Biomedical Engineering Reference
In-Depth Information
were revealed with SHG imaging in our lab [51], with the myosin thick filaments generating strong
harmonic signal and actin filaments not producing contrast. This arises due to the strong and weak
birefringence of myosin and actin, respectively. We also successfully acquired SHG images of micro-
tubules in mitotic spindles and centrosomes [6]. Microtubules are polymers of α- and β-tubulin
dimers of high polarity, with the α-subunit exposed at one end and β-subunit at the other. The
SHG emission is much weaker compared to the coiled-coil myosin and collagen (~10-50-fold). It is
also very interesting to note that SHG signal disappears at the midzone of mitotic spindles and the
center of centrosomes, where microtubules are aligned antiparallel, so there is no net dipole and the
harmonic emission from individual filaments interfere destructively as described by Equation 4.3.
In contrast, fluorescence from GFP:tubulin was observed throughout these regions as no symmetry
constraints exist.
The assembly of intermediate astroglial filaments from the central nervous system were also studied
by SHG imaging by Cheng et al. [52]. However, the mechanism of the harmonic generation remains
unknown, as the basic units of an intermediate filament are antiparallel tetramers, which possess an
inversion symmetry [53], and no SHG should be expected. On the other hand, SHG imaging of a mouse
ear skin containing hair follicles indicates complete absence of SHG signal from the keratin intermedi-
ate filaments of the hair, consistent with the molecular orientation of intermediate filaments [6]. We
note that fluorescence and CARS/SRS are not constrained by these symmetry considerations. Indeed,
we observed strong keratin autofluorescence.
4.4.2 tPeF and SHG comparison
The symmetry constraints of SHG discussed earlier allow the extraction of structural details that can-
not be obtained by other optical methods. But on the other side, it limits the scope of applicability for
this technique. For example, in contrast to SHG, two-photon absorption is a noncoherent process, and
it does not require the target sample to contain molecules with specific structural alignment, which
enables TPEF microscopy to have broader applicability. For example, autofluorescence from endog-
enous biomolecules such as NADH, flavins, or tetinol is commonly used for TPEF imaging [54]. In
addition to the ability to image endogenous species, TPEF has become widespread more due to the wide
range of fluorophores with desired spectral and chemical properties as well as labeling essentially any
protein of interest [55]. This can be achieved via the well-developed technology of immunostaining, or
via the vast palette of fluorescent proteins [56].
Compared to SHG microscopy, TPEF has several other disadvantages. First is the lack of sensitivity
to structural information, where fluorescent tags are reporters of protein assembly, whereas SHG visual-
ized the proteins directly. Second, photoexcitation for TPEF is a real photon absorption process, where
molecules are promoted to the electronic excited state. Heat release and photobleaching (in-plane) are
two undesired consequences that lead to sample damage and image quality deterioration. For SHG,
there is no energy exchange between light and the materials under study. Lastly, SHG imaging tar-
gets are endogenous protein structures (with the exception of dye-labeled membranes), which require
no special sample preparation, while TPEF often involves adding external or genetically encoded fluo-
rophores. Adding foreign species to the system often changes the property of the system. For exam-
ple, immunostaining requires that the tissues or cells be fixed, which makes
in vivo
study impossible.
Additionally, genetic reporters can alter the function of some proteins, especially those that actively
polymerize/depolymerize.
4.4.3 tHG and SHG comparison
Unlike TPEF microscopy, THG shares similar structure sensitivity of SHG for biomedical imaging of
tissues. However, THG and SHG operate on significantly different mechanisms. While SHG requires
