The increasing use of tissue clearing techniques underscores the urgent need for cost-effective and simplified deep imaging methods. While traditional inverted confocal microscopes excel in high-resolution imaging of tissue sections and cultured cells, they face limitations in deep imaging of cleared tissues due to refractive index mismatches between the immersion media of objectives and sample container. To overcome these challenges, the RIM-Deep was developed to significantly improve deep imaging capabilities without compromising the normal function of the confocal microscope. This system facilitates deep immunofluorescence imaging of the prefrontal cortex in cleared macaque tissue, extending imaging depth from 2 mm to 5 mm. Applied to an intact and cleared Thy1-EGFP mouse brain, the system allowed for clear axonal visualization at high imaging depth. Moreover, this advancement enables large-scale, deep 3D imaging of intact tissues. In principle, this concept can be extended to any imaging modality, including existing inverted wide-field, confocal, and two-photon microscopy. This would significantly upgrade traditional laboratory configurations and facilitate the study of connectomes in the brain and other tissues.

Neurons generate and propagate electrical pulses called action potentials which annihilate on arrival at the axon terminal. We measure the extracellular electric field generated by propagating and annihilating action potentials and find that on annihilation, action potentials expel a local discharge. The discharge at the axon terminal generates an inhomogeneous electric field that immediately influences target neurons and thus provokes ephaptic coupling. Our measurements are quantitatively verified by a powerful analytical model which reveals excitation and inhibition in target neurons, depending on position and morphology of the source-target arrangement. Our model is in full agreement with experimental findings on ephaptic coupling at the well-studied Basket cell-Purkinje cell synapse. It is able to predict ephaptic coupling for any other synaptic geometry as illustrated by a few examples.