In vivo multiphoton fluorescence microscopy allows imaging of cellular structures in brain tissue to depths of hundreds of micrometers and, when combined with the use of activity-dependent indicator dyes, opens the possibility of observing intact, functioning neural circuitry. We have developed tools for analyzing in vivo multiphoton data sets to identify responding structures and events in single cells as well as patterns of activity within the neural ensemble. Data were analyzed from populations of cerebellar Purkinje cell dendrites, which generate calcium-based complex action potentials. For image segmentation, active dendrites were identified using a correlation-based method to group covarying pixels. Firing events were extracted from dendritic fluorescence signals with a 95% detection rate and an 8% false-positive rate. Because an event that begins in one movie frame is sometimes not detected until the next frame, detection delays were compensated using a likelihood-based correction procedure. To identify groups of dendrites that tended to fire synchronously, a k-means-based procedure was developed to analyze pairwise correlations across the population. Because repeated runs of k-means often generated dissimilar clusterings, the runs were combined to determine a consensus cluster number and composition. This procedure, termed meta-k-means, gave clusterings as good as individual runs of k-means, was independent of random initial seeding, and allowed the exclusion of outliers. Our methods should be generally useful for analyzing multicellular activity recordings in a variety of brain structures.
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