Quantifying nanoscale T-cell membrane protein distributions across complex topography large depth-of-field localisation microscopy

Session

Session 5 - Super-resolution and Nanoscale Imaging

Authors

Aleks Ponjavic (1)

Affiliations

1. University of Leeds

Abstract text

The nanoscale topography of cells plays a key role in a variety of biological processes, particularly for T cells, where finger-like structures scan the environment to sense pathogens. Imaging this topography (Fig. 1a) with localisation microscopy in 3D is important, but challenging due to the relatively large scales involved and the requirement to achieve sufficient localisation densities. PAINT can be used overcome these challenges, but is typically hampered by high background, particularly for large depth-of-field 3D imaging.

 

Here we use actively controlled probes to greatly increase the effective concentration in PAINT, without influencing the background. By building up a concentrated and reversible reservoir of photoactivatable or spontaneously blinking probes, we increase the localisation rate of PAINT up to 50-fold (Fig. 1b). We combine this method with large depth-of-field microscopy to demonstrate volumetric super-resolution imaging of entire T-cell membranes (Fig. 1c). Finally, the new method is combined with conventional membrane protein localisation microscopy to characterise nanoscale T-cell protein distributions across the complex topography of the cell membrane (Fig 1d). By considering the local distribution of neighbouring proteins we demonstrate how eigenvector analysis can be used to decipher preferential organisation and clustering of membrane proteins.