Quantitative detection of optical anisotropy of single microtubules by polarization-sensitive interferometric scattering microscopy

Abstract

Microtubules (MTs) are ubiquitous cytoskeletal biopolymers essential for diverse cellular processes. MTs consist of strictly ordered tubulin dimers arranged into hollow cylindrical filaments and are known to be optically anisotropic, which enables their direct observation in microscopes based on polarization contrast. However, there are no experimental data to quantify the relation between the momentary optical anisotropy of the MT and the immediate arrangement of proteins in the MT structure. In this work, we introduce polarization-sensitive microscopy based on interferometric detection of scattering to quantify the scattering anisotropy of single unlabeled MTs with high precision. Our data explain the structural origin of MT anisotropy with a marginal contribution of the intrinsic asymmetry of a single tubulin molecule. We monitor changes in the MT scattering resulting from the binding of tau proteins to single MTs with a resolution of several proteins per diffraction-limited spot. We associate the changes in the contrast as well as in the scattering anisotropy with the formation of a shell around the MT formed by densely packed tau proteins. Our experimental results match closely with the theoretical model of the MT and include quantitative details about the polarization-dependent interaction of light with biological matter.

Single-Shot Quantitative Polarization Imaging of Live Cancer Cells

“…Oldenburg and co-workers demonstrated quantified two-dimensional (2D) distributions of the birefringence by replacing the polarization compensators with liquid crystal (LC)-based variable retarders in the conventional Pol-scopes, , and they used polarization microscopy in the reproductive biology and other areas. , However, the requirement to acquire multiple images at different retarder delays has constrained the usefulness of such pol-scopes for fast and live imaging. Few single-shot polarization measurement techniques have been developed using specialized optical elements such as Wollaston prism, grating, etc. , Recently, single-shot off-axis holographic imaging systems have been developed by using a fixed carrier frequency by polarization beam splitter or Wollaston prism. , Spatially resolved polarization mapping has also been demonstrated using a digital holography approach where two orthogonal polarized light fields are recorded with different carrier frequencies in the single recording of the hologram. , This technique brings tunability in the carrier frequencies in the hologram recording process and is, hence, suitable for objects with higher bandwidths.…”

“…31,23 Recently, single-shot off-axis holographic imaging systems have been developed by using a fixed carrier frequency by polarization beam splitter or Wollaston prism. 32,33 Spatially resolved polarization mapping has also been demonstrated using a digital holography approach where two orthogonal polarized light fields are recorded with different carrier frequencies in the single recording of the hologram. 34,35 This technique brings tunability in the carrier frequencies in the hologram recording process and is, hence, suitable for objects with higher bandwidths.…”