AbstractTwo‐photon imaging is an innovative optical technique that has quickly become state of the art for imaging fluorescent signals in a variety of organisms. With many advantages over conventional confocal microscopy, such as greater image resolution, deeper access (∼400 µm), and much less photo damage, two‐photon microscopy has already proven to be an extremely useful tool for imaging live cells or tissue. Due to its tremendous versatility, recent efforts have adapted this technique to allow visualization of fluorescent cells directly in living animals. This unit describes a basic procedure for performing two‐photon imaging in vivo as applied to the dorsal surface of the brain in live anesthetized mice or rats. The protocol outlines a surgical preparation to enable the capture of stable, high‐resolution (<1 µm) images of fluorescently labeled neurons in intact brain with very little detrimental effect to either cells or tissue.
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