Imaging of Myelin by Coherent Anti-Stokes Raman Scattering Microscopy - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 269

probe fi eld E ′ come from the same laser beam. When the beating
frequency ( w p − w s ) is in resonance with a vibration of the molecular
chemical bonds, the mixed pump and Stokes fi eld can effectively
excite the molecule to a vibrationally excited state. The effi ciency
of generated CARS process is then signifi cantly enhanced because
the molecular vibration facilitates the energy exchange between
light fi elds. Therefore, the molecules in resonance will show higher
signal than those of resonance, providing a vibrational contrast in
a CARS image. Meanwhile, as a coherent process, the CARS signal
fi elds from different molecules have a well-defi ned phase relation-
ship and they add up like the emission from an antenna array. The
emission is concentrated in one direction defi ned by the construc-
tive interference, which greatly facilitates the signal collection. The
coherent addition also results in a quadratic signal increase with
the respect to the density of molecular chemical bonds.
In the nervous system, myelin sheath is a multiple layer
membrane which comprises 70% lipid by weight. Membrane lipid
contains a huge amount of ordered CH 2 groups in the lipid chains.
The vibrations of these ordered CH 2 groups produce strong CARS
signal. Figure 1a shows a CARS image of myelin sheath in the fresh
spinal tissue isolated from guinea pig and a CARS spectrum of
myelin ( 12 ). The strong peak in the CARS spectrum represents
the symmetric stretching vibration at 2,840 cm −1 (Fig. 1b ). CARS
microscopy offers several unique advantages for myelin imaging.
1. Label-free detection of myelin
CARS microscopy directly probes the symmetrical stretching
vibrations of CH 2 groups in the myelin lipids, thus no label is
necessary for CARS imaging of myelin. Nonlabeling property
simplifi es the sample preparation and avoids the photobleach-
ing problem occurring at the imaging using fl uorescent labels.
Therefore, CARS imaging is nondestructive and can be applied

a b

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Wavenumber (cm -1 )

Fig. 1. CARS image and CARS spectrum. ( a ) A typical CARS image of myelin sheath in the fresh
spinal tissue isolated from guinea pig. ( b ) CARS spectrum of myelin sheath showing a strong
peak at 2,840 cm −1 , which represents symmetrical stretching vibration of CH 2 groups.