Abstract
Recent advances in in vivo two-photon imaging have extended the technique to permit the detection of action potentials (APs) in populations of spatially resolved neurons in awake animals. Although experimentally demanding, this technique's potential applications include experiments to investigate perception, behavior, and other awake states. Here we outline experimental procedures for imaging neuronal populations in awake and anesthetized rodents. Details are provided on habituation to head fixation, surgery, head plate design, and dye injection. Determination of AP detection accuracy through simultaneous optical and electrophysiological recordings is also discussed. Basic problems of data analysis are considered, such as correction of signal background and baseline drift, AP detection, and motion correction. As an application of the method, the comparison of neuronal activity across arousal states is considered in detail, and some future directions are discussed.
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