image: a Rotary-linear-polarized illumination scheme of OLID. b Top: the sync signals of camera readout (blue line) and motor rotation pulse (red line). Middle: intensity curve of the laser after passing through a polarizer parallel to the X-axis of the system on the focal plane. Bottom: the emission intensity curve of a GFP molecule on the focal plane under polarization modulated excitation. There are four modulation cycles in one motor rotation cycle. c Alexa Flour 561 labeled F-actin in fixed mouse kidney sections, with wide-field image on the left, OLID image on the right. Scale bar: 5 μm. d The flowchart of OLID-SDOM. The data of four modulation cycles at each pixel were transformed by 1D Fourier transform, and then we extracted the modulated frequency component and generated a 1D single period data. After processing each pixel, 3D deconvolution of the obtained single period image, named as OLID image, was carried out to obtain the , and , and orientation mapping was extracted by FFT phase extraction. view more
Credit: by Meiling Guan, Miaoyan Wang, Karl Zhanghao, Xu Zhang, Meiqi Li, Wenhui Liu, Jing Niu, Xusan Yang, Long Chen, Zhenli Jing, Micheal Q Zhang, Dayong Jin, Peng Xi, Juntao Gao
The dipole orientation of a fluorescent molecule is an inherent physical nature in the fluorescent excitation/emission process, and orientation measurement has found widespread use in studying biological mechanisms associated with molecular arrangement and rotation. Additionally, the diffraction limit restricts the application of conventional wide-field fluorescence microscopy in studying the fine structural changes, interactions, and function of subcellular. Some researchers have utilized polarization excitation modulation and deconvolution to achieve super-resolution and orientation measurement. However, the methods above only were applied to a handful of samples with apparent fluorescence anisotropy. In samples with low fluorescence anisotropy, the read-out signal is usually buried under the system noise, which appears to be polarization invariant. Biologists often hesitate on the analysis of fluorescent dipole orientation, because they could hardly observe it at a low signal-to-noise level.
In a new paper published in Light Science & Application, a team of scientists, led by Professor Juntao Gao from Beijing National Research Center for Information Science and Technology, Tsinghua University, China and Professor Peng Xi from College of Future Technology, Peking University, China and co-workers have developed a comprehensive method termed Optical Lock-in Detection Super-resolution Dipole Orientation Mapping (OLID-SDOM) for weak fluorescence anisotropy mapping in universal subcellular ultrastructure. A frequency-domain Optical Lock-in Detection (OLID) was employed to extract the dipole signal, which was modulated by polarized excitation at a given frequency. Super-resolution reconstruction was then applied to reduce spatial averaging of the polarized signal, which would also increase the fluorescence anisotropy. In contrast to other ON-OFF modulation based OLID, polarization modulation based OLID applies to most organic dyes and fluorescent proteins, especially for the most common Green Fluorescent Protein (GFP) which could be easily labeled to proteins for live-cell imaging. As such, their OLID-SDOM method achieves a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguishes distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo.
In their work, fluorescence anisotropy was first observed in various subcellular organelles (e.g. lysosomes, endosomes, Golgi, etc.) in live yeast cells and live mammalian cells. Next, they investigated the dynamic orientation alteration of the spine head during outreaching, which only happens on the mono-budding spine. These scientists summarize the advantages of their method with SDOM:
“1) the preprocessing of OLID reduces the influence of noise, and speed up the convergence; 2) the sparsity constraint is avoided in OLID-SDOM, comparing with that in the SDOM process; 3) 3D deconvolution of OLID-SDOM has a higher resolution than the 2D deconvolution of SDOM; 4) the orientation mapping method by FFTPE is faster than the least-squared fitting of SDOM with similar or higher accuracy.”
“These results on live subcellular organelles and live neuron spines indicate that OLID-SDOM holds great potential for a wide range of biological applications, particularly in which the dipole nature is neglected previously.” The scientists forecast.
Journal
Light Science & Applications