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

Abstract

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.

Selective flexible packaging pathways of the segmented genome of influenza A virus

“…However, distances were still within the cylinder radius (300 nm, see above) that allowed colocalisation to be detected. Finally, to verify the confocal image-based MSC analysis further, we carried out super-resolution multicolour STED microscopy (STED) 44 with a resolution of 79 nm for a selected set of segments ( Supplementary Fig. 13).…”

Spaser Nanoparticles for Ultranarrow Bandwidth STED Super‐Resolution Imaging

“…Thus, conventional fluorophores, like fluorescent dyes and proteins, semiconductor quantum dots, and upconversion nanoparticles, typically have a spectral linewidth in range of 30–100 nm, which normally allow four to five spectra in visible region without overlap. To alleviate this obstacle, concentrating efforts have been made on aspects of instrument modification, probe design, and data processing, such as single molecular fluorescence spectrum localization, sequence exchange fluorescence probes, and online fluorescence linear split . Nevertheless, solutions to the problem of spectral crosstalk are still highly demanded and yet to be demonstrated.…”

Toxicology in the Super‐Resolution Era

“…Standard staining methods and dyes/ fluorophores can usually be used (based on the depletion laser in the system), and there are no special buffers required (Winter et al, 2017). However, it is often necessary to decrease the secondary dye/fluorophore concentration and blocks appropriately.…”