12 Nonlinear Label-Free Super-Resolution Microscopy Using Structured Illumination
297
Since many molecules in biological structures show intrinsic fluorescence, i.e., are
autofluorescent, 2PEF and 3PEF processes can be also used to study and visualize
such molecules in label-free manner.
In the early days of multiphoton microscopy, it was believed that the resolution
could be also improved due to the nonlinear nature of the interaction. However, the
lateral resolution improvement while using conventional laser-scanning microscope
was soon found to be only moderate [53], since the increased resolution is almost
counterbalanced by the fact that the excitation occurs at longer wavelengths. For
example, typical lateral resolutions in SHG and THG microscopy (NA = 0.9, λ
= 1230 nm) are often around 400–500 nm whereas the axial resolution is around
1 µm [54]. While the relative axial resolution can be slightly improved when compared to linear modalities, the main advantage of using multiphoton modalities have
not been the achievable resolution. However, this fact might be about to change,
since several nonlinear super-resolution techniques, with many of them based on the
SIM approach, have been recently proposed and demonstrated [18–23, 55]. These
nonlinear super-resolution techniques are the topic of the next section.
12.5 Super-Resolution Using Nonlinear Processes
and Structured Illumination
In this section, we discuss the recent developments in nonlinear super-resolution
techniques. Some of the discussed techniques (mainly based on 2PEF and 3PEF) have
not yet been used for label-free imaging as such, but the techniques have demonstrated
their potential by using fluorescent probes. We emphasize that these techniques could
be used also in label-free manner if they would make use of the autofluorescence
of molecules. First, we will discuss super-resolution techniques based on incoherent
processes, such as 2PEF and 3PEF. Second, we will discuss techniques based on
coherent processes, such as SHG, THG and CARS.
12.5.1 Super-Resolution Using Incoherent Nonlinear
Processes
A while after the conventional SIM was demonstrated for the first time, the approach
was also applied to 2PEF and 3PEF microscopy [28, 56]. Implementation of these
modalities were not trivial since the decrease in the achievable axial resolution of
the conventional wide-field technique was seen as a major drawback [57]. A viable
solution to improve the axial resolution and to enable optical sectioning was to
utilize a technique known as temporal focusing, where ultrashort pulses are made to
temporally overlap only at the focal plane of the microscope [58–60]. In other words,
the incident ultrashort pulse is dispersed using a diffraction grating into different
297
Since many molecules in biological structures show intrinsic fluorescence, i.e., are
autofluorescent, 2PEF and 3PEF processes can be also used to study and visualize
such molecules in label-free manner.
In the early days of multiphoton microscopy, it was believed that the resolution
could be also improved due to the nonlinear nature of the interaction. However, the
lateral resolution improvement while using conventional laser-scanning microscope
was soon found to be only moderate [53], since the increased resolution is almost
counterbalanced by the fact that the excitation occurs at longer wavelengths. For
example, typical lateral resolutions in SHG and THG microscopy (NA = 0.9, λ
= 1230 nm) are often around 400–500 nm whereas the axial resolution is around
1 µm [54]. While the relative axial resolution can be slightly improved when compared to linear modalities, the main advantage of using multiphoton modalities have
not been the achievable resolution. However, this fact might be about to change,
since several nonlinear super-resolution techniques, with many of them based on the
SIM approach, have been recently proposed and demonstrated [18–23, 55]. These
nonlinear super-resolution techniques are the topic of the next section.
12.5 Super-Resolution Using Nonlinear Processes
and Structured Illumination
In this section, we discuss the recent developments in nonlinear super-resolution
techniques. Some of the discussed techniques (mainly based on 2PEF and 3PEF) have
not yet been used for label-free imaging as such, but the techniques have demonstrated
their potential by using fluorescent probes. We emphasize that these techniques could
be used also in label-free manner if they would make use of the autofluorescence
of molecules. First, we will discuss super-resolution techniques based on incoherent
processes, such as 2PEF and 3PEF. Second, we will discuss techniques based on
coherent processes, such as SHG, THG and CARS.
12.5.1 Super-Resolution Using Incoherent Nonlinear
Processes
A while after the conventional SIM was demonstrated for the first time, the approach
was also applied to 2PEF and 3PEF microscopy [28, 56]. Implementation of these
modalities were not trivial since the decrease in the achievable axial resolution of
the conventional wide-field technique was seen as a major drawback [57]. A viable
solution to improve the axial resolution and to enable optical sectioning was to
utilize a technique known as temporal focusing, where ultrashort pulses are made to
temporally overlap only at the focal plane of the microscope [58–60]. In other words,
the incident ultrashort pulse is dispersed using a diffraction grating into different