Quantitative mapping of membrane nano-environments through single-molecule imaging of solvatochromic probes

Session

Session 5 - Super-resolution and Nanoscale Imaging

Authors

Luca Panconi (3), Jonas Euchner (1), Maria Makarova (2), Dirk-Peter Herten (1), Dylan Owen (3), Daniel Nieves (3)

Affiliations

1. Centre of Membrane Proteins and Receptors, Institute of Cardiovascular Sciences, School of Chemistry, University of Birmingham, Birmingham
2. Centre of Membrane Proteins and Receptors, School of Biosciences, Institute of Metabolism and Systems Research, University of Birmingham, Birmingham
3. Institute of Immunology and Immunotherapy, Centre of Membrane Proteins and Receptors, University of Birmingham, Birmingham

Keywords

lipid order, membrane domains, super-resolution, single molecule fluorescence

Abstract text

It is hypothesised that the mammalian cell plasma membrane contains domains with varying lipid composition and biophysical properties. Studying membrane lipid domains is challenging due to their predicted morphological similarity to the bulk membrane and length scales below the diffraction-limited resolution limit. To address this, we combine the fluorogenic, solvatochromic probe di-4-ANEPPDHQ which reports on its biophysical environment through changes in its fluorescence emission, with spectrally-resolved single-molecule localisation microscopy (SMLM). Data takes the form of a marked point pattern comprising nanometre-precision localisation coordinates and a generalised polarisation (GP) value related to the probe’s environment. Further, we introduce Point Label Analysis of Super-resolved Membrane Attributes (PLASMA), a software package for analysing marked point patterns, with particular emphasis on probing SMLM data. We find that quantification algorithms which exploit topological data analysis allow the detection and mapping of membrane lipid nano-domains in both artificial membranes and live cells. The combination of environmentally-sensitive fluorophores, multi-modal SMLM and new analysis methods represents a powerful tool for mapping membrane biophysical properties at nanometre resolution, and cellular nano-environments in general.

Figure caption: Marked point patterns expressing Generalised Polarisation (GP) marks are acquired through SMLM. Novel quantitative analysis techniques highlight regions of atypical membrane order. Domains are identified at sub-300nm radii with average GP values of ~0.25 above the global mean.

References

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2. Owen, D. M., G. Ashdown, 2015. Imaging Membrane Order Using Environmentally Sensitive Fluorophores. Methods. Mol. Biol. 1232:115–122.

3. Parasassi, T., et al, 1990. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys. J. 57:1179–1186.

4. Sezgin, E., et al 2015. Spectral imaging to measure heterogeneity in membrane lipid packing. Chemphyschem. 16:1387–94.