field super-resolution microscopy,
or the so-called “far-field fluorescence nanoscopy”. Among them, photoactivated
localization microscopy and stochastic optical reconstruction microscopy use the
on/off blinking and switching behavior of fluorescent proteins to partially excite and
localize the fluorophores with ~10 nm precision [4–7]. Stimulated emission depletion microscopy [8–10], along with ground-state depletion (GSD) microscopy [11]
and saturated structured illumination microscopy [12, 13] are based on nonlinear
optical effects that reduce the point spread function (PSF) of the focused light by
controlling the excitation beam geometry.
Although the power of far-field fluorescence nanoscopy approaches enabled many
breakthrough and discoveries in cell biology and biomedical sciences, due to the
necessity of employing fluorescent labeling, many important intracellular processes
that could be perturbed by the fluorophores remain unexplored [14–18]. In addition, many intrinsic biomolecules and nanomaterials, such as hemes, nanotubes, and
graphene, have strong absorption yet low quantum efficiency [19–21], which makes
it hard to visualize these species using far-field fluorescence nanoscopy. Label-free
super-resolution microscopy (LFSRM), which uses other inherent chemical or physical properties from the samples to provide the contrast, thus shows advantages over
fluorescence-based approaches for in vivo imaging of cells and organisms and draws
increasing interests in the recent years.
One way to provide contrast mechanism in the label-free imaging manner is to
use the intrinsic molecular absorption that is prevalent from inorganic materials to
biomolecules. Linear absorption has been well studied since the early twentieth century and inspired various widely used measurement methods, including infrared (IR)
spectroscopy (vibrational state absorption) [22] and UV–Vis spectroscopy (electronic
state absorption) [23]. On comparing with scattering or multiphoton processes, we
found that the large cross-section of linear absorption allows researchers to push
the sensitivity of absorption spectroscopy to single-molecule detection [24, 25].
The application of Fourier transform in the interferometric spectroscopy enables
the extraction of absorption spectra with high spectral resolution from the measured
interferogram [26–29]. The development of bright coherent sources, like synchrotron
radiation or quantum cascade lasers [30, 31], along with the deployment of fast electronics or sensitive detectors [27, 32] has improved not only the signal-to-noise ratio
to an extreme but also the spectral resolution and has reduced the data acquisition
time. These advancements in linear absorption spectroscopy have all facilitated the
deployment of spectroscopic imaging in chemistry, materials science, pharmaceutics, and polymer sciences [33–36]. Meanwhile, the advancements in nonlinear optics
have also seen the growth of nonlinear absorption-based spectroscopy, such as transient absorption spectroscopy and stimulated Raman scattering spectroscopy [15, 19,
37–41]. Transient absorption microscopy, being able to provide the time-resolved
spectroscopic imaging of various ultra-fast processes, is deployed in the field of nanomaterials, semiconductors, catalysts, and so on [42–46]. Stimulated Raman scattering