In Vivo Imaging of All Cortical Layers and Hippocampal CA1 Pyramidal Cells by Two-Photon Excitation Microscopy

Abstract

Conventional in vivo two-photon microscopy has revealed vital information about neural activity in relation to brain function, despite its limitations in imaging events at depths greater than several hundred micrometers from the surface of the brain. Here, we developed a novel two-photon microscope consisting of a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5 ps, repetition rate of 10 MHz, and a high-sensitivity photomultiplier tube for efficient detection of fluorescence. By applying this newly developed two-photon microscope to in vivo imaging, we were able to successfully visualize hippocampal neurons in dentate gyrus and panoramic views of CA1 pyramidal neurons and cerebral cortex, in both young adult and adult mice. Fine structures of dendrites in CA1 neurons could be visualized with a high peak signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. We hope that our two-photon microscopy system will be applicable to investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.