IntroductionÂ
Two-photon excitation (TPE) microscopy1 has evolved as an alternative to conventional single-photon confocal microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly scattering samples, and reduced phototoxicity, enabling long-term imaging of photosensitive biological specimens. The inherent three-dimensional resolution of TPE microscopy has been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and submicrometer scale within thick biological specimens proved critical. For example, TPE of the calcium-sensitive fluorophore rhod-2 has been used to resolve differences in the kinetics of intracellular calcium ([Ca2+]i) transients in donor myocytes and juxtaposed host cardiomyocytes deep in Langendorff-perfused mouse hearts following intracardiac transplantation of fetal cardiomyocytes and skeletal myoblasts.2, 3 For neuroscientists, TPE microscopy has become an invaluable tool for studying calcium dynamics in thick brain slices and live animals4, 5 and for long-term imaging of neuronal development.6 The spatial confinement of TPE has also been used for three-dimensional photolysis of caged compounds in femtoliter volumes7–9 or diffusion measurements by two-photon fluorescence photobleaching recovery.10,11 This article describes the basic physical principles of TPE and reviews the advantages and limitations of its use in laser scanning fluorescence microscopy of cells and tissue. Illustrative examples will demonstrate how the unique features of this imaging technique have provided novel insights into cellular and subcellular mechanisms that could not have been obtained otherwise.
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