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measurements are needed, attaching a small weight to the bottom of the intestine and
hanging the intestine is a more precise method for determination of intestine length.
Do not let the tissue sit out at room temperature or dry out which will significantly
affect the morphology for imaging and quality of tissue sample for biochemical
analysis.
Gently wash the lumen of the small intestine with ice-cold PBS. To do this, fill a
10-ml syringe with ice-cold PBS, and attach a blunt needle or a pipette tip to the
syringe. Gently flush out all the chyme from the lumen of the tissue. Continue to
flush the tissue until the liquid coming out is clear.
9.1.2 Imaging CLDs in enterocytes
In this section, we describe different methods of visualizing intestinal CLDs. As
mentioned above, intestinal CLDs are dynamic cellular organelles, and their visual-
ization requires attention to timing after a meal or gavage, meal or diet composition,
and location of the intestine sampled.
9.1.2.1 Coherent anti-Stokes Raman scattering (CARS) microscopy
CARS microscopy is a label-free imaging technique that is based on molecular
vibrational spectroscopy. Vibrational spectroscopy relies on the intrinsic chemical
properties of the molecule of interest. In a CARS process, pump (
o p ), Stokes
o 0 p ) frequencies interact with the sample to generate a coherent
optical signal at the anti-Stokes (
(
o s ), and probe (
o as ) frequency. When the difference in frequency
between the pump and the Stokes beams (
o p o s ) is in resonance with a molecular
vibration, the CARS signal can be significantly enhanced. The CARS energy dia-
gram and schematic of the CARS microscope are shown in Fig. 9.1 . The advantages
of CARS are that there is no need for exogenous labeling, the samples do not have to
be fixed, and the signals are highly specific and sensitive to the molecule of interest
( Cheng, 2007; Evans & Xie, 2008 ). For imaging TAG in intact intestinal tissue, the
symmetric CH 2 vibration was measured.
9.1.2.1.1 Materials
1. 35 mm glass bottom petri dish
2. Dulbecco's modified Eagle's medium (DMEM) with 20 mM HEPES, 100 U/ml
penicillin-streptomycin, and 10% fetal bovine serum
3. 12-well tissue culture plates
4. Micro/spring scissors and fine tip tweezers
5. Pipette and pipette tips
9.1.2.1.2 Methods
1. Collect and clean the intestine as described in Section 1.1 .
2. Cut a 5 mm section of interest and place into a 12-well tissue culture plate with
DMEM. Keep tissue plate on ice.
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