多种超分辨荧光成像技术比较和进展评述

doi:10.3724/SP.J.1123.2021.06015

摘要/Abstract

摘要：

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。

关键词: 超分辨荧光成像, 纳米尺度, 可视化, 活细胞结构和动态

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.

Key words: super-resolution fluorescence imaging, nanometer scale, visualization, cellular structure and dynamics

中图分类号:

O658

陈婕, 刘文娟, 徐兆超. 多种超分辨荧光成像技术比较和进展评述[J]. 色谱, 2021, 39(10): 1055-1064.

CHEN Jie, LIU Wenjuan, XU Zhaochao. Comparison and progress review of various super-resolution fluorescence imaging techniques[J]. Chinese Journal of Chromatography, 2021, 39(10): 1055-1064.

图/表 4

图1 主要超分辨率技术原理

Fig. 1 Principle of three super-resolution technologies a. The principle of structured illumination microscopy (SIM). The sample is illuminated by a sinusoidal pattern to produce moiré fringes, which can be detected using a SIM microscope. After acquiring approximately 15 images with different phases and orientations, the algorithm was used to remove the known illumination patterns to reconstruct a high-resolution image. b. The principle of stimulated emission depletion (STED). In STED imaging, an excitation laser is used to excite the fluorophore, and the sample is irradiated by a second, doughnut-shaped laser beam aligned with the excitation laser. The excited fluorophores are depleted by stimulated emission and driven to the ground state before emitting fluorescence, thereby improving the imaging resolution. c. The principle of single molecule localization microscopy (SMLM) is to stochastically activate a small portion of single-molecule fluorophores, which are acquired, located, and inactivated. This cycle is repeated to accurately locate each fluorophore and reconstruct a super-resolution image. Picture based on a reported paper[7], obtained with permission from the copyright of the Multidisciplinary Digital Publishing Institute.

图2 3D-SIM成像

Fig. 2 3D-SIM imaging a. Simultaneous imaging of 3D-SIM in C2C12 with DAPI-stained DNA (blue), nuclear membrane stained with anti-lamin B (green), and epitope NPC stained with an antibody that specifically recognizes the epitope NPC (red)[25]. Scale bar: 1 μm. b. 3D-SIM imaging of the FtsZ structure fused with GFP in living Bacillus subtilis cells[29]. The higher image resolution provided by 3D-SIM allows the observation of the contracted Z ring and the inside of the cell membrane during division (indicated by the white arrow). Pictures based on reported papers[25,29], obtained with permission from the American Association for the Advancement of Science and PLOS. CLSM: confocal laser scanning microscopy; NPC: the nuclear pore complex; GFP: green fluorescent protein.

图3 活细胞STED成像

Fig. 3 STED imaging of live cells a. STED imaging COS-7 cells expressing Halo-Sec61b (endoplasmic reticulum membrane) and SNAP-KDEL (endoplasmic reticulum cavity) fusion proteins labeled with ATTO590 and SiR, respectively. Scale bar: 2 μm. b. STED imaging HeLa cells expressing ARF1-Halo and β-COP-SNAP labeled with ATTO590 and SiR, respectively. Scale bar: 5 μm. c. The ARF1 tubular structure is the main membrane outflow of the Golgi body. Picture based on reported papers[38, 39], obtained with permission from copyrights of Springer Nature and The American Society for Cell Biology.

图4 哺乳动物细胞BS-C-1中(a)微管和CCPs的双色STORM成像和(b) 3D-STORM成像

Fig. 4 (a) Two-color stochastically optical reconstruction microscopy (STORM) imaging of microtubules and CCPs and (b) 3D-STORM imaging of microtubules in mammalian cells BS-C-1 Picture based on reported papers[42,43], obtained with permission from copyrights of the American Association for the Advancement of Science.

