Biomedical Engineering Reference
In-Depth Information
7
Second-Harmonic
Generation Imaging of
Microtubules
7.1 Why Study Microtubules with SHG Imaging? ............................154
SHG Intensity Reflects Microtubule Polarity • Endogenous Signal
in Scattering Tissues and Whole Embryos •
Combining with Other
Imaging Modalities • Drawbacks
7.2 Application 1: Mapping the Distribution of Polarized
Microtubule Bundles in Native Brain Tissues .............................158
7.3 Application 2: Endogenous Time Stamp and Marker for
Cell Division for Whole-Embryo Developmental Studies .........159
7.4 Technical Notes for SHG Imaging of Microtubules.................... 162
7.5 Summary............................................................................................163
References......................................................................................................165
Alex C. Kwan
University of California,
Berkeley
Cornell University
Microtubules are a well-studied class of cytoskeleton and serve numerous vital functions for the cell.
They are the mechanical supports for cellular compartments and the roadways for active intracellu-
lar transport. A single microtubule consists of 13 parallel protofilaments, each a linear chain of the
tubulin proteins connected end to end. The protofilaments are attracted to each other lengthwise; so,
each microtubule is a 25-μm-diameter rod with a hollow core. Tubulin, the basic repeating unit, is a
heterodimer of α- and β-tubulins. One end of the tubulin dimer has an accessible, bound guanosine-5′-
triphosphate (GTP). This GTP can be hydrolyzed readily so that the end can bond with another tubulin,
and therefore, this is the fast-growing end, or the “plus end.” Since tubulins are joined end to end to form
protofilaments, each microtubule is structurally asymmetric. This polarity is particularly important for
active transport because it dictates the travel direction of molecular motors, which carry cargoes from
the soma to distal cellular compartments (Hirokawa and Takemura, 2005).
The structural polarity of tubulin also manifests itself as hyperpolarizability. As a result, microtu-
bules can participate in second-order nonlinear optical processes such as second-harmonic generation
(SHG). Presumably, because the hyperpolarizability of tubulin is small, SHG imaging at single-microtu-
bule resolution is not yet possible. However, if the neighboring microtubules have similar polarity, such
that the SHG amplitudes coherently add, then the SHG intensity increases as the square of the number
of scattering tubulins. SHG signals can be reliably observed from a single axon, that is, ~50 microtubules
within the focal volume.
SHG from microtubules has been imaged in a variety of preparations (Figures 7.1 and 7.2), includ-
ing mitotic spindles in
Caenorhabditis elegans
embryos (Campagnola et al., 2002, Mohler et al., 2003),
sea urchin embryos (Mohler et al., 2003), mouse oocytes and embryos (Hsieh et al., 2008a), zebra fish
embryos (Chu et al., 2003, Chen et al., 2006, Hsieh et al., 2008b, Olivier et al., 2010), and cultured rat
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