Recently, a new polarization-dipole azimuth-based super-resolution technique has been proposed by Dr. Peng Xi's group in the College of Engineering, which not only provides a new dimension for super-resolution, but also provides a timely solution to a recent hot debate in the field.
Since fluorescence polarization was discovered on 1926, various fluorescence anisotropy techniques have been developed to study dipole orientation of fluorophores. However, in the case of super resolution, while other properties of fluorescence, such as intensity, spectrum, fluorescence lifetime, etc., have been well applied, little attention is paid to the direction of the fluorescence dipole (polarization). In 2014, Walla team published an article on Nature Methods to achieve sparse reconstructed super-resolution imaging by polarization-modulating lasers. In the beginning of this year, Keller group has published a comment on this article on Nature Methods journal: fluorescence polarization adds little additional information to (fluorescence intensity) super-resolution. This raised an interesting debate: whether the polarization modulation can bring super-resolution information?
However, both the Walla and Keller groups looked at this problem from a conventional fluorescence intensity point of view. Taking into account fluorescence intensity and fluorescence anisotropy, this work introduces the dipole angle to distinguish fluorescence through the fourth dimension of the fluorescence, and perfectly answers this controversy.
Traditional fluorescence anisotropy techniques are limited to samples of relative uniform polarization. Fluorescence polarization would be affected by a bulk of fluorophores due to Abbe’s diffraction limit, when it comes to complex samples. SDOM utilizes polarization modulation of excitation laser and demodulation of both intensity and polarization, which improves spatial resolution as well as detection accuracy of dipole orientation. With the additional information of fluorescence polarization imposed on the original super-resolution intensity image, Xi group has observed several interesting findings in biological samples. At the same time, SDOM technology has a very fast imaging speed (up to 5 frames per second super resolution), the excitation light power requirements are very low (milliWatts level), is ideal for live cell observation. The observation of living yeast cells is achieved herein.
Figure 1 Principle of SDOM. SDOM not only generates better spatial resolution, but more importantly, it brings a new dimension to super-resolution microscopy.
This work has been published on Light: Science & Application on Oct. 21, 2016. The co-first authors Mr. Hao Zhang is a Ph.D. candidate from College of Engineering, Peking University and Long Chen is from TNLIST, Tsinghua University. The corresponding author of this paper, Prof. Peng Xi, is an Associate Professor in the College of Engineering, Peking University.