Abstract
Single-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules’ orientation in addition to their localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization-splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules’ localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility.
Highlights
Single-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, it does not provide information on their conformation and orientation, which are key functional signatures
We focused on ventral stress fibers, both ends of which associate with focal adhesions (FAs) on the ventral surface of the cell; on dorsal stress fibers, with one end associating with FAs on the ventral surface and the other end extending upwards toward the dorsal cell surface; and on meshworks (Fig. 2a). 4polar-Stochastic Optical Reconstruction Microscopy (STORM) images of an actin-stained cell are shown in Fig. 2b–d, which depict, respectively, the single-molecule localization image (STORM), the ρ and the δ images from the same cell
To test the hypothesis of possible filaments oriented off-plane in the observed regions of interest (ROI), we investigated the correlation of δ and ρ with the single-molecule detection parameters, in particular their intensity, which is expected to decrease when fluorophores are tilted off plane due to their lower photo-excitation, and the point spread function (PSF)
Summary
Fluorophores attached to a protein act as emission dipoles. While their position is directly defined as the center of their PSF image, their orientation is not directly extractable. A fluorophore is represented by its mean orientation (ρ,η) averaged over the imaging integration time, and its wobbling angle (δ3D) explored during this integration time (Fig. 1a). The goal of 4polar-STORM is to measure the fluorophore’s orientation and wobbling projected in the sample plane (ρ,δ). (Fig. 1a), based on the projection of the fluorescence signal on four polarizations channels along the directions 0°, 45°, 90°, and 135°. Respectively (0° corresponding here to the horizontal direction of the sample) (Fig. 1b), under total internal reflection with close to isotropic excitation in the sample plane.
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