Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
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Pawley, J. B. Handbook of biological confocal microscopy. 3rd edn, (Springer US, New York, 2006).
Sauer, M. & Heilemann, M. Single-molecule localization microscopy in eukaryotes. Chem. Rev. 117, 7478–7509 (2017).
Fornasiero, E. F. & Opazo, F. Super-resolution imaging for cell biologists: concepts, applications, current challenges and developments. Bioessays 37, 436–451 (2015).
Turkowyd, B., Virant, D. & Endesfelder, U. From single molecules to life: microscopy at the nanoscale. Anal. Bioanal. Chem. 408, 6885–6911 (2016).
Eggeling, C., Willig, K. I., Sahl, S. J. & Hell, S. W. Lens-based fluorescence nanoscopy. Q. Rev. Biophys. 48, 178–243 (2015).
Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 18, 685–701 (2017).
Heintzmann, R. & Huser, T. Super-resolution structured illumination microscopy. Chem. Rev. 117, 13890–13908 (2017).
Wu, Y. & Shroff, H. Faster, sharper, and deeper: structured illumination microscopy for biological imaging. Nat. Methods 15, 1011–1019 (2018).
Gustafsson, M. G. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).
Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L. & Gustafsson, M. G. Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).
Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).
Muller, C. B. & Enderlein, J. Image scanning microscopy. Phys. Rev. Lett. 104, 198101 (2010).
York, A. G. et al. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat. Methods 9, 749–754 (2012).
Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proc. Natl Acad. Sci. USA 110, 21000–21005 (2013).
York, A. G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat. Methods 10, 1122–1126 (2013).
Shao, L., Kner, P., Rego, E. H. & Gustafsson, M. G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).
Fiolka, R., Shao, L., Rego, E. H., Davidson, M. W. & Gustafsson, M. G. Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc. Natl Acad. Sci. USA 109, 5311–5315 (2012).
Demmerle, J. et al. Strategic and practical guidelines for successful structured illumination microscopy. Nat. Protoc. 12, 988–1010 (2017).
De Luca, G. M. et al. Re-scan confocal microscopy: scanning twice for better resolution. Biomed. Opt. Express 4, 2644–2656 (2013).
Huang, X. S. et al. Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy. Nat. Biotechnol. 36, 451–459 (2018).
Wegel, E. et al. Imaging cellular structures in super-resolution with SIM, STED and localisation microscopy: a practical comparison. Sci. Rep. 6, 27290 (2016).
Göttfert, F. et al. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys. J. 105, L01–03 (2013).
Bottanelli, F. et al. Two-colour live-cell nanoscale imaging of intracellular targets. Nat. Commun. 7, 10778 (2016).
Urban, N. T., Willig, K. I., Hell, S. W. & Nagerl, U. V. STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J. 101, 1277–1284 (2011).
Heine, J. et al. Adaptive-illumination STED nanoscopy. Proc. Natl Acad. Sci. USA 114, 9797–9802 (2017).
van de Linde, S., Heilemann, M. & Sauer, M. Live-cell super-resolution imaging with synthetic fluorophores. Annu. Rev. Phys. Chem. 63, 519–540 (2012).
van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 991–1009 (2011).
Demmerle, J., Wegel, E., Schermelleh, L. & Dobbie, I. M. Assessing resolution in super-resolution imaging. Methods 88, 3–10 (2015).
Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253–266 (2014).
Baddeley, D. & Bewersdorf, J. Biological insight from super-resolution microscopy: what we can learn from localization-based images. Annu. Rev. Biochem. 87, 965–989 (2018).
Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).
Burgert, A., Letschert, S., Doose, S. & Sauer, M. Artifacts in single-molecule localization microscopy. Histochem. Cell Biol. 144, 123–131 (2015).
Ishitsuka, Y., Nienhaus, K. & Nienhaus, G. U. Photoactivatable fluorescent proteins for super-resolution microscopy. Methods Mol. Biol. 1148, 239–260 (2014).
Heilemann, M., Margeat, E., Kasper, R., Sauer, M. & Tinnefeld, P. Carbocyanine dyes as efficient reversible single-molecule optical switch. J. Am. Chem. Soc. 127, 3801–3806 (2005).
Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nat. Methods 8, 499–508 (2011).
Wombacher, R. et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nat. Methods 7, 717–719 (2010).
Takakura, H. et al. Long time-lapse nanoscopy with spontaneously blinking membrane probes. Nat. Biotechnol. 35, 773–780 (2017).
Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106, 22287–22292 (2009).
Gustafsson, N. et al. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat. Commun. 7, 12471 (2016).
Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).
Keller, P. J., Schmidt, A. D., Wittbrodt, J. & Stelzer, E. H. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).
Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).
Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).
Chang, B. J., Perez Meza, V. D. & Stelzer, E. H. K. csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm. Proc. Natl Acad. Sci. USA 114, 4869–4874 (2017).
