page_title: Super-resolution dipole orientation mapping
section_title: Fluorescence polarization microscopy
hierarchical_section_title: Super-resolution dipole orientation mapping / Fluorescence polarization microscopy
caption_attribution_description: English: (a) A typical setup of fluorescence anisotropy (FA). Circularly polarized light source is used, with either epi-illumination (displayed in the figure) or confocal illumination (together with a point detector, not displayed). Wollaston Prism (WP) could allow simultaneous detection of two polarization channels utilizing two halves of the camera. (b) A typical setup of linear dichroism (LD). The linearly polarized light source passes a polarization modulator (PM), resulting in varying polarization of excitation light. The time trace image of fluorescence intensity changing with the excitation polarization would be recorded. Both epi-illumination or confocal illumination could be used and the polarization modulator could be a rotary half wave plate, an electro-optical modulator, etc. (c) A typical setup of defocused pattern recognition (DPR). The sample is usually defocused with several hundred nanometers away from the focal plane of the objective. The diffraction pattern of the dipoles would be recorded and be used for orientation measurement. Circularly polarized light source is used for effective excitation of all the dipoles. Abbreviations: S – light source; L – collimating lens; DM – dichroic mirror; Obj – objective; TL – tube lens; CCD – CCD camera; WP – Wollaston Prism; PM – polarization modulator.
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: As a fundamental physical dimension of fluorescence, polarization has been applied extensively in biological researches. Through fluorescence polarization microscopy (FPM), the dipole orientation as well as the intensity of fluorescent probes could be measured. Compared with X-ray crystallography or electron microscopy which could elucidate ultra-high resolution of individual proteins or macromolecule assemblies, FPM don't  require complex sample preparation which made it suitable for live cell imaging. Near-field imaging techniques, such as Atomic Force Microscopy (AFM) could also provide with structural information, which however, is limited only to samples on the surface. FPM is capable of imaging orientations in dynamic samples at the time scale of seconds or milliseconds, thus it can serve as a complementary method for measurement of subcellular organelle structures.
  FPM has been evolving during past decades, from manual or mechanical switching of polarization detection or excitation to simultaneously detection and fast polarization modulation via electro-optic devices. With faster imaging speed and higher imaging quality, FPM has been incorporated with various imaging modalities, such as wide-field, confocal microscopy, two-photon confocal, total internal reflection fluorescence microscope, FRAP, etc. However, as an optical imaging technique, the development of fluorescence polarization microscopy (FPM) is barricaded by the diffraction limit. Compared to the abundant super-resolution techniques on fluorescence intensity imaging, super-resolution techniques in FPM is still in its infancy.
  Recently, three forms of FPM emerged and has been proved that they could achieve super-resolution. They are SPoD, SDOM and polar-dSTORM(polarization-resolved direct stochastic optical reconstruction microscopy).
  Polar-dSTORM used On-Off modulation of the fluorescent probes and acquired adequate frames for a reconstruction of super resolution image. The imaging resolution of polar-dSTORM is high, with localization precision of tens of nanometers. Single dipole average orientation is directly measured separately and the wobbling angle is statistically calculated from neighboring emitters.  The drawback of polar-dSTORM is long imaging time of 2 - 40 min, which requires a stationary sample during the imaging period. The sample preparation of dSTORM also makes it hard for live cell samples.
  SDOM has achieved super resolution dipole orientation mapping with a spatial resolution of 150 nm and sub-second temporal resolution. It has been applied to both fixed cell and live cell imaging, which shows great advantages over diffraction limited FPM techniques on both revealing sub-diffrational structures and measuring local dipole orientations. In comparison with polar-dSTROM, SDOM still measures average dipoles and could not separate the signal of the wobbling of single fluorophores from the variation of orientation distribution of fluorophores with the resolvable area. Same with SPoD, the power of SDOM would be weakened if the fluorescent probes are distributed too homogeneously or too dense.
  Thanks to the intrinsic polarization of chromophores, fluorescence polarization reveals the structures and functions of the biological macromolecules. With incorporation with various optical imaging modalities, FPM has played an irreplaceable role in solving many questions. Fast and non-invasive imaging of the samples makes it a complementary tool for X-crystallography which typically applies to individual proteins, or sub-complexes, or EM which requires invasive sample preparation, or AFM which could measure the surface of the sample. Compared to these methods, the specific labeling of the fluorescent probes provides better focus on the structure of interest.
  As the development of FPM techniques, its power has spread from uniform oriented fluorophores to fluorescent dipoles with organized orientation or on complex bio-structures. The detection accuracy has improved from measuring the bulk volume polarization to sub-diffraction area measurement and single
caption: Schematic setup of different types of FPM.
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