Biology Reference
In-Depth Information
FIGURE 9.1
Schematic of the CARS microscope. The pump and Stokes lasers generated from two
tightly synchronized Ti:sapphire lasers (Tsunami, Spectra-Physics) are collinearly combined
and directed into a laser scanning confocal microscope (FV300
IX71, Olympus Inc.).
External PMTs are used to detect the CARS signal. The CARS energy diagram is shown above
the setup.
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3.
Cut the section of interest longitudinally with a sharp pair of spring scissors being
careful not to crush the villi in surrounding areas.
4.
On a glass bottom petri dish for imaging, pipet 100
l of DMEM and using a
fine tip tweezer gently lay open the intestine flat on the DMEM droplet for
luminal imaging. Gently remove most of the DMEM from the petri dish, and
allow the tissue to lightly attach to the surface of the petri dish.
5.
Small intestine tissue is ready for imaging. All imaging should be completed
within 3 h of harvesting the tissue.
6.
CARS imaging procedures (
Zhu et al., 2009
): Pump and Stokes lasers are
generated from two synchronized Ti:sapphire lasers (Tsunami, Spectra-Physics,
Mountain View, CA), with a pulse width of 5 ps. These two lasers are tightly
synchronized (Lock-to-Clock, Spectra-Physics), collinearly combined, and
directed into a laser scanning confocal microscope (FV300/IX71, Olympus
America, Center Valley, PA). The average powers of the pump and Stokes beams
at the sample are 40 and 30 mW, respectively. The epi-detected CARS (E-CARS)
signals are collected by the same objective for focusing, while the
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