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Description:Multiphoton Microscopy Fundamentals and Applications in Multiphoton Excitation Microscopy Two-photon excitation microscopy also referred to as non-linear, multiphoton, or two-photon laser scanning microscopy is an alternative to confocal and deconvolution microscopy that provides distinct advantages for three-dimensional imaging. In particular, two-photon excitation excels at imaging of living cells, especially within intact tissues such as brain slices, embryos, whole organs, and even entire animals. The effective sensitivity of fluorescence microscopy, especially with thick specimens, is generally limited by out-of-focus flare. This limitation is greatly reduced in a confocal microscope, through the use of a confocal pinhole to reject out-of-focus background fluorescence and produce thin less than 1 micrometer , unblurred optical sections. Figure 1 - Two-Photon Jablonski Energy Diagram This article presents a description of the basic physical principles of multiphoton excitation and discusses the advantages and limitations of its use in laser-scanning microscopy. Practical considerations are highlighted in order to illustrate the utility of this technique and demonstrate some of its physical limitations.