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

• DOI: 10.1038/srep46492
• Publication Date: 2017-04-18T09:41:27Z
• Authors: Franziska R. Winter; Maria Loidolt; Volker Westphal; Alexey N. Butkevich; Carola Gregor; Steffen J. Sahl; Stefan W. Hell
• Keywords: STED microscopy

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