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Full system considerations for multiphoton microscopy

Wokosin, D.L. and Girkin, J.M. (2001) Full system considerations for multiphoton microscopy. In: Multiphoton Microscopy in the Biomedical Sciences (Proceedings of SPIE Volume: 4262). SPIE, pp. 231-237.

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A most-important-variables analysis of practical, successful multiphoton excitation fluorescence microscopy is presented. The key strength of multiphoton imaging -- localization of the excitation volume -- helps to decouple the excitation from the emission; the emission no longer needs to be imaged. The presentation starts with maximizing the detected signal and proceeds to the laser source considerations. The main goal is to match the instrumentation to the biological problem. The main aspects covered are... Emission collection: potential signal; Focal plane mismatch: chromatic aberration; Light detected: actual signal; Live cell imaging: preservation of biological function; Deep imaging: preservation of image contrast; Signal production: potential limitations.