Figure 1

Schematic diagram of SDOM. (a) SDOM is based on a wide-field epi-fluorescence illumination microscope. The rotary linear polarized excitation is realized by continuously rotating a half-wave plate in front of a laser. Then, the illumination beam is focused onto the back focal plane of the objective to generate uniform illumination with rotating polarization light. The series of fluorescence images excited from different angles of polarized excitation are collected by an EMCCD camera. As illustrated in the inset schematic figure (b), the fluorophores (such as GFP) are linked to the target protein via the C terminus (connected to GFP’s N terminus); the dipole angle of the fluorophore will reflect the orientation of the target protein. (c) Illustration of the principle of the SDOM super-resolution technique. Two neighboring fluorophores with 100 nm distance and different dipole orientations (pseudocolor in red and green) emit periodic signals excited by rotating polarized light. By rotating the polarization of excitation, the emission ratio between the two molecules is modulated accordingly, resulting in their separation in the polarization domain. The sparsity deconvolution can achieve a super-resolution image of effective dipole intensities under polarization modulation; with least-squares fitting, the dipole orientation can be determined. Arrows indicate the directions of dipole orientations. (d) The SDOM result of two intersecting lines, with arrows on top of the super-resolution image, illustrating the dipole orientation and OUF. (e) The corresponding data are represented in (X, Y, θ) coordinates, in which the XY plane is the super-resolved intensity image. From both d and e, we can see that as SDOM introduces a new dimension, the molecules that are not able to be resolved in the super-resolution intensity image can be completely separated in the dipole orientation domain. Scale bar=200 nm.