Chen, F., Tillberg, P. W. & Boyden, E. S. Optical imaging. Expansion microscopy. Science 347, 543–548 (2015).
Tillberg, P. W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987–992 (2016).
Zhao, Y. et al. Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy. Nat. Biotechnol. 35, 757–764 (2017).
Chang, J. B. et al. Iterative expansion microscopy. Nat. Methods 14, 593–599 (2017).
Cahoon, C. K. et al. Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex. Proc. Natl Acad. Sci. USA 114, E6857–E6866 (2017).
Wang, Y. F. et al. Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes. Nat. Protoc. 13, 1869–1895 (2018).
Stelzer, E. H. K. Contrast, resolution, pixelation, dynamic range and signal‐to‐noise ratio: fundamental limits to resolution in fluorescence light microscopy. J. Microsc. 189, 15–24 (1998).
Endesfelder, U. et al. Chemically induced photoswitching of fluorescent probes: a general concept for super-resolution microscopy. Molecules 16, 3106–3118 (2011).
Fernandez-Suarez, M. & Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929–943 (2008).
Yang, Z. et al. Super-resolution fluorescent materials: an insight into design and bioimaging applications. Chem. Soc. Rev. 45, 4651–4667 (2016).
Uno, S. N. et al. A guide to use photocontrollable fluorescent proteins and synthetic smart fluorophores for nanoscopy. Microscopy 64, 263–277 (2015).
Nienhaus, K. & Nienhaus, G. U. Fluorescent proteins for live-cell imaging with super-resolution. Chem. Soc. Rev. 43, 1088–1106 (2014).
van de Linde, S. et al. Investigating cellular structures at the nanoscale with organic fluorophores. Chem. Biol. 20, 8–18 (2013).
Stepanenko, O. V., Stepanenko, O. V., Kuznetsova, I. M., Verkhusha, V. V. & Turoverov, K. K. β-barrel scaffold of fluorescent proteins: folding, stability and role in chromophore formation. Int. Rev. Cell Mol. Biol. 302, 221–278 (2013).
Lukinavicius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).
Lukinavicius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).
Yan, Q. & Bruchez, M. P. Advances in chemical labeling of proteins in living cells. Cell Tissue Res. 360, 179–194 (2015).
Grimm, J. B. et al. Bright photoactivatable fluorophores for single-molecule imaging. Nat. Methods 13, 985–988 (2016).
Shim, S. H. et al. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc. Natl Acad. Sci. USA 109, 13978–13983 (2012).
Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).
Mikhaylova, M. et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nat. Commun. 6, 7933 (2015).
Melak, M., Plessner, M. & Grosse, R. Actin visualization at a glance. J. Cell Sci. 130, 525–530 (2017).
Simonson, P. D., Rothenberg, E. & Selvin, P. R. Single-molecule-based super-resolution images in the presence of multiple fluorophores. Nano Lett. 11, 5090–5096 (2011).
Zhang, G., Zheng, S., Liu, H. & Chen, P. R. Illuminating biological processes through site-specific protein labeling. Chem. Soc. Rev. 44, 3405–3417 (2015).
Stanly, T. A. et al. Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters. Biol. Open 5, 1343–1350 (2016).
Ehmann, N. et al. Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states. Nat. Commun. 5, 4650 (2014).
Jungmann, R. et al. Quantitative super-resolution imaging with qPAINT. Nat. Methods 13, 439–442 (2016).
Waldchen, S., Lehmann, J., Klein, T., van de Linde, S. & Sauer, M. Light-induced cell damage in live-cell super-resolution microscopy. Sci. Rep. 5, 15348 (2015).
Lando, D. et al. Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast. Open Biol. 2, 120078 (2012).
Loschberger, A. et al. Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J. Cell Sci. 125, 570–575 (2012).
Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013).
Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).
Galiani, S. et al. Super-resolution microscopy reveals compartmentalization of peroxisomal membrane proteins. J. Biol. Chem. 291, 16948–16962 (2016).
Berning, S., Willig, K. I., Steffens, H., Dibaj, P. & Hell, S. W. Nanoscopy in a living mouse brain. Science 335, 551 (2012).
Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).
Sonnen, K. F., Schermelleh, L., Leonhardt, H. & Nigg, E. A. 3D-structured illumination microscopy provides novel insight into architecture of human centrosomes. Biol. Open 1, 965–976 (2012).
Mennella, V. et al. Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat. Cell Biol. 14, 1159–1168 (2012).
Lawo, S., Hasegan, M., Gupta, G. D. & Pelletier, L. Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat. Cell Biol. 14, 1148–1158 (2012).
Conduit, P. T. et al. A molecular mechanism of mitotic centrosome assembly in Drosophila. eLife 3, e03399 (2014).
