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.

Figure 1. Setup and DNA origami imaging.

(a) Schematic drawing of the main optical parts of the STED microscope: S Sample, OL objective lens, P z-piezo translator, BS beam scanner, λ/2 half-wave plate, λ/4 quarter-wave plate, DM dichroic mirror (DM1: 460DCXRU, DM2: ZT594RDC, DM3: T525LPXR), NF notch filter (532 nm Notch), PH pinhole, VPP vortex phase plate, AOM acousto-optical modulator, F filter (F1: 514/30, F2: 562/40, F3: 532 nm Notch, F4: 620/14), APD avalanche photodiode detector. (b) Normalized absorption and emission spectra of the dyes Atto430LS, AlexaFluor488 and Atto532 utilized for three-colour imaging, as well as related laser lines and detection windows. (c) Schematic drawing of a DNA origami molecule immobilized on a glass surface through biotin-neutravidin links. Two positions on the origami located at a distance d from each other are labelled with dye molecules. (d) Inset shows STED image of a single DNA origami molecule with spots marked by Atto532 molecules at d = 50 ± 5 nm. The line profile drawn across the DNA origami shown in the inset was fitted by two Lorentzian functions. Fitting yielded a spot separation distance of 51 nm with single spot full width at half maxima (FWHM) of <35 nm. (e) Same as (d), but for an origami with d = 70 ± 5 nm labelled with AlexaFluor488. The corresponding fit yielded a separation distance of 71 nm and single-peak FWHM of <50 nm. (f) Same as (e) for an Atto430LS-labelled origami. The corresponding fit yielded a separation distance of 66 nm and peak FWHM of <40 nm.

Figure 2. Dual-colour STED nanoscopy.

(a) STED image of a living HeLa cell expressing vimentin-EGFP (green) and Pex3-Halo-tag stained with 540R-Halo (red). The confocal counterpart is shown in the lower-right corner. (b) Close-ups of the region marked in (a) of the two colour channels, demonstrating the absence of cross-talk and the high signal-to-background ratio in both channels. (c) STED image of a fixed hippocampal axon stained against neurofascin with AlexaFluor488 (left) and actin with phalloidin-Atto532 (centre) reveals the characteristic periodic lattice. Corresponding confocal images are shown in the lower-right corner. (d) Line profiles drawn parallel to the line shown in (c) for the two channels illustrate the interplay of neurofascin and actin. Counts of three pixels perpendicular to the line were summed up. Data of all figure panels were smoothed with a 1.0 pixel wide Gaussian. No linear unmixing was performed.

Figure 3. The subcortical periodic actin/betaII spectrin lattice is discontinued at synaptic sites.

(a) Three-colour STED image of a dendrite decorated with spines and stained with betaII spectrin (AlexaFluor488, yellow), phalloidin (Atto532, magenta), and Homer (Atto430LS, cyan) shows the periodic spectrin organization. (b) Single-channel images of the spine indicated in (a). Magenta and cyan dashed lines highlight the shape of the spine and the position of the PSD, respectively. BetaII spectrin enters into the spine neck but does not reach the PSD. (c) Same as (a), but for the presynaptic site, identified by Bassoon staining instead of Homer. (d) Single channel images of the boxed area in (c). Magenta and cyan dashed lines highlight the position of the actin cage and of Bassoon, respectively. All panels of the figure show linear unmixed STED data, smoothed with a 1.0 pixel wide Gaussian function. Raw data of the image in panel (a) is shown in Supplementary Fig. 4d.