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Abstract

Multiphoton laser−scanning microscopy is still developing rapidly, both technologically and by broadening its range of application. Technical progress has been made in the optimization of fluorophores, in increasing the imaging depth of multiphoton microscopy, and in microscope miniaturization. These advances further facilitate the study of neuronal structure and signaling in living and even in behaving animals, in particular in combination with the expression of fluorescent proteins. In addition, nonlinear optical contrast mechanisms other than multiphoton excitation of fluorescence are being explored. Nonlinear optical microscopy, especially two−photon microscopy, has found widespread applications in neuroscience. Ongoing technological improvements continue to improve existing microscopy tools and open the way for experiments previously considered impossible