Imaging of Myelin by Coherent Anti-Stokes Raman Scattering Microscopy - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 270

to image myelin in its natural state, such as in biologically
active tissues and live animals ( 13 ).
2. 3D submicron resolution
The nonlinear dependence on excitation intensity ensures that
the CARS signal is only generated in the focal center, provid-
ing an inherent submicron 3D spatial resolution. For myelin
imaging, the lateral resolution was measured to be 0.28 μ m

m ( 12 ).
Such resolution allows detecting the detailed structure in
myelin, such as a node of Ranvier.
3. Multimodal nonlinear optical imaging
The laser beams for CARS could be used for two-photon excited
fl uorescence (TPEF) and sum-frequency generation (SFG)/sec-
ond harmonic generation (SHG) so that TPEF and SFG/SHG
imaging can be performed simultaneously with CARS imaging
in the same platform. In this multimodal nonlinear optical
imaging platform, CARS is used for imaging of myelin; TPEF
is used for imaging of fl uorescent probe-labeled ions or proteins,
such as Ca 2+ ion indicators; SFG/SHG is used for imaging of
astroglial fi laments ( 14 ). Therefore, multiple components in
the nervous tissues can be visualized at the same time.
4. Raman spectral information of myelin lipid
In addition to morphology of myelin, conformation of the
hydrocarbon chains of myelin lipid is also able to be deter-
mined by confocal Raman spectroscopy in the CARS platform
( 15 ). Raman spectra of myelin lipid from the C-C and C-H
vibrational bands can be used to determine the intermolecular
chain disordering degree of myelin lipid and Raman spectra
from C = C bonds and C-H bonds in lipid acyl chains can be
used to determine the unsaturation degree of myelin lipid
chains. The information obtained from Raman spectra will
help to analyze the change of myelin lipid in the diseased
condition.

and the axial resolution was calculated to be 0.70 μ

3. CARS
Microscope

Figure 2 shows a schematic of a CARS microscope which includes
a laser source, optical pathway, a laser-scanning microscope, and
signal collection components. The details of these four parts are
described below.

1. A laser source: The production of a CARS process requires a
pump and a Stokes laser beam. In Fig. 2 , both lasers are gener-
ated from two Ti:sapphire oscillators (Mira 900, Coherent
Inc, Santa Clara, CA) and they are tunable from 700 nm to