The Xu Tao/Ji Wei team has made new breakthroughs in the field of multicolor super-resolution microscopy imaging technology

Single molecule localization microscopy (SMLM) improves the resolution of fluorescence microscopy by an order of magnitude by combining light-controlled fluorescence molecular labeling, centroid fitting algorithm, single molecule detection and other technologies, and realizes the analysis of cellular nanostructures, which has been widely used in the observation and research of cell biology. However, the expansion and application of multicolor imaging has been limited, mainly because the current light-controlled fluorescent molecular probes with excellent performance (such as Alexa Fluor 647, CF660C, etc.) are mostly concentrated in the far-red band, and it is difficult to use conventional bandpass filter selection methods to achieve multi-channel imaging.

Recently, Academician Xu Tao’s research group and Ji Wei’s research group published a research paper entitled “Tetra-color superresolution microscopy based on excitation spectral demixing” in the journal Light: Science & Applications, proposing a multicolor super-resolution imaging technology based on excitation spectrum splitting ( Excitation-resolved stochastic optical reconstruction microscopy, ExR-STORM), a technique that identifies different far-red fluorescent probes through differences in excitation efficiency, enabling four-color single-molecule localization super-resolution imaging.

ExR-STORM microscopy provides a new method for single-molecule identification and further improves single-molecule spectral splitting capabilities. This technique enables multicolor imaging by excitation of far-red fluorescent probes with three wavelengths of 620 nm, 639 nm and 671 nm, using the characteristics of the excitation spectrum as the “fingerprint” of single molecules. The key technological innovation of this method is the design of a time-sharing synchronous rapid imaging optical path of high-frequency galvanometer, which can eliminate the effect of intensity changes caused by single-molecule scintillation (Figure 1). This design also provides new ideas for the measurement of single-molecule excitation spectra. Using ExR-STORM microscopy combined with the labeling of four far-red probes, four-color super-resolution imaging of intracellular mitochondria, intermediate filament, endoplasmic reticulum, microtubules, and other structures was successfully achieved (Figure 2). The imaging results show that this technology has the advantages of strong spectral splitting ability and small localization error caused by chromatic aberration, and has wide application prospects in biological research fields such as organelle interaction and biomacromolecular colocalization analysis. At the same time, the sample preparation method and imaging process of this technology are compatible with conventional STORM technology, which makes the technology have high performance and ease of use, which is conducive to the promotion and application of this technology, and provides a powerful imaging tool for research in the field of biology and medicine.

Figure 1. ExR-STORM works by using galvanometer to project images excited by three lasers into different areas of the camera to achieve high-frequency time-sharing synchronous and fast imaging.

Figure 2. Label mitochondria, intermediate filament, endoplasmic reticulum, and microtubules with CF660C, Alexa Fluor 647, Dyomics 654, and DyLight 633 for super-resolution imaging of four-color single-molecule localization.

Academician Xu Tao, researcher Ji Wei and senior engineer Gu Lushengzheng are the co-corresponding authors of the paper, and Dr. Wu Wanyan, doctoral student Luo Shixing and assistant researcher Fan Chunyan from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences are co-first authors. Professor Hu Junjie and Professor Meng Wenxiang from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences provided constructive suggestions for the cell biology imaging verification of this paper. This work was supported by the National Key Research and Development Program, the National Natural Science Foundation of China, and the Strategic Leading Science and Technology Project of the Chinese Academy of Sciences (Class B) “Cross-scale Research on the Structure and Function of Biomacromolecular Complexes”. (Source: LightScience Applications WeChat public account)

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