Extended Data Fig. 8: Correlating structure and morphodynamics with Ca²⁺-activity.

a, Confocal overview images in organotypic hippocampal slice cultures from Prox1-cre::Ai95 mice with mossy fibers conveying excitatory input from DG granule cells via the DG hilus (right) to CA3 pyramidal neurons (left). Scale bar: 25 µm. b, Isotropically super-resolved, volumetric LIONESS acquisitions in the stratum lucidum of CA3 at two timepoints (left: 0 minutes, right: 10 minutes) revealed morphodynamics of the complex interface between pre- and postsynaptic structures at mossy fiber to CA3 pyramidal neuron synapses. The black arrowhead marks a structure changing over time. The dashed frame in panel a indicates the position of the LIONESS volume. White arrowheads at image edges indicate the corresponding positions of xy- and xz-views. Scale bar: 2 µm. c, Plane from the LIONESS volume overlaid with diffraction-limited (confocal) signal from the calcium indicator GCaMP6f (green). LIONESS images are identical replicates providing structural context to the time-varying Ca²⁺-signals. Scale bar: 1 µm. The GABA_A antagonist gabazine was applied to increase activity. d, GCaMP signal of the conspicuous mossy fiber bouton shown in panel c as a function of time. Total signal from a rectangular region enclosing the mossy fiber bouton (roughly corresponding to the upper right quadrant of the image in panel c) was integrated and normalized to the first frame. e, GCaMP signal as a function of time (and position) recorded as an additional color channel during the volumetric LIONESS acquisition for timepoint 0 min in panel b. The images are representative of n = 4 technical replicates recorded from 3 biological specimens. LIONESS images are maximum intensity projections spanning 150 nm.