Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 232

When excited with ultrashort laser pulses in a microscope, the amplitude of
SHG is proportional to the square of the incident light intensity. The nonlinear
process of SHG takes place through an interaction between the electric field and
its spatial derivative. In SHG, two photons are converted into a single photon at
twice the excitation energy emitted coherently in materials lacking a center of
symmetry. Light emission by in this process is anisotropic and coherent, and its
phase is coupled to the excitation field and hence requires phase-matching effects
between the electric fields associated with the process. In biological materials,
the asymmetrical structures required for efficient second harmonic generation
are cellular membranes that possess such asymmetrically distributed molecular
structures. Other structures within cells and tissues that can produce SHG signal
are collagen and actin filaments. SHG imaging greatly reduces photobleaching
and phototoxicity. Near infrared wavelength excitation allows excellent depth
penetration in biological material and makes live tissue imaging possible. SHG
signals have characteristic polarization, and, therefore, polarization anisotropy can
also be used to determine the absolute orientation and degree of organization of
proteins in a tissue.
Third harmonic generation is another multiphoton process that can be used to
image materials that have some third-order nonlinearity depending on the material
property, symmetry, and the incident light. At the focus of lens, if there is a
discontinuity or inhomogeneity, like an interface between two media, the symmetry
along the optical axis is broken, and a third harmonic signal can be obtained. Better
optical sectioning can be achieved due to the nonlinear process of THG taking place
only at the focal plane. Being the property of all materials, THG can be used for
noninvasive imaging of biological material without the need of labeling.
All of these multiphoton imaging methods have benefited from the recent
advancement in femtosecond laser technology. The state-of-the-art femtosecond
lasers are available in the wavelength range of 750-1,300 nm with ultrashort pulse
widths from a few 100 fs to less than 10 fs, providing a unique light source for
a range of multiphoton processes to be observed and therefore making it easier
than before to characterize and image intracellular and molecular features that are
otherwise unobservable in a biological material.

7.2 Principle of Multiphoton Process

Nonlinear optical processes take place when the response of a material to the
applied optical field depends nonlinearly on the strength of the field. The applied
field changes the distribution of internal charges like electrons, ions, or nuclei
within a molecular system, resulting in a field-induced electric dipole moment
that, in turn, becomes a new source to emit radiation. This is the fundamental