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T issue Imaging with CARS Micr oscopy 335

(a) (b)

Tissue

Glass

FIGURE 11.8 Effect of linear scattering on quality of CARS tissue images.
(a) Two-photon-excited autofl uorescence xz-image of chicken breast tissue,
excited at 800 nm using a 1.2-NA water immersion lens. Total scan depth is
80 μm. (b) Simultaneously detected CARS signal from water. Note that the
penetration depth of CARS is less than for two-photon-excited fl uorescence,
and that the coherent third-order signal is more sensitive to linear scattering
at dense objects. Linear scattering is evident from the apparent shadowing
streaks in the image.

understanding of these effects will help the interpretation of the
images.

11.6.2 Backscattering in Tissues
In addition affecting the incident light and the CARS signal genera-
tion process, scattering also acts on the generated CARS light.
Although postgeneration does not decrease the number of CARS
photons produced, it changes the propagation direction of the CARS
emission by redistributing it over a large cone angle. Since the unaf-
fected CARS signal is predominantly propagating in the forward
direction, 93,94 postgeneration scattering will generally lower the
amount of photons that can be captured in the forward direction and
increase the number of photons that can be intercepted in the back-
ward direction. The latter notion is particularly relevant for CARS
imaging in thick tissues when forward detection is limited due to the
opacity of the tissue and the signal can only be detected in the back-
ward direction.
In Fig. 11.9 the amount of CARS light that is scattered back from
a tissue phantom is plotted as a function of phantom thickness. It is
clear that for tissues thicker than a couple of hundred micrometers, a
significant fraction of the forward propagating light is redirected into
the epidirection. Backward scattering of forward propagating CARS
95