Extended Data Fig. 4: Multi-Z imaging minimizes axial motion artifacts.

a. During periods of brain motion, fluorescence is redistributed across planes, with cells becoming brighter in some planes and dimmer in others. Top, average change in fluorescence during a period of movement indicated by arrow below in adjacent planes. Bottom, average fluorescence time series corresponding to the cell indicated (yellow arrowhead). b. Top, mean fluorescence (left), difference image prior to (middle) and following (right)lateral motion correction. Sum of 4 image planes shown. During periods of brain motion (indicated by black arrow, bottom), lateral motion correction compensates for brain motion. Bottom, fluorescence time series corresponding to the cell indicated (yellow arrowhead, top). Time series extracted from a single plane(plane 2) before lateral motion correction (light blue, top), after lateral motion correction (dark blue, middle), and from a region of interest (ROI) spanning all four image planes following lateral motion correction (gray, bottom). Artifacts introduced by brain motion are suppressed when extracting time series from multiplane ROIs. Data are representative of axial motion across 34 fields, 5 animals.