Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena. 1. Introduction Scientific research keeps on developing new technologies to enable a high resolution optical diagnosis based on in vivo imaging of the skin and its components, aiming both at avoiding scars due to unnecessary biopsies and skin resections and providing a support for histopathology, that, in spite of remaining the Gold Standard for diagnosis, does not always show a satisfactory interobserver agreement. One of the most recent clinical imaging technologies is multiphoton tomography (MPT), which is becoming established as the preferred method for image living cells with submicron resolution [1–10]. 2. Principles of Functioning of Multiphoton Laser Tomography Multiphoton laser microscopy (multiphoton laser tomography, MPT) provides instant imaging of living skin at a cellular and subcellular level. MPT can exploit autofluorescence of intrinsic tissue fluorophores and nonlinear harmonic generation from tissue matrix components such as collagen, thereby enabling functional and structural imaging of unstained biological tissue [1–10]. Whereas, for conventional confocal fluorescence microscopy, fluorophores are excited by absorption of individual photons in the visible or ultraviolet spectrum, MPT excitation entails the simultaneous absorption of two or more photons of longer wavelength, usually in the near-infrared spectrum. This longer wavelength infrared radiation undergoes less scattering than visible light and can thus facilitate high-resolution imaging deeper into biological tissue. Efficient MPT excitation usually requires ultrashort femtosecond laser pulses, and these are also efficient in
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