7 Label-Free Pump–Probe Nanoscopy
183
In particular, in pump–probe microscopy the spatial resolution is related to the
product of the IPSFs of the pump and probe beams. With the condition of the pump
and probe processes involving one-photon transition, a resolution improvement of
√
2 is expected compared to the resolution achieved by linear optical microscopy at
the same wavelengths [24, 28]. This improvement is comparable to the one obtained
in confocal fluorescence microscopy [78].
The improvement of the spatial resolution of optical microscopes has been a
central challenge in the last few decades, and a lot of effort has been put to circumvent this limitation. Exploiting interference by two opposing high aperture lenses
[79–82] or by structured illumination patterns [83], an isotropic two-fold resolution
improvement down to 100 nm was demonstrated in fluorescence microscopy. A step
forward toward the real circumvention of the diffraction barrier was achieved with
the exploitation of nonlinearities in order to prevent the simultaneous signaling of
adjacent identical target molecules.
In fluorescence microscopy, two families of super-resolution techniques, based on
the switching between ON (bright) and OFF (dark) molecular states, were introduced
in the 1990s.
The first family is based on stochastic switching and read-out [84, 85], where
single molecules are stochastically switched on in a sparse ensemble and localized
with a precision given by the inverse of the square root of the number of detected
photons (here the nonlinearity). Techniques like stochastic optical reconstruction
microscopy (STORM) and photo-activatable localization microscopy (PALM) use
the stochastic approach and achieved resolution of <20 nm [86].
The second super-resolution family is based on targeted switching and read-out,
and on the general RESOLFT (reversible saturable optical fluorescence transition)
concept [87]. In this approach, the diffraction-limited excitation volume that actually
fluoresces is engineered by superimposing a second beam, for example, a doughnutshaped one that switches off the molecules at the periphery, leaving in the on state
only the ones at the very center. The central region can be made arbitrarily small
by saturating—that is the nonlinearity—the off transition. One of the main realizations of this technique is stimulated emission depletion (STED) [88–90], which uses
the stimulated emission transition to quench the fluorescent molecules. In cellular
imaging, a resolution down to 20 nm was demonstrated [91].
7.3.1 The Generalized RESOLFT Concept
The RESOLFT concept is very general, and it can be applied to any saturable transition; that is fluorescence is not mandatory. In fact, the RESOLFT principle consists
of switching the target molecules between an ON state, which gives a detectable
signal, and an OFF state, which is optically dark. In order to prevent the simultaneous signaling of adjacent identical target molecules, the volume that actually gives
a detectable signal is manipulated. First, the OFF → ON transition is achieved in a
diffraction-limited focal spot. Second, the ON → OFF transition (called depletion)
183
In particular, in pump–probe microscopy the spatial resolution is related to the
product of the IPSFs of the pump and probe beams. With the condition of the pump
and probe processes involving one-photon transition, a resolution improvement of
√
2 is expected compared to the resolution achieved by linear optical microscopy at
the same wavelengths [24, 28]. This improvement is comparable to the one obtained
in confocal fluorescence microscopy [78].
The improvement of the spatial resolution of optical microscopes has been a
central challenge in the last few decades, and a lot of effort has been put to circumvent this limitation. Exploiting interference by two opposing high aperture lenses
[79–82] or by structured illumination patterns [83], an isotropic two-fold resolution
improvement down to 100 nm was demonstrated in fluorescence microscopy. A step
forward toward the real circumvention of the diffraction barrier was achieved with
the exploitation of nonlinearities in order to prevent the simultaneous signaling of
adjacent identical target molecules.
In fluorescence microscopy, two families of super-resolution techniques, based on
the switching between ON (bright) and OFF (dark) molecular states, were introduced
in the 1990s.
The first family is based on stochastic switching and read-out [84, 85], where
single molecules are stochastically switched on in a sparse ensemble and localized
with a precision given by the inverse of the square root of the number of detected
photons (here the nonlinearity). Techniques like stochastic optical reconstruction
microscopy (STORM) and photo-activatable localization microscopy (PALM) use
the stochastic approach and achieved resolution of <20 nm [86].
The second super-resolution family is based on targeted switching and read-out,
and on the general RESOLFT (reversible saturable optical fluorescence transition)
concept [87]. In this approach, the diffraction-limited excitation volume that actually
fluoresces is engineered by superimposing a second beam, for example, a doughnutshaped one that switches off the molecules at the periphery, leaving in the on state
only the ones at the very center. The central region can be made arbitrarily small
by saturating—that is the nonlinearity—the off transition. One of the main realizations of this technique is stimulated emission depletion (STED) [88–90], which uses
the stimulated emission transition to quench the fluorescent molecules. In cellular
imaging, a resolution down to 20 nm was demonstrated [91].
7.3.1 The Generalized RESOLFT Concept
The RESOLFT concept is very general, and it can be applied to any saturable transition; that is fluorescence is not mandatory. In fact, the RESOLFT principle consists
of switching the target molecules between an ON state, which gives a detectable
signal, and an OFF state, which is optically dark. In order to prevent the simultaneous signaling of adjacent identical target molecules, the volume that actually gives
a detectable signal is manipulated. First, the OFF → ON transition is achieved in a
diffraction-limited focal spot. Second, the ON → OFF transition (called depletion)