Long-term, high-resolution imaging in the neocortex in vivo

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.