Biomedical Engineering Reference
In-Depth Information
(a1)
(a2)
(b)
(a3)
(a4)
(c)
(d)
(e)
60
30
0
-30
-60
60
30
0
-30
-60
-60 -30 0 30 60
-60 -30 0
ω
30 60
ω
FIgurE 7.8
Polarization dependence of SHG intensity from microtubules. (a1 through a4) Fresh axonemes were
prepared from sea urchin sperms. SHG images were collected using linearly polarized excitation at four different
angles of polarization. (b) The mean image. Scale bar = 10 μm. (c) From the intensity recorded at each of the polar-
ization angles, a direction map for hyperpolarizability is computed, which matches well with the physical orienta-
tion of the axonemes. (d, e) Quantification of the correlation between hyperpolarizability direction, φ, and physical
orientation, ω, for images obtained with four or six different polarization angles. (Reprinted from Odin, C. et al.,
2009. Second harmonic microscopy of axonemes.
Optics Express,
17, 9235-9240. Copyright 2009, with permissions
from the Optical Society of America.)
SHG imaging, where the forward emission is maximal at two lobes that are ~25° away from the forward
axis, the emission from microtubules is almost entirely along the forward axis. Moreover, the calcu-
lated forward-over-backward emission intensity ratio for microtubule bundles is significantly higher
than those calculated for collagen, another well-characterized biological SHG source. This agrees with
the observation that it is easier to image collagen in the epipathway and to suggest a possible method
for separating different sources of SHG signals. For example, forward-to-backward emission intensity
ratios have been used to estimate the overall polarity of the microtubule arrays within axons and apical
dendrites (Kwan et al., 2008).
7.5 Summary
SHG imaging of microtubules has been applied to a large variety of preparations, particularly for visual-
izing mitotic spindles and microtubule bundles in neurons. The SHG signal offers unique advantages:
the method is sensitive to polarized microtubule ensembles and is applicable to scattering tissues.
The development of SHG imaging of microtubules has so far focused on a few biological applications,
including mapping polarized microtubules in neuronal compartments and marking time of cell divi-
sion during embryonic development. Because of its unique capabilities, SHG imaging of microtubules
should continue to thrive in areas where no comparable technique exists.
