Super resolution imaging of proteins’ organization in 3D using polarized single molecule localization

ABSTRACT:

Imaging molecular orientation at the nanoscale in live cells and tissues is fundamental in the understanding of proteins’ organization, which is driven by their structural and conformational properties. Measuring fluorescent molecules’ orientation is an interesting way to approach this problem, providing that the label is rigidly attached to the protein of interest. Despite the great progresses in fluorescence imaging down to nanometric scales, in particular by the use of single molecule (SM) localization-based super resolution imaging, orientations imaging is still only at its early stage. Measuring single molecules’ 3D orientations in addition to their 3D spatial localization is in particular, still today, a challenge, due to the intrinsic coupling of both spatial and orientational parameters in the SM point spread function (PSF) image formation. We present polarized fluorescence microscopy methods that are able to report both orientational and spatial information from single molecules in a non-ambiguous way. These methods, based on polarized splitting imaging or Fourier plane polarization and phase manipulation, give access to orientation parameters including the label wobbling extent, with a few degrees precision, in combination with 10’s nm spatial localization precision. We present the potential of these approaches for the imaging of the nanoscale organization of actin in cells, from very organized stress fiber bundles to complex networks in the cell lamellipodia.