Extended Data Fig. 8: Analysis of motion artifacts.
From: Miniature three-photon microscopy maximized for scattered fluorescence collection
(a) Three-dimension reconstruction of a 1038-μm stack of the YFP-labeled neuronal structure in PPC L5/6 and underlying hippocampal CA1 in Thy1-YFPH transgenic mouse. The data was acquired at 2-μm intervals at a frame rate of 8.35 Hz (200 × 200 pixels), and 50 frames were averaged at each slice. (b) Representative 50-frame averaged x-y images at L5 and hippocampal CA1. The experiment was independently repeated n = 4 times. (c) Lateral motion artifacts in PPC L5 and hippocampus CA1 in anaesthetized, head-fixed and freely behaving mice. The anesthesia and head-fixed data were 2,000 frames and the freely behaving data were 13,900 frames. The experiment was independently repeated n = 2 times. (d) Schematic diagram of the analysis method for axial motion artifacts. We obtained a 100-μm stack at 2-μm intervals near the focal plane prior to time lapse imaging. Then, we performed correlation analysis between a time lapse frame with each layer of the volumetric stack. The axial position with the greatest correlation coefficient was regarded as the current focal plane. (e, f) Axial motion artifacts in PPC L5 (e) and hippocampus CA1 (f) under different imaging paradigms. The data source of (e, f) were the same as in (c), the experiment was independently repeated n = 2 times. (g) Calcium imaging in a mouse under head-fixed and freely behaving conditions. Left, a 50-frame averaged frame showing the hippocampal CA1 region, depth and laser power used. Right, time courses of calcium activities of the indexed neurons expressing GCaMP6s in x-y image under head-fixed and freely behaving conditions. The experiment was independently repeated n = 4 times. (h) Relative change of the averaged amplitudes, transient decay time constants and SNRs of single neurons of the freely behaving to head-fixed condition (n = 15 neurons). The box shows 5th and 95th percentile of data in (c, e, f) and 25th and 75th data in (h). Black center line in (c, h) and blue dash line in (e, f), median; whiskers, maximum and minimum.
