page_title: Super-resolution dipole orientation mapping
section_title: Principle of SDOM
hierarchical_section_title: Super-resolution dipole orientation mapping / Principle of SDOM
caption_attribution_description: English: The priniciple of SDOM
context_page_description: Super-resolution dipole orientation mapping is a form of fluorescence polarization microscopy that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.
context_section_description: Unlike other super-resolution methods,such as STED, SIM, PLAM and STORM, SDOM could achieve super-resolution based on a wide-field epi-fluorescence illumination microscope. The key point of SDOM is polarized excitation. We can see the SDOM imaging system on figure(a). 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.
  All organic fluorescent dyes and fluorescent proteins are dipoles, whose orientations are closely related to the structure of their labeled target proteins. Because both the excitation absorption and fluorescence emission of dipoles have polarization features, FPM has been widely used to study dipole orientation. 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),  and the dipole angle of the fluorophore will reflect the orientation of the target protein. Therefore, we can use the SDOM to study the structure of the protein.
  Figure(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. The super-resolution was achieved in the polarization domain.
  The SDOM result of two intersecting lines was shown in figure(d), with arrows on top of the super-resolution image, illustrating the dipole orientation. Figure(e)showed 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.
caption: Schematic diagram of SDOM.
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