Supplementary Figure 7: Motion correction of MiniLFM data.

(a) Lateral motion-induced image shift versus time during a 10-minute, regular (non-LFM) Miniscope recording from hippocampal CA1 of a mouse moving back and forth on a linear track. One pixel corresponds to ~1 µm in the sample. Quasi-periodic peaks in traces occur when animal initiates track traversal after reward administration at track ends. Right panels: Histograms of shifts in the lateral-medial (L-M) and anterior-posterior (A-P) directions. (b) Illustration of motion correction pipeline (see Supplementary Note 3 and Online Methods): A motion metric (magenta trace) is calculated on the raw data and thresholded at a high value (black horizontal line) to detect strong motion bursts. The time series of raw data frames (illustrated as a film strip) is split into low-motion segments (black rectangles) that lie in between the motion bursts. The low-motion segments are SID-processed individually, which yields a set of neuron footprints (blue images) and corresponding activity traces (green) for each segment. The neurons from each segment are then pooled, merging strongly overlapping footprints and extracting the corresponding activity traces across the full dataset. To further reduce motion artefacts and interpolate motion-affected frames, the same motion metric is now thresholded at a lower value, and the time steps above threshold are masked out (grey areas) from the activity traces. For each neuron, a model is trained on the motion-masked activity traces that estimates the underlying firing rate (black trace) and GECI response time constants. The model is then used to interpolate the masked-out time steps, yielding a motion-corrected estimate of the physiological neural activity (blue trace). (c) Examples of motion-corrected activity traces (see also Supplementary Fig. 8). Top: Motion metric calculated from the raw data (magenta trace), with black horizontal line indicating the threshold above which frames are considered motion-affected. Bottom: Three examples of motion-corrected Ca2+ activity signals (blue) together with maximum-likelihood-estimate of underlying firing rate (black), each corresponding to a line of the heatmap shown in Fig. 2c.