Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 240

in refractive index or third-order nonlinear susceptibility, third harmonic light
is produced, and as a result of this interface effect, third harmonic imaging is
possible and can be applied to study transparent samples having low contrast
[ 49 ]. Volumetric imaging has also been done in both biological and nonbiological
specimens demonstrating the dynamical imaging properties of THG in live samples
[ 50 , 51 ]. Third harmonic generation near the focal point of a tightly focused laser
beam can be used to probe small structures of transparent samples at the interfaces
and inhomogeneities. Due to the coherent nature of third harmonic process, the axial
resolution of THG microscopy is, however, equal to the confocal parameter of the
fundamental beam [ 49 ]. The signal level in THG microscopy can be optimized by
the influence of sample structure and beam focusing [ 52 ], where they controlled the
signal level by modifying the Rayleigh range of the excitation beam and applied
this method for the contrast modulation in THG images of Drosophila embryos.
Besides structural and beam shape dependence, the THG is also sensitive to the
local differences in the refractive index, third-order susceptibility, and dispersion.
This aspect of THG was used to image lipids that are present in many biological
cells and tissues [ 53 ]. In this study, a multimodal technique for microscopy was used
to image lipid bodies, fluorescent compounds in tissues, and extracellular matrix
by combining two-photon fluorescence, SHG, and THG microscopy. Another
multimodal nonlinear microscopic technique based on a femtosecond Cr:forsterite
laser was used to simultaneously generate SHG, THG, two-photon, and three-
photon fluorescence images [ 45 ].
Third harmonic generation microscopy is shown to be particularly suitable for
imaging biogenic crystals and polarization sensitive imaging of crystalline structure
in biological samples [ 54 , 55 ]. THG has been used for the characterization of
saline solutions and structural changes in collagen [ 56 ]. It is shown that THG
epidetection is generally possible when the sample structure is embedded in a
scattering, nonabsorbing tissue with thickness greater than the reduced scattering
mean free path [ 57 ]. A combination of THG, SHG, and TPEF image contrast
methods on the same microscope is also being used for simultaneously imaging
the biological material [ 58 , 59 ].

7.3.4 Coherent Anti-Stokes Raman Scattering (CARS)

CARS microscopy is based on the use of two different laser beams, pump and the
Stokes beams, that are tuned to match the energy gap between two vibrational levels
in a molecule. CARS is used to probe chemical bond vibration levels and provides
molecular specificity, particularly for small molecules [ 5 , 12 ].
Stronger vibrational signals can be obtained with CARS microscopy that is
a nonlinear Raman scattering method. CARS is a four-wave mixing process as
shown in Fig. 7.1 f, in which a pump beam at frequency ! P and a Stokes beam
at frequency ! S interact with a sample to generate an anti-Stokes signal at
frequency ! AS D 2! P ! S . This expression can be written as ! AS D ! P C ! vib that