Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 236

nonlinear excitation process is indistinguishable from the characteristic fluorescence
in single photon excitation. TPEF microscopy has great impact in the field of
laser scanning microscopy and provides a versatile tool for biological imaging.
The basic principle of TPEF is shown schematically in Fig. 7.1 b. Two photons
from the laser source are absorbed simultaneously by an intrinsic or exogenous
fluorophore molecule. A fluorescent photon is emitted after a short delay and can
be collected to generate an image. TPEF also suffers from photobleaching like all
other contrast mechanisms based on fluorescence and the use of very high intensities
for excitation can lead to higher order 2-photon interactions in the focal volume,
excitation saturation and photodamage [ 17 ].
In TPEF imaging, optical sections can be obtained at greater depths within a
tissue as compared to confocal or wide-field imaging due to the use of longer
wavelengths hence less scattering. The excitation light is not attenuated by a
fluorophore above and below the focal plane of the lens. Since very small amount
of fluorescence is generated away from the point of illumination, therefore, all the
detected photons may be used for imaging regardless of whether they have been
scattered or not. TPEF microscopy has wide range of applications [ 18 - 20 ]. TPEF
has been used to image live tissue at depths of up to 1 mm [ 21 , 22 ], to imaging
cerebral blood flow [ 23 ] and neuronal activity [ 24 ] providing greater understanding
of the function and disorders in the brain. Now, TPEF imaging is being used in
compact and versatile microscopes for in vivo imaging in clinical settings. Fiber-
optic-based imaging methods have also been used to build miniaturized two-photon
microscopes and endoscopes [ 25 - 27 ].

7.3.2 Second Harmonic Generation (SHG)

In SHG process, two photons are converted into a single photon at twice the
excitation energy in materials lacking a center of symmetry. Light emission in this
process is anisotropic and coherent, and the phase of scattered light field 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. The nonlinearity of the SHG process results in a signal that is maximum
at the focus of a microscope, resulting in the intrinsic three-dimensional sectioning
without the use of a confocal aperture. The SHG process greatly reduces out-of-
focus plane photobleaching and phototoxicity. Near infrared wavelength excitation
allows greater depth penetration in biological material, making this method well
suited for studying intact tissue in biomedical field. Information about the organi-
zation of chromophores, including dyes and structural proteins, at the molecular
level can be extracted from SHG imaging data in several ways. SHG signals have
well-defined polarizations, and hence, SHG polarization anisotropy can also be