参考文献

[1]	Kner P, Chhun B B, Griffis E R, et al. Nat Methods, 2009, 6(5):339
[2]	Hell S W. Angew Chem Int Ed, 2015, 54(28):8054
[3]	Moerner W E. Angew Chem Int Ed, 2015, 54(28):8067
[4]	Shroff H, Galbraith C G, Galbraith J A, et al. Nat Methods, 2008, 5(5):417
[5]	Huang B, Jones S A, Brandenburg B, et al. Nat Methods, 2008, 5(12):1047
[6]	Gustafsson M G L. PNAS, 2005, 102(37):13081
[7]	Georgieva M, Nöllmann M. Res Rep Biol, 2015, 6:157
[8]	Hirano Y, Matsuda A, Hiraoka Y. Microscopy, 2015, 64(4):237
[9]	Heintzmann R, Huser T. Chem Rev, 2017, 117(23):13890
[10]	Sonnen K F, Schermelleh L, Leonhardt H, et al. Biol Open, 2012, 1(10):965
[11]	Hell S W, Sahl S J, Bates M, et al. J Phys D, 2015, 48(44):443001
[12]	Muller T, Schumann C, Kraegeloh A. ChemPhysChem, 2012, 13(8):1986
[13]	Blom H, Widengren J. Chem Rev, 2017, 117(11):7377
[14]	Willig K I, Kellner R R, Medda R, et al. Nat Methods, 2006, 3(9):721
[15]	Wang L, Frei M S, Salim A, et al. J Am Chem Soc, 2019, 141(7):2770
[16]	Fernandez-Suarez M, Ting A Y. Nat Rev Mol Cell Biol, 2008, 9(12):929
[17]	Samanta S, He Y, Sharma A, et al. Chem, 2019, 5(7):1697
[18]	Yang Z, Sharma A, Qi J, et al. Chem Soc Rev, 2016, 45(17):4651
[19]	Betzig E, Patterson G H, Sougrat R, et al. Science, 2006, 313(5793):1642
[20]	Jradi F M, Lavis L D. ACS Chem Biol, 2019, 14(6):1077
[21]	Zou G, Liu C, Fang Z, et al. Sens Actuators B Chem, 2019, 288:113
[22]	Vicidomini G, Bianchini P, Diaspro A. Nat Methods, 2018, 15(3):173
[23]	Sahl S J, Hell S W, Jakobs S. Nat Rev Mol Cell Biol, 2017, 18(11):685
[24]	Huang B, Bates M, Zhuang X. Annu Rev Biochem, 2009, 78:993
[25]	Schermelleh L, Carlton P M, Haase S, et al. Science, 2008, 320(5881):1332
[26]	Wang C J, Carlton P M, Golubovskaya I N, et al. Genetics, 2009, 183(3):905
[27]	Brown A C N, Oddos S, Dobbie I M, et al. PLOS Biol, 2011, 9(9):e1001152
[28]	Cogger V C, McNerney G P, Nyunt T, et al. J Struct Biol, 2010, 171(3):382
[29]	Strauss M P, Liew A T, Turnbull L, et al. PLOS Biol, 2012, 10(9):e1001389
[30]	Horsington J, Turnbull L, Whitchurch C B, et al. J Virol Methods, 2012, 186(1/2):132
[31]	Hirvonen L M, Wicker K, Mandula O, et al. Eur Biophys J, 2009, 38(6):807
[32]	Shao L, Kner P, Rego E H, et al. Nat Methods, 2011, 8(12):1044
[33]	Huang X, Fan J, Li L, et al. Nat Biotechnol, 2018, 36(5):451
[34]	Guo Y, Li D, Zhang S, et al. Cell, 2018, 175(5):1430
[35]	Donnert G, Keller J, Medda R, et al. PNAS, 2006, 103(31):11440
[36]	Westphal V, Rizzoli S O, Lauterbach M A, et al. Science, 2008, 320(5873):246
[37]	Lukinavicius G, Reymond L, D'Este E , et al. Nat Methods, 2014, 11(7):731
[38]	Bottanelli F, Kromann E B, Allgeyer E S, et al. Nat Commun, 2016, 7:10778
[39]	Bottanelli F, Kilian N, Ernst A M, et al. Mol Biol Cell, 2017, 28(12):1676
[40]	Neuman K C, Block S M. Rev Sci Instrum, 2004, 75(9):2787
[41]	Gelles J, Schnapp B J, Sheetz M P. Nature, 1988, 331(6155):450
[42]	Bates M, Huang B, Dempsey G T, et al. Science, 2007, 317(5845):1749
[43]	Huang B, Wang W, Bates M, et al. Science, 2008, 319(5864):810
[44]	Juette M F, Gould T J, Lessard M D, et al. Nat Methods, 2008, 5(6):527
[45]	Jones S A, Shim S-H, He J, et al. Nat Methods, 2011, 8(6):499
[46]	Shim S H, Xia C, Zhong G, et al. PNAS, 2012, 109(35):13978
[47]	Uno S-N, Kamiya M, Yoshihara T, et al. Nat Chem, 2014, 6(8):681
[48]	Takakura H, Zhang Y, Erdmann R S, et al. Nat Biotechnol, 2017, 35(8):773
[49]	Fornasiero E F, Opazo F. BioEssays, 2015, 37(4):436