Biomedical Engineering Reference
In-Depth Information
7.1.3 combining with other imaging Modalities
An SHG microscope has nearly identical requirements as a two-photon-excited fluorescence microscope;
so, the two modalities can be easily applied together on the same specimen. For studying microtubules,
it is often useful to visualize the surrounding cellular environment. For example, Thy1-GFP mice have
sparsely labeled neurites (Feng et al., 2000), which enable the identification of neurite types that contain
polarized microtubules (Kwan et al., 2008). Another example is using fluorescently tagged histones to
label the chromosomes, providing more clues for classifying the different phases of cell division (Olivier
et al., 2010). SHG imaging of microtubules can also be combined with third-harmonic generation
imaging. The triple detection of two-photon-excited fluorescence, third-harmonic generation, and SHG
have been used to visualize the various aspects of cellular architectures during embryonic development
(Chu et al., 2003, Chen et al., 2006, Olivier et al., 2010). Mechanical manipulation can be applied via
laser dissection of microtubules
in situ
(Tolic-Norrelykke et al., 2004). Complementary modalities such
as coherent anti-Stokes Raman scattering can also be simultaneously used, for example, to visualize the
myelin fibers (Fu et al., 2008).
Separating the emissions of SHG from other imaging modalities is straightforward. The SHG signal
from microtubules is more strongly excited by the low end of the tuning range of a typical Ti:sapphire
laser (Figure 7.5a). This is opposite to some common fluorophores such as GFP that are optimally excited
by higher wavelengths. Moreover, SHG emission is narrowband, centering at half the excitation wave-
length with a bandwidth of ~1/√2 of the excitation bandwidth. For a Ti:sapphire laser, the SHG emission
bandwidth is ~10 nm. The background broadband fluorescence signal can be reduced by using nar-
rowband emission filters (Figure 7.5b). By tuning the excitation wavelength, the SHG emission can be
shifted to avoid the peak wavelength of the fluorescence emission.
7.1.4 Drawbacks
Despite evidence that tubulin is the basic unit responsible for SHG, the factors that affect molecular
hyperpolarizability remain unclear (Gualtieri et al., 2008). As a result, it is difficult to interpret the
observed SHG intensity quantitatively in terms of the properties of the microtubules. The main ques-
tion is how do structural and conformational changes, for example, via posttranslational modification
of tubulin or addition of microtubule-associated proteins, influence the SHG intensity? For structural
changes, numerical simulation can predict how differences in the number density, intermicrotubule
distances, and polarity can affect the SHG intensity. For molecular and conformational changes, which
are more relevant to many biological questions, the answer is less obvious and requires correlated
imaging and biochemical analysis. One study has shown that the expression of tau, a microtubule-
binding protein, can lead to an increase in SHG intensity (Stoothoff et al., 2008). More studies will be
required before SHG intensity can be a quantitative probe for characterizing
in situ
modifications to
microtubules.
Compared to other methods for measuring microtubule polarity, SHG imaging is sensitive to the
magnitude but not to the sign. This drawback may be remedied by comparing the phase of SHG ampli-
tude from microtubules with a reference to extract the sign of polarity (Kemnitz et al., 1986). The weak
SHG signal from microtubules would make such calibration a challenging task. Another consequence of
weak intensity is the signal contamination from other endogenous SHG sources. For example, muscles
and skin contain collagen, which generates an SHG signal that would overwhelm the signal from micro-
tubules. Therefore, SHG imaging of microtubules is possible only when other dominant SHG sources
are absent.
Despite the drawbacks, SHG microscopy is uniquely capable of identifying polarized microtu-
bule ensembles and is applicable to native, scattering tissues. As a result, SHG imaging of microtu-
bules has found a niche role in several fields of study. The following sections highlight two specific
applications.
