12 Nonlinear Label-Free Super-Resolution Microscopy Using Structured Illumination
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12.6 Current Applications of Nonlinear Microscopy
We start this section by reminding the reader of the advantages of performing multiphoton microscopy [16, 49]. After that, we will briefly review the current applications
of multiphoton microscopy which could all benefit from super-resolution techniques.
First, compared to conventional fluorescence microscopy, multiphoton modalities
enable to study objects deeper inside intact tissues. This is due to the fact, that longer
excitation wavelengths can be used, which do not scatter as strongly from tissues
[63, 64]. For example, in vivo 3PEF imaging of subcortical structures within intact
mouse brain have been recently demonstrated reporting very impressive imaging
depths of over 1 nm [52]. Second, multiphoton modalities provide intrinsic optical
sectioning, which in fact makes it straightforward to perform three-dimensional imaging [16]. Third, when carefully performed, the use of multiphoton excitation leads to
smaller phototoxicity and bleaching effects, because the excitation occurs at longer
wavelengths where single-photon fluorescence is negligible. The efficient excitation
of fluorophores via multiphoton processes occurs only at the focal point [49].
When biological tissues are imaged using multiphoton modalities, it is important
to understand the molecular origins for the contrast. In the case of label-free imaging
techniques, the sources for contrast become more limited because external fluorescent
molecules cannot be utilized. Fortunately, biological tissues contain many intrinsic
fluorophores which can be used in 2PEF and 3PEF microscopies [49]. By far, the
two most often utilized fluorophores are reduced nicotinamide adenine dinucleotide
phosphates [NAD(P)H] and flavin adenine dinucleotides (FAD). In particular, assessment of the ratio of the relative amounts of NADH and FAD molecules, also known
as the redox ratio, has been found to be very useful for monitoring metabolic activity of cells, which is known to be an important factor in carcinogenesis [65, 66].
An excellent source for further reading on intrinsic fluorophores and their use in
multiphoton microscopy is found in [49].
In addition to 2PEF and 3PEF, also other nonlinear processes can be utilized
for label-free contrast. Since the pioneering studies, SHG and THG microscopies
have evolved into highly useful tools to study biological samples [44–46, 67]. For
example, SHG microscopy can provide useful information of tissue morphology for
diagnostic applications [68, 69], while THG microscopy can be used to study the
organization of lipid molecules [70–72].
The detected nonlinear signal, and thus the contrast, originates in SHG microscopy
mostly from ordered molecular structures, such as from fibrous collagen, elastin,
myosin and actin proteins, and from microtubules [73]. The fact that SHG microscopy
is particularly sensitive to the order of SHG-active molecules, and not just to their
concentration, is because coherent second-order nonlinear processes are strongly
affected by symmetry issues [17]. As a consequence, SHG imaging can be used
to provide additional structural information of objects. This has been found to be
very useful, for example, for performing collagen scoring in fibrotic tissues [68], in
303
12.6 Current Applications of Nonlinear Microscopy
We start this section by reminding the reader of the advantages of performing multiphoton microscopy [16, 49]. After that, we will briefly review the current applications
of multiphoton microscopy which could all benefit from super-resolution techniques.
First, compared to conventional fluorescence microscopy, multiphoton modalities
enable to study objects deeper inside intact tissues. This is due to the fact, that longer
excitation wavelengths can be used, which do not scatter as strongly from tissues
[63, 64]. For example, in vivo 3PEF imaging of subcortical structures within intact
mouse brain have been recently demonstrated reporting very impressive imaging
depths of over 1 nm [52]. Second, multiphoton modalities provide intrinsic optical
sectioning, which in fact makes it straightforward to perform three-dimensional imaging [16]. Third, when carefully performed, the use of multiphoton excitation leads to
smaller phototoxicity and bleaching effects, because the excitation occurs at longer
wavelengths where single-photon fluorescence is negligible. The efficient excitation
of fluorophores via multiphoton processes occurs only at the focal point [49].
When biological tissues are imaged using multiphoton modalities, it is important
to understand the molecular origins for the contrast. In the case of label-free imaging
techniques, the sources for contrast become more limited because external fluorescent
molecules cannot be utilized. Fortunately, biological tissues contain many intrinsic
fluorophores which can be used in 2PEF and 3PEF microscopies [49]. By far, the
two most often utilized fluorophores are reduced nicotinamide adenine dinucleotide
phosphates [NAD(P)H] and flavin adenine dinucleotides (FAD). In particular, assessment of the ratio of the relative amounts of NADH and FAD molecules, also known
as the redox ratio, has been found to be very useful for monitoring metabolic activity of cells, which is known to be an important factor in carcinogenesis [65, 66].
An excellent source for further reading on intrinsic fluorophores and their use in
multiphoton microscopy is found in [49].
In addition to 2PEF and 3PEF, also other nonlinear processes can be utilized
for label-free contrast. Since the pioneering studies, SHG and THG microscopies
have evolved into highly useful tools to study biological samples [44–46, 67]. For
example, SHG microscopy can provide useful information of tissue morphology for
diagnostic applications [68, 69], while THG microscopy can be used to study the
organization of lipid molecules [70–72].
The detected nonlinear signal, and thus the contrast, originates in SHG microscopy
mostly from ordered molecular structures, such as from fibrous collagen, elastin,
myosin and actin proteins, and from microtubules [73]. The fact that SHG microscopy
is particularly sensitive to the order of SHG-active molecules, and not just to their
concentration, is because coherent second-order nonlinear processes are strongly
affected by symmetry issues [17]. As a consequence, SHG imaging can be used
to provide additional structural information of objects. This has been found to be
very useful, for example, for performing collagen scoring in fibrotic tissues [68], in