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The introduction of the confocal microscope and its subsequent development provided an important tool for the biologist and the clinician: a means to achieve in vivo microscopy of cells, tissues, and organs, resulting in images with enhanced resolution, depth discrimination, and contrast. However, as researchers and clinicians pushed the limits of confocal microscopy, it became clear that new microscopes were required to image deeper into thick, highly scattering and absorbing specimens. Furthermore, as new fluorescent probes with absorption bands in the ultraviolet region were being developed for molecular biology, developmental biology, and neurobiology, there was an increasing need for confocal microscopes to operate in the UV region. In addition to the problems of obtaining microscope objectives and other optical components suitable for UV light, it was apparent that the light was toxic to living cells.
The partial solution to these increasingly important limitations came from an unexpected source: the field of nonlinear optics. Part III of the book describes and analyzes the history of nonlinear optics, the development of nonlinear microscopy, and the theory and instrumentation of multiphoton excitation microscopy.
Nonlinear optical spectroscopy preceded the development of and served as the foundation for nonlinear microscopy, which is why it is included in this textbook. Multiphoton excitation microscopy is one type of nonlinear microscopy, and so it is instructive and important to review its antecedents.
The emphasis in this chapter is on experimental studies following the invention of the ruby laser by Theodore Maiman in 1960. While these nonlinear techniques were developed to explore the symmetry-forbidden excited states of molecules that are not realized by linear excitation, they also provided new modes of contrast in microscopic imaging. Together, these technical advances and the advent of lasers that produced femtosecond (fs) pulses made possible the invention of microscopes based on multiphoton excitation processes. Note that picosecond (ps) lasers can also be used with a multiphoton microscope, although they require much higher average power to be equally effective.
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