Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 234

Fig. 7.1 Energy diagrams of various optical excitation methods that are used for imaging contrast
in biomedical field. The incoherent (nonresonant) modes of excitation resulting in fluorescence are
( a ) 1-photon excitation, ( b ) 2-photon excitation, and ( c ) 3-photon excitation. The resonant coherent
modes of multiphoton excitation are ( d ) SHG, ( e ) THG, and ( f ) CARS resulting in a coherent signal

7.3 Multiphoton Imaging Modes

SHG, THG, and TPEF have become the most widely used contrast modes of
multiphoton imaging. The nonlinear contrast mechanism depends on the second-
and third-order nonlinearities as described by Eq. 7.1 , which can be produced by
the light-matter interaction at the focus of a high numerical aperture microscope
objective. As the nonlinear effects are proportional to the second or third power of
the fundamental light intensity, light only at a very tiny focal volume interacts with
the sample, eliminating the out-of-focus light, resulting in an inherent optical sec-
tioning. Selecting a nonlinear contrast method of choice, a multiphoton microscope
can be made by raster scanning a laser beam across the focal plane of a microscope
objective and measuring the signal intensity as a function of the focal spot position.
The 3-D images can be obtained by moving the sample or the objective along the
laser beam axis. A typical multiphoton imaging system is shown in Fig. 7.2 .