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pattern. The strongest SHG signals came from the area between the poles and the equator, where nearly
parallel microtubules had the same orientation. At the equator, overlapping microtubules from the two
poles had opposite orientations, leading to destructive interference and no SHG signal. Away from the
spindle poles, the radially projecting astral microtubules were just weakly detected. SHG intensity has a
quadratic dependence on the number of scattering units; therefore, it falls quickly for low tubulin con-
centration, unlike fluorescence that varies linearly with the fluorophore concentration.
An optical method for finding polarized microtubules is desirable because the conventional approach,
the hook method, has severe limitations. To use the hook method, cells are lysed and permeated so that,
when exogenously added tubulin would adhere to the existing microtubules, it forms hook-like append-
ages (Heidemann and McIntosh, 1980). Following fixation and using transmission electron microscopy,
the chirality of individual hooks can be measured in a cross-sectional view of the microtubules. The
number of clockwise and counterclockwise hooks corresponds to the number of plus-end microtubules
pointing out of and into the cross section. Therefore, unlike SHG intensity that reports only the polarity
magnitude, the hook method can be used to determine the magnitude and sign of the polarity. The hook
method has been applied to characterize microtubule polarity in axons and dendrites of primary cul-
tured neurons (Baas et al., 1988, 1989), excised nerves (Burton and Paige, 1981, Heidemann et al., 1981),
and thinly sectioned brain tissues (Burton, 1988, Rakic et al., 1996). One drawback for the hook method
is the large fraction of microtubules with ambiguously oriented hooks. Exogenous tubulin must be able
to enter the lysed cells so that the hook method is also limited to thin and fixed specimens.
The recent discovery and characterization of plus-end-tracking proteins (Schuyler and Pellman,
2001) have added a new tool for characterizing the microtubule polarity. As their name suggests, these
proteins are exclusively bounded to the fast-growing plus ends. By fluorescently tagging the plus-end-
tracking proteins, it is possible to optically track their motion and locate the plus ends of growing
microtubules (Stepanova et al., 2003). The large arsenal of genetic approaches means that this method
can be easily applied to in vitro as well as in vivo preparations (Rolls et  al., 2007, Stone et  al., 2008).
Moreover, the sign and magnitude of the polarity can be estimated. One potential problem for using
fluorescently labeled plus-end-tracking protein is that many microtubules are stabilized by capping
proteins and are less dynamic in vivo ; therefore, polarity estimates may be skewed by mostly observing
the dynamic fraction.
7.1.2 endogenous Signal in Scattering tissues and Whole embryos
A key advantage for SHG microscopy is its applicability to scattering tissues (Figure 7.4). Similar to
two-photon-excited fluorescence microscopy, the excitation volume is confined by the quadratic depen-
dence of emission on the laser intensity; so, nondescanned detection can be used to collect all scat-
tered photons as the signal and enables an imaging depth of >500 μm in scattering tissues (Denk et al.,
1990, Helmchen and Denk, 2005). The ability to image deeply in the scattering tissue distinguishes
SHG microscopy from other optical imaging techniques that have been heavily used to characterize the
microtubules, such as fluorescence-speckle microscopy (Waterman-Storer et al., 1998) and dark-field
microscopy (Horio and Hotani, 1986). Furthermore, the microtubule is an intrinsic SHG source that
does not require staining with dyes. This simplifies the experimental procedure and protects delicate
specimens. Maintaining the native state is critical for certain applications such as time-lapse imaging of
embryos, where the developmental process can be easily stunned.
One restrictive requirement for SHG imaging of microtubules is the need for detecting the forward-
directed emission, which places a practical limit on imaging depth. SHG intensity from microtubules
is mostly forward directed and the forward-over-backward intensity ratio is ~5-100 depending on the
spatial distribution of tubulins (Kwan et  al., 2008). Since the backward emission from microtubules
is small, two approaches have been used. One, the forward emission can be directly imaged with a
photomultiplier tube behind the condenser lens. Two, it is possible to use the epipathway to detect
back-scattered forward emission, in addition to the direct backward emission (Legare et  al., 2007).
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