title: Long-term, high-resolution imaging in the neocortex in vivo.
abstract: INTRODUCTIONIn the neocortex, elucidating the mechanisms of structural plasticity is essential to an understanding of the emergent network properties and fundamental cognitive phenomena, such as memory formation. Time-lapse imaging microscopy has revealed a remarkable array of dynamic activities in dendritic structures in developing cortical tissue in vitro, the developing cortex in vivo, and even the adult neocortex. Chronic high-resolution in vivo imaging of the structure of neurons in the cortex became possible with the invention of two-photon laser scanning microscopy (2PLSM). This technique has key advantages over conventional, single-photon excitation techniques, such as confocal microscopy. Two-photon excitation is the near-simultaneous (within femtoseconds) absorption of two photons coinciding on a fluorophore. The absorption rate depends quadratically on the illumination intensity and is therefore confined to a small volume around the focal point. Scattered excitation light is too weak to generate fluorescence. Thus, the signal is generated exclusively in a tiny focal volume, and all emitted fluorescence photons constitute useful signals. A related advantage is that the longer wavelengths used to generate two-photon excitation penetrate scattering tissue more efficiently than the shorter wavelengths used to generate single-photon excitation of the same fluorophores. The upper layers (1-4) of neocortex are ideal for high-resolution 2PLSM imaging. This protocol describes experimental procedures for in vivo imaging in the neocortex.
authors.forename: Brian E
authors.initials: BE
authors.lastname: Chen
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Trachtenberg
authors.forename: Anthony J G D
authors.initials: AJ
authors.lastname: Holtmaat
authors.forename: Karel
authors.initials: K
authors.lastname: Svoboda