Biomedical Engineering Reference
In-Depth Information
FIgurE 7.4
SHG imaging of microtubules in scattering tissues. (a) SHG from a transverse hippocampal slice,
typically 250-400 μm thick and prepared from 14- to 20-day-old rat pups. The neurons were kept healthy by
perfusing with oxygenated artificial cerebrospinal fluid. SHG signals were clearly seen from the mossy fibers,
an axonal bundle that connects the dentate gyrus (DG) to the CA3 region of the hippocampus. (b) In a magni-
fied view, SHG signals were also observed from thin processes, putative single axons, in the CA3 region. Scale
bar = (a) 200 μm and (b) 100 μm. (Reprinted from Dombeck, D. A. et al., 2003. Uniform polarity microtubule
assemblies imaged in native brain tissue by second-harmonic generation microscopy.
Proceedings of the National
Academy of Sciences of the United States of America,
100, 7081-7086. Copyright 2003, with permission from the
National Academy of Sciences, USA.)
Furthermore, by measuring SHG signal from different angular acceptance angles, the fraction of the
direct and scattered fractions may be separated (Han and Brown, 2010). In practice, detecting backscat-
tered signal is more suitable for strong SHG sources such as collagen; so, all reported imaging studies
of microtubules have focused on collecting the forward emission. The need to detect forward emissions
limits the utility of SHG imaging of microtubules in en bloc tissues or intact animals.
Interestingly, fixatives such as paraformaldehyde abolish the SHG signal from microtubules. This
is likely because protein cross-linking due to fixation has altered the hyperpolarizability or the spatial
arrangement of tubulins. Moreover, in acute brain slices where the tissue health deteriorates over the
course of several hours, the SHG signal from microtubules also gradually decreases. These anecdotal
observations suggest that SHG is possible only in intact microtubules within their native environment.
SHG intensity could potentially be used to assess the structural and functional integrity of microtubules
in situ
(Barnes et al., 2010).
