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  • Title: [Comparison and progress review of various super-resolution fluorescence imaging techniques].
    Author: Chen J, Liu W, Xu Z.
    Journal: Se Pu; 2021 Oct; 39(10):1055-1064. PubMed ID: 34505427.
    Abstract:
    “Seeing is believing” is the central philosophy of life science research, which runs through the continuous understanding of individual molecules, molecular complexes, molecular dynamic behavior, and the entire molecular network. Living and dynamic molecules are functional in nature; therefore, fluorescence microscopy has emerged as an irreplaceable tool in life science research. However, when fluorescence imaging is performed at the molecular level, some artificial signals may lead to erroneous experimental results. This obstacle is due to the limitation of the optical diffraction limit, and the fluorescence microscope cannot distinguish the target in the diffraction-limited space. Super-resolution fluorescence imaging technology breaks through the diffraction limit, allows visualization of biomolecules at the nanometer scale to the single-molecule level, and allows us to study the structure and dynamic processes of living cells with unprecedented spatial and temporal resolution. It has become a powerful tool for life science research and is gradually being applied to material science, catalytic reaction processes, and photolithography as well. The principle of super-resolution imaging technologies is different; therefore, it has different technical performances, thus limiting their specific technical characteristics and application scope. Current mainstream super-resolution imaging technologies can be classified into three types: structured illumination microscopy (SIM), stimulated emission depletion (STED), and single-molecule localization microscopy (SMLM). These microscopes use different complex technologies, but the strategy is the same and simple, i.e. two adjacent luminous points in a diffraction-limited space can be spatially resolved by time resolution. SIM has been used for three-dimensional real-time imaging in multicellular organisms; however, compared with other technologies, its lower horizontal and vertical resolutions need to be further optimized. STED is limited by its small imaging field of view and high photobleaching; however, the best time resolution can be considered at a high spatial resolution, and it has been proven that three-color STED imaging can be performed. In SMLM super-resolution imaging, the time resolution is affected by the time required to locate all fluorophores, which is closely related to the switching and luminescence properties of the fluorophore. With the improvement in horizontal and vertical resolution of imaging, the image acquisition speed, photobleaching characteristics, and the possibility of multi-color and dynamic imaging have increasingly become the key determinants of super-resolution fluorescence imaging. Thus far, the main use of super-resolution imaging technology has been focused on biological applications for studying structural changes less than 200 nm in dimension. In addition to the combination of structural and morphological characterization with biomolecular detection and identification, super-resolution imaging technology is rapidly expanding into the fields of interaction mapping, multi-target detection, and real-time imaging. In the latter applications, super-resolution imaging technology is particularly advantageous because of more flexible sample staining, higher labeling efficiency, faster and simpler readings, and gentler sample preparation procedures. In this article, we compare the principles of these three technologies and introduce their application progress in biology. We expect the results described herein will help researchers clarify the technical advantages and applicable application directions of different super-resolution imaging technologies, thus facilitating researchers in making reasonable choices in future research. 所见即所得是生命科学研究的中心哲学,贯穿在不断认识单个分子、分子复合体、分子动态行为和整个分子网络的历程中。活的动态的分子才是有功能的,这决定了荧光显微成像在生命科学研究中成为不可替代的工具。但是当荧光成像聚焦到分子水平的时候,所见并不能给出想要得到的。这个障碍是由于受光学衍射极限的限制,荧光显微镜无法在衍射受限的空间内分辨出目标物。超分辨荧光成像技术突破衍射极限的限制,在纳米尺度至单分子水平可视化生物分子,以前所未有的时空分辨率研究活细胞结构和动态过程,已成为生命科学研究的有力工具,并逐渐应用到材料科学、催化反应过程和光刻等领域。超分辨成像技术原理不同,其具有的技术性能各异,限制了各自特定的技术特色和应用范围。目前主流的超分辨成像技术包括3种:结构光照明显微镜技术(structured illumination microscopy, SIM)、受激发射损耗显微技术(stimulated emission depletion, STED)和单分子定位成像技术(single molecule localization microscopy, SMLM)。这些显微镜采用不同的复杂技术,但是策略却是相同和简单的,即通过牺牲时间分辨率来提升衍射受限的空间内相邻两个发光点的空间分辨。该文通过对这3种技术的原理比较和在生物研究中的应用进展介绍,明确了不同超分辨成像技术的技术优势和适用的应用方向,以方便研究者在未来研究中做合理的选择。
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