Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam. Unmixing of the different marker signals based on the simultaneous readout of all channels is performed with a non-negative matrix factorization algorithm. We illustrate the approach showing four-colour nanoscopy of fixed and living cellular samples.

. Unmixed STED data of a three-colour fluorescent bead sample.
The sample consisted of 100 nm crimson (a), 200 nm dark red (b) and 100 nm far red (c) beads.
(d) overlay of the individual unmixed channels. grey: crimson, green: dark red, red: far red beads. Excitation wavelength was 656 nm (5 µW) and STED wavelength was 775 nm (200 mW). For more details see 1 . Data is counts registered (colourmaps indicated). Scale bars: 1 µm. Figure S3. Unmixed STED data of a two-colour live-cell sample for calibration. HeLa cells were stained with 580CP-Halo-Sec61β (ER) and 620CP-Snap-OMP25 (outer mitochondrial membrane). (a,b) unmixed STED data for 580CP (b) and 620CP (b). (c) overlay of (a) and (b). Blue: 580CP -ER, yellow: 620CP -mitochondria. Excitation was at 612 nm with 5 µW and a STED power of around 60 mW at 775 nm. Scale bars: 2 µm. Based on the absorption and emission spectra as well as the detection windows indicated in Supplementary Figs. S5 and S6, bleedthrough can be rationalized by integration. Note however that the spectra may be subject to changes due to coupling to an antibody or embedding in a mounting medium; the spectra shown here represent the dyes in solution as specified by the manufacturers. For the percentage distribution of a certain dye in a specific detection channel see Supplementary Tables S6-S9, where the transfer matrices A for the respective samples are stated.

Supplementary
Supplementary Table S1. Minima and maxima of the adjusted colourmaps of the fourcolour fixed-cell sample of Fig. 4  Note that the maximum of Atto594 is higher in STED than in confocal imaging, in contrast to the other dyes. This is due to the seven-fold longer acquisition time during STED compared to confocal imaging and the fact that Atto594, being the bluest of the four dyes, has the lowest STED switch-off efficiency, which leaves a larger number of signaling on-state dyes per scanning step.

SiR-Halo
The dye SiR and its tagged derivative SiR-Halo were prepared according to the reported procedure 4 .