Spectral STED microscopy improves spectral sensitivity with polarity-sensitive probes and enables correlative measurements of membrane order and anomalous lipid diffusion

Molecular plasma membrane organization and dynamics play an important role in cellular signalling. 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 spatio-temporal resolution and sensitivity to local molecular properties. However, it remains difficult to combine several of them for a multimodal characterisation that would reduce the possibility of misinterpretations. Here, we integrated a spectral detector into a super-resolution stimulated emission depletion (STED) microscope, achieving three goals. First, we show that compared to the standard ratiometric detection using fixed bandpass filters, the spectrally resolved acquisition together with spectral fitting or phasor analysis improves the accuracy of experiments determining membrane lipid order with polarity-sensitive probes multifold. Secondly, we 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 correlate the obtained membrane lipid order with the anomalous trapped diffusion properties of a fluorescent sphingomyelin lipid analogue in the plasma membrane of living cells, as determined by STED fluorescence correlation spectroscopy, and highlight that some of the most apparent trapping sites locate at the boundaries of local ordered environments discernible by the introduced spectral STED microscopy. With additional measurements in model membranes and Monte-Carlo simulations we conclude that for sub-100 nm ordered environments uneven probe partitioning cannot by itself explain the trapping diffusion of SM in cells.