Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses

Abstract

Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels (“multilevelSTED”) optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.

Structural organization of the actin-spectrin–based membrane skeleton in dendrites and soma of neurons

“…It is worth noting that, because a dendrite often contained both periodic and irregular regions, the fraction of dendrites exhibiting at least one periodic region was quite large (>80%), consistent with previous results (15,16). We also observed heterogeneity among dendrites with some dendrites being covered by the 1D MPS structure substantially more extensively than the others.…”

“…We and others have also observed the 1D MPS in dendrites (9,10,13,15,16). Here, we systematically investigated the distributions of four MPS components, βII-spectrin, βIII-spectrin, actin and adducin, in cultured hippocampal neurons.…”

“…Although the MPS is generally observed throughout the entire axonal shaft of all mature neurons (1,9,10,12,13), the extent to which this structure is present in dendrites is unclear and varies from study to study (9,10,13,15,16). In the two early studies reporting the 1D MPS in dendrites, a relatively small fraction of dendrites in wild-type neurons were observed to exhibit this structure (9, 10), whereas later studies reported either sparse or extensive presence of the 1D MPS in dendrites (13,15,16). It has been shown that the MPS develops early in axons, starting to appear as early as day 2 in vitro (DIV 2) (9, 10).…”

“…In this structure, short actin filaments capped by adducin are organized into repetitive, ring-like structures that wrap around the circumference of the axon underneath the axonal membrane, and adjacent actin rings are connected by spectrin tetramers, forming a long-range quasi-1D periodic structure with a periodicity of ∼190 nm (1). This periodic structure is formed extensively throughout the axonal shaft, including the axon initial segment and the unmyelinated distal axons, as well as the nodes of Ranvier and internodal segments in myelinated axons (1, 9-14), but appears to be perturbed at synaptic sites (13,15,16). It is a highly prevalent structure observed in many different types of neurons, including both excitatory and inhibitory neurons, as well as neurons in both central and peripheral nervous systems (12, 13), and is evolutionarily conserved across diverse animal species, ranging from Caenorhabditis elegans and Drosophila to rodents and humans (13).…”

Super-resolution microscopy and its applications in neuroscience

“…27 Despite its short time from the invention, STED microscopy has made many contributions to the observation of subcellular structures such as endoplasmic reticulum, 28 mitochondria 29,30 and cytoskeletons. 31,32 Because of complicated components involved in signal transduction between cells, dualcolor STED [33][34][35] and even multi-color STED 36,37 have been established to study several active complexes at the same time. Besides, 3D STED is also available for biologists which provides 3D imaging with the resolution of 45 Â 45 Â 108 nm in a cell originally.…”