Imaging of Myelin by Coherent Anti-Stokes Raman Scattering Microscopy - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 275

sandwich chamber using a double sided tape (Fig. 3b ). And
the fi xed slice and one drop of Krebs' solution or PBS are
placed into the space between two pieces of cover glass. If
CARS imaging needs to be performed for a long time, a dou-
ble sided tape should be made into a closed loop to prevent the
water around the sample in the center from evaporation. The
slices are handled with a brush.
6. Imaging of frozen slices
Cryosections attached on the gelatin-coated slides are fi rst
dried in the air for half an hour to stick on the slides and then
washed with PBS to remove OCT. Now, cryosections are ready
to image or use for other procedures, such as immunolabeling.
In immunolabeling, dehydration and use of mounting solution
should be avoided. Prior to imaging, a piece of cover glass is
placed on the top of slides using a double-sided tape when the
cryosections are still wet. When imaging, the cover glass side
faces to the objective.

6. CARS Imaging
of Sciatic Nerve
In Vivo

1. Surgery to expose the mouse sciatic nerve
The skin of the mouse upper thigh is shaved and a small longi-
tudinal incision is made after the mouse is deeply anesthetized.
Dissection scissors are used to open the skin. The exposed tis-
sue is gently rinsed with Milli-Q water in order to remove hair
clippings from the incision. The sciatic nerve is clearly visible
through the musculature and no further dissection is required
for imaging. To minimize image distortion caused by blood
pulsing, the femoral artery is temporarily ligated near the upper
thigh with silk sutures during the imaging period. A heating
pad is placed beneath the animal's chest to maintain body
temperature.
2. CARS imaging of sciatic nerve
The anesthetized mouse is placed on its stomach on a home-
built stage which contains a cover glass chamber to facilitate
imaging on an inverted microscope. The animal is positioned
such that the exposed tissue makes direct contact with the
cover glass. Tissue hydration is maintained by adding a small
amount of PBS to the chamber. In in vivo imaging, a 40× water
immersion objective (LUM PlanFl/IR, numerical aper-
ture = 0.8, Olympus) is used to focus the excitation beams into
the sample. This objective has a working distance of 3.3 mm
and relatively large fi eld of view, 353 × 353 m, which facili-

tates in vivo imaging of specifi c structures on an inverted
microscope. The E-CARS signal was collected by the same

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