Imaging of Myelin by Coherent Anti-Stokes Raman Scattering Microscopy - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 272

4. Signal collection: The forward-detected CARS (F-CARS) signal
is collected by an air condenser (NA = 0.55). The epi-detected
CARS (E-CARS) signal is collected by the same objective. In
multimodal nonlinear optical imaging, the epi-detected TPEF
or SFG/SHG signal is spectrally separated from the E-CARS
signal by a dichroic splitter. CARS signal from relatively thin
fi xed slices or cryosections is favorably collected in forward
direction. CARS signal from fresh tissues is able to be collected
in both forward and epi-direction. CARS signal from live animals
can only be collected in epi-direction. Both F-CARS and E-CARS
signals are detected with the same type of photomultiplier tube
(PMT, R3896, Hamamatsu, Japan). On the CARS microscope,
a spectrometer equipped with an electron multiplying charge
coupled device (EMCCD) can also be attached to the side port
with the slit replaced with a pinhole for confocal Raman
analysis.

4. Polarization
Dependence of
CARS Signal

CARS signal is sensitive to the polarization of excitation beams.
CARS signal from myelin ( I II ) is maximized when the polarization
of two excitation beams is parallel to the symmetrical axis of CH 2
groups in myelin lipid and CARS signal ( I ⊥ ) is minimized when
the polarization is perpendicular to the symmetrical axis of CH 2
groups. Therefore, in a CARS microscope, half way plates are
installed to adjust the polarization of excitation beams according
to the orientation of myelin fi bers in the sample.
The polarization dependence of CARS signal is able to be used
to determine the orientation and ordering degree of lipid in myelin
based on the ratio of CARS signals ( I II / I ⊥ ). The information of
ordering degree of lipid in myelin can be used to determine
the myelin condition during the degradation process ( 17 ). On the
other hand, the infl uence of polarization dependence of CARS sig-
nal is able to be eliminated through reconstruction of the two
complementary images with perpendicular excitation polarizations
( 18 ) or by using circularly polarized laser beams ( 19 ).

5. Ex Vivo CARS
Imaging of Spinal
and Brain Tissues

Materials used for CARS imaging of myelin in fresh, fi xed, and
frozen tissues:

(a) Krebs' solution: NaCl 124 mM, KCl 2 mM, KH 2 PO 4 1.2 mM,
MgSO 4 1.3 mM, CaCl 2 2 mM, dextrose 10 mM, NaHCO 3
26 mM, and sodium ascorbate 10 mM, equilibrated with 95%
O 2 , 5% CO 2 ; or other artifi cial cerebrospinal fl uid;