Correlative spectral STED nanoscopy and STED-FCS elucidate the origin of anomalous diffusion in cellular membranes

Advances in our understanding of the nanoscale architecture of the plasma membrane heavily rely on the development of non-invasive experimental methods, particularly of advanced fluorescence microscopy and spectroscopy techniques with high spatiotemporal resolution and sensitivity to local molecular properties. By integrating a spectral detector into a super-resolution stimulated emission depletion (STED) microscope, we unlocked further potential for multifaceted characterisation of membrane heterogeneities. First, we show that compared to the standard ratiometric detection, the spectrally resolved acquisition (together with spectral fitting or phasor analysis) can improve the spectral sensitivity of experiments with STED-compatible polarity-sensitive probes several fold. We further demonstrate that this acquisition scheme allows the use of such probes in combination with other dyes with overlapping spectra, enabling co-localisation of the membrane order maps with other cellular structures of interest, e.g. fluorescently labelled proteins. Finally, we can correlate the obtained membrane order with the detailed diffusion properties, reported by STED fluorescence correlation spectroscopy (STEDFCS). We find that for fluorescent lipid analogues with inhomogeneous partitioning between ordered and disordered membrane domains, including sphingomyelin-based probes, the most apparent trapping sites locate at the boundaries of ordered domains.