3D d STORM imaging of fixed brain tissue

Abstract

Central nervous system tissue contains a high density of synapses each composed of an intricate molecular machinery mediating precise transmission of information. Deciphering the molecular nanostructure of pre- and postsynaptic specializations within such a complex tissue architecture poses a particular challenge for light microscopy. Here, we describe two approaches suitable to examine the molecular nanostructure of synapses at 20–30 nm lateral and 50–70 nm axial resolution within an area of 500 μm × 500 μm and a depth of 0.6 μm to several micrometers. We employ single-molecule localization microscopy (SMLM) on immunolabeled fixed brain tissue slices. tomoSTORM utilizes array tomography to achieve SMLM in 40 nm thick resin-embedded sections. dSTORM of cryo-sectioned slices uses optical sectioning in 0.1–4 μm thick hydrated sections. Both approaches deliver 3D nanolocalization of two or more labeled proteins within a defined tissue volume. We review sample preparation, data acquisition, analysis, and interpretation.