Journal Article

Super-resolution dipole orientation mapping via polarization demodulation

Xi, Peng, Wang, Miao-Yan, Jing, Zhen-Li, Zhang, Michael Q, Chen, Long, Yang, Xu-San, Jin, Dayong, Han, Hong-Bin, Gao, Jun-Tao, Zhanghao, Karl

2016

Overview

Fluorescence polarization microscopy (FPM) aims to detect the dipole orientation of fluorophores and to resolve structural information for labeled organelles via wide-field or confocal microscopy. Conventional FPM often suffers from the presence of a large number of molecules within the diffraction-limited volume, with averaged fluorescence polarization collected from a group of dipoles with different orientations. Here, we apply sparse deconvolution and least-squares estimation to fluorescence polarization modulation data and demonstrate a super-resolution dipole orientation mapping (SDOM) method that resolves the effective dipole orientation from a much smaller number of fluorescent molecules within a sub-diffraction focal area. We further apply this method to resolve structural details in both fixed and live cells. For the first time, we show that different borders of a dendritic spine neck exhibit a heterogeneous distribution of dipole orientation. Furthermore, we illustrate that the dipole is always perpendicular to the direction of actin filaments in mammalian kidney cells and radially distributed in the hourglass structure of the septin protein under specific labelling. The accuracy of the dipole orientation can be further mapped using the orientation uniform factor, which shows the superiority of SDOM compared with its wide-field counterpart as the number of molecules is decreased within the smaller focal area. Using the inherent feature of the orientation dipole, the SDOM technique, with its fast imaging speed (at sub-second scale), can be applied to a broad range of fluorescently labeled biological systems to simultaneously resolve the valuable dipole orientation information with super-resolution imaging. Fluorescence polarization microscopy: super resolution imaging of dipoles The ability to map the orientation of fluorescent dipoles with super-resolution will open up new opportunities in cellular microscopy. Peng Xi and co-workers have developed a scheme that obtains information about fluorescent dipole orientation on a super-resolution spatial scale by applying sparse deconvolution and least-squares estimation algorithms to modulated polarization data. They used this scheme to glean information about the structures of various fixed and live fluorescently labelled cells. The team discovered that actin filaments in mammalian kidney cells are always arranged perpendicular to the dipole direction, whereas dipoles are radially distributed for the septin protein in live yeast cells under specific labelling. The fast imaging speed of the technique, about 5 frames per second, potentially makes it possible to observe and study the dynamics of living samples.

• Publisher: Nature Publishing Group UK,Springer Nature B.V,Nature Publishing Group
• Subject: 639/624/1107/328/1652 / 639/624/1107/328/2236 / 639/624/1107/328/2238 / 639/624/1107/510 / Applied and Technical Physics / Atomic / Classical and Continuum Physics / Fluorescence / Lasers / Mapping / Microscopy / Molecular / Optical and Plasma Physics / Optical Devices / Optics / Original / original-article / Photonics / Physics / Physics and Astronomy