Fig. 4: Molecularly informed reconstruction of living brain tissue.

a, Confocal image of CA1 neuropil in organotypic hippocampal slice culture, with PSD95-HaloTag expressed via a genetic knock-in (PSD95-HaloTag mouse line), labeling all excitatory postsynapses (orange) and virus-assisted delivery of synaptophysin 1-eGFP (SYP1, blue), highlighting a subset of presynaptic terminals. Denoising (Noise2Void⁵¹) applied (top). Scale bar, 2 µm. Overlay with isotropically super-resolved volumetric LIONESS data (middle and bottom). Orthogonal planes in xy and xz directions represented as maximum intensity projections spanning 150 nm, with positions of corresponding planes indicated by arrowheads at image edges. Confocal SYP1 and PSD95 signals extend beyond the 3D super-resolved cellular structures defined by LIONESS. Data are representative for n = 4 biological replicates. b, 3D reconstruction of a selected dendrite (gold) from the same LIONESS volume with all synaptically connected axons as defined by postsynaptic presence of PSD95 (top). Magnified views as indicated by the boxes in the top panel, highlighting diverse geometric arrangements of synaptically connected boutons and spines together with renderings of PSD95 and SYP1 signals (middle). Renderings of molecule locations are based on thresholded confocal signals compressed in z direction to account for anisotropy of the confocal point-spread function. LIONESS planes from the corresponding subvolumes together with molecular information (bottom). Maximum intensity projections spanning 150 nm. Synaptic labeling and imaging together with LIONESS is representative of n = 4 biological replicates. Proofreading of automated segmentation and 3D visualizations were conducted for one dataset. Scale bars, 500 nm (middle and bottom). Scale bars refer to the center of the respective renderings.