Original Title: Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein
Corresponding Authors:Xuping Yong, Institute of Biophysics, Chinese Academy of Sciences; Xu Tao, Institute of Biophysics, Chinese Academy of Sciences
Authors: Mingshu Zhang, Zhifei Fu, Changqing Li, Anyuan Liu, Dingming Peng, Fudong Xue, Wenting He, Shan Gao, Fan Xu, Dan Xu, Ling Yuan, Fa Zhang, Zhiheng Xu, Tao Xu, Pingyong Xu.The super-resolution imaging technique PALM (Photo-Activated Localization Microscopy) breaks the optical diffraction limit and won the Nobel Prize in Chemistry in 2014. Compared to other widely used super-resolution imaging techniques, this method has the highest spatial resolution (~20 nm), leading to broad applications in biology. However, this technique requires thousands of raw images to reconstruct a super-resolution image, resulting in low temporal resolution, which poses challenges for practical applications in live cells.Additionally, the limitations of existing photo-switchable fluorescent proteins make observing early developmental structures a current challenge in ultra-high-resolution imaging. Fluorescent protein labeling of developing organisms is characterized by non-invasiveness, low toxicity, and low background. However, the folding, maturation, and accumulation of fluorescent proteins take time, causing their fluorescence signals to lag behind certain early events in development. Fluorescent proteins that emit light earlier can capture structures or events that appear earlier in development, making quantitative analysis and data interpretation based on fluorescence signals more accurate and reliable. However, currently, commonly used photo-convertible fluorescent proteins in super-resolution imaging do not exhibit early emission characteristics.To address these two issues in super-resolution imaging, the research groups of Xu Tao from the Institute of Biophysics, Chinese Academy of Sciences, and Xuping Yong collaborated to develop a novel photo-convertible fluorescent protein probe pcStar and a new live-cell super-resolution imaging method Quick-SIMBA. Compared to the widely used mEos3.2 published by the collaborative team (Nature Methods, 2012), the pcStar fluorescent protein probe has early emission and high photo-conversion efficiency, which helps improve the temporal resolution and labeling density of single-molecule super-resolution imaging techniques (Figure 1), and can be applied to super-resolution imaging of short-lived biological molecules/structures. Using the next-generation single-molecule localization super-resolution imaging probe pcStar, the team achieved ultra-early labeling in bacteria, eukaryotic cell lines, fetal neural stem cells, and Drosophila embryos. Quick-SIMBA is a new generation of live-cell super-resolution imaging method based on the single-molecule localization super-resolution technique SIMBA (Cell Research, 2017), combining the pcStar probe, sCMOS camera, and improved algorithms. This technology has the highest spatiotemporal resolution (0.1-0.25 s, 50 nm) among current live-cell single-molecule localization imaging techniques, allowing for the clear resolution of dense tubular endoplasmic reticulum in live cells (Figure 2), which has long been regarded as a continuous sheet structure due to insufficient spatiotemporal resolution in traditional imaging. Moreover, by combining pcStar and SIMBA imaging techniques, the team labeled and analyzed the ultra-early structures during the development of Drosophila embryos (Figure 3), providing new evidence and perspectives on the developmental formation process of these structures.
Figure 1. Compared to mEos3.2, the pcStar fluorescent protein probe has higher photo-conversion efficiency and labeling density
Figure 2. Super-resolution structure of endoplasmic reticulum (pcStar labeled) resolved by Quick-SIMBA
