Principles of Multiphoton-Excitation Fluorescence Microscopy
Adapted from “Principles of Multiphoton-Excitation Fluorescence Microscopy,” Chapter 7, in Imaging in Neuroscience and Development (eds. Yuste and Konnerth). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.INTRODUCTION
Fluorescence microscopy has been gaining importance in quantitative biological research due to dramatic improvements in fluorophores, optical systems, light sources, and detectors. In particular, confocal fluorescence microscopy, usually by laser scanning, has for the first time allowed the observation of biological processes with high spatial resolution inside intact living tissue. Often called “optical sectioning,” this method allows spatial reconstruction of 3D specimens without the use of a microtome. This article presents the physical mechanisms upon which the properties of multiphoton microscopy are based and discusses some practical aspects of its implementation.
