The Importance of Axial Resolution in Fluorescence Colocalization

Abstract

Array Tomography (ATomo) is a optical microscopy technique that uses a combination of resin embedding and physical sectioning to facilitate uniform labeling throughout a tissue volume with a high degree of label multiplexing. ATomo‘s thin (70 nm) sections produce super-resolution imaging along the z-axis, at the cost of an involved imaging and volume reconstruction process. We have recently published a related technique, REMI, which embeds cell cultures in resin and images them directly without physical sectioning. While this simplifies the imaging process without sacrificing the multiplexing ability of ATomo, the method is limited to optical sectioning procedures, i.e. ∼1 μm under confocal conditions. The extent to which increased or decreased axial resolution influences multiplexed label localizations has yet to be determined. Here we compare these two methods directly using a described neuronal feature, the hippocampal synapse.