Burnette, D. T. et al. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells. J. Cell Biol. 205, 83–96 (2014).
Baddeley, D. et al. Measurement of replication structures at the nanometer scale using super-resolution light microscopy. Nucleic Acids Res. 38, e8 (2010).
Chagin, V. O. et al. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons. Nat. Commun. 7, 11231 (2016).
Smeets, D. et al. Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci. Epigenet. Chromatin 7, 8 (2014).
Ball, G. et al. SIMcheck: A toolbox for successful super-resolution sructured illumination microscopy. Sci. Rep. 5, 15915 (2015).
Culley, S. et al. Quantitative mapping and minimization of super-resolution optical imaging artifacts. Nat. Methods 15, 263–266 (2018).
Nieuwenhuizen, R. P. et al. Measuring image resolution in optical nanoscopy. Nat. Methods 10, 557–562 (2013).
Tortarolo, G., Castello, M., Diaspro, A., Koho, S. & Vicidomini, G. Evaluating image resolution in stimulated emission depletion microscopy. Optica 5, 32–35 (2018).
Steinhauer, C., Jungmann, R., Sobey, T. L., Simmel, F. C. & Tinnefeld, P. DNA origami as a nanoscopic ruler for super-resolution microscopy. Angew. Chem. Int. Edit. 48, 8870–8873 (2009).
Schmied, J. J. et al. DNA origami-based standards for quantitative fluorescence microscopy. Nat. Protoc. 9, 1367–1391 (2014).
Komis, G. et al. Superresolution live imaging of plant cells using structured illumination microscopy. Nat. Protoc. 10, 1248–1263 (2015).
Kraus, F. et al. Quantitative 3D structured illumination microscopy of nuclear structures. Nat. Protoc. 12, 1011–1028 (2017).
Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).
Gould, T. J., Verkhusha, V. V. & Hess, S. T. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat. Protoc. 4, 291–308 (2009).
Lovelace, M. D. et al. The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells. Mol. Biol. Cell 28, 1066–1078 (2017).
Chang, G. H. et al. ARHGAP18: an endogenous inhibitor of angiogenesis, limiting tip formation and stabilizing junctions. Small GTPases 5, 1–15 (2014).
Crittenden, J. R. et al. Striosome-dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons. Proc. Natl Acad. Sci. USA 113, 11318–11323 (2016).
Nozaki, T. et al. Dynamic organization of chromatin domains revealed by super-resolution live-cell imaging. Mol. Cell 67, 282–293 (2017).
Große, L. et al. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. EMBO J. 35, 402–413 (2016).
Ramdas Nair, A. et al. The microcephaly-associated protein Wdr62/CG7337 is required to maintain centrosome asymmetry in Drosophila neuroblasts. Cell Rep. 14, 1100–1113 (2016).
Boettiger, A. N. et al. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422 (2016).
Cattoni, D. I. et al. Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions. Nat. Commun. 8, 1753 (2017).
Ricci, M. A., Manzo, C., Garcia-Parajo, M. F., Lakadamyali, M. & Cosma, M. P. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo. Cell 160, 1145–1158 (2015).
Wurm, C. A. et al. Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient. Proc. Natl Acad. Sci. USA 108, 13546–13551 (2011).
Mönkemoller, V., Oie, C., Hubner, W., Huser, T. & McCourt, P. Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations. Sci. Rep. 5, 16279 (2015).
Fritzsche, M. et al. Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation. Sci. Adv. 3, e1603032 (2017).
Colin-York, H. et al. Super-resolved traction force microscopy (STFM). Nano Lett. 16, 2633–2638 (2016).
Jung, Y. et al. Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies. Proc. Natl Acad. Sci. USA 113, E5916–E5924 (2016).
Poulter, N. S. et al. Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex. Nat. Commun. 6, 7254 (2015).
Guizetti, J. et al. Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331, 1616–1620 (2011).
Saka, S. & Rizzoli, S. O. Super-resolution imaging prompts re-thinking of cell biology mechanisms: selected cases using stimulated emission depletion microscopy. Bioessays 34, 386–395 (2012).
Booth, M. J. Adaptive optical microscopy: the ongoing quest for a perfect image. Light Sci. Appl. 3, e165 (2014).
Gould, T. J., Burke, D., Bewersdorf, J. & Booth, M. J. Adaptive optics enables 3D STED microscopy in aberrating specimens. Opt. Express 20, 20998–21009 (2012).
Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1028–1040 (2016).
Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).
Gao, L. et al. Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell 151, 1370–1385 (2012).
Gustavsson, A. K., Petrov, P. N., Lee, M. Y., Shechtman, Y. & Moerner, W. E. 3D single-molecule super-resolution microscopy with a tilted light sheet. Nat. Commun. 9, 123 (2018).
Geissbuehler, S. et al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging. Nat. Commun. 5, 5830 (2014).
Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2013).
Rego, E. H. et al. Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution. Proc. Natl Acad. Sci. USA 109, E135–143 (2012).
Chmyrov, A. et al. Nanoscopy with more than 100,000 ‘doughnuts’. Nat. Methods 10, 737–740 (2013).
Chmyrov, A. et al. Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy. Sci. Rep. 7, 44619 (2017).
Chang, Y. W. et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat. Methods 11, 737–739 (2014).
Kaufmann, R. et al. Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Lett. 14, 4171–4175 (2014).
Liu, B. et al. Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Sci. Rep. 5, 13017 (2015).
Conduit, P. T., Wainman, A., Novak, Z. A., Weil, T. T. & Raff, J. W. Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP. eLife 4, e08483 (2015).
Tonnesen, J., Katona, G., Rozsa, B. & Nagerl, U. V. Spine neck plasticity regulates compartmentalization of synapses. Nat. Neurosci. 17, 678–685 (2014).
Deng, S. et al. Effects of donor and acceptor’s fluorescence lifetimes on the method of applying Forster resonance energy transfer in STED microscopy. J. Microsc. 269, 59–65 (2018).
Winckler, P. et al. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. Sci. Rep. 3, 2387 (2013).
Honigmann, A. et al. Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells. Nat. Commun. 5, 5412 (2014).
Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).
Valm, A. M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162–167 (2017).
Moffitt, J. R., Pandey, S., Boettiger, A. N., Wang, S. & Zhuang, X. Spatial organization shapes the turnover of a bacterial transcriptome. eLife 5, e13065 (2016).
Nehme, E., Weiss, L. E., Michaeli, T. & Shechtman, Y. Deep-STORM: Super resolution single molecule microscopy by deep learning. Optica 5, 458–464 (2018).
Ouyang, W., Aristov, A., Lelek, M., Hao, X. & Zimmer, C. Deep learning massively accelerates super-resolution localization microscopy. Nat. Biotechnol 36, 460–468 (2018).
Kraus, O. Z. et al. Automated analysis of high-content microscopy data with deep learning. Mol. Syst. Biol. 13, 924 (2017).
Williams, E. et al. The image data resource: a bioimage data integration and publication platform. Nat. Methods 14, 775–781 (2017).
Ma, H., Fu, R., Xu, J. & Liu, Y. A simple and cost-effective setup for super-resolution localization microscopy. Sci. Rep. 7, 1542 (2017).
Kwakwa, K. et al. easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy. J. Biophotonics 9, 948–957 (2016).
Holm, T. et al. A blueprint for cost-efficient localization microscopy. ChemPhysChem 15, 651–654 (2014).
Diekmann, R. et al. Chip-based wide field-of-view nanoscopy. Nat. Photonics 11, 322–328 (2017).
Diekmann, R. et al. Nanoscopy of bacterial cells immobilized by holographic optical tweezers. Nat. Commun. 7, 13711 (2016).
Sheppard, C. J. R., Mehta, S. B. & Heintzmann, R. Superresolution by image scanning microscopy using pixel reassignment. Opt. Lett. 38, 2889–2892 (2013).
Huff, J. The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat. Methods 12, 1205 (2015).
Korobchevskaya, K., Colin-York, H., Lagerholm, B. & Fritzsche, M. Exploring the potential of Airyscan microscopy for live cell imaging. Photonics 4, 41 (2017).
Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).
Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S. W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl Acad. Sci. USA 102, 17565–17569 (2005).
Grotjohann, T. et al. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478, 204–208 (2011).
Chozinski, T. J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485–488 (2016).
Holden, S. J., Uphoff, S. & Kapanidis, A. N. DAOSTORM: an algorithm for high-density super-resolution microscopy. Nat. Methods 8, 279–280 (2011).
Marsh, R. J. et al. Artifact-free high-density localization microscopy analysis. Nat. Methods 15, 689–692 (2018).
Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).
Pavani, S. R. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl Acad. Sci. USA 106, 2995–2999 (2009).
Juette, M. F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).
Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V. Binding-activated localization microscopy of DNA structures. Nano Lett. 11, 4008–4011 (2011).
Szczurek, A. et al. Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations. Nucleic Acids Res. 45, e56 (2017).
Liu, W. et al. Breaking the axial diffraction limit: a guide to axial super-resolution fluorescence microscopy. Laser Photonics Rev. https://doi.org/10.1002/lpor.201700333 (2018).
We apologize to the many researchers whose work we were unable to cite owing to space constraints. Furthermore, we thank I. Dobbie, C. Lagerholm and J. Demmerle for their valuable comments on the manuscript. L.S. is supported by the Wellcome Trust Strategic Award 107457 supporting advanced microscopy at Micron Oxford. L.S. and T.H. acknowledge support by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 766181. G.D. is supported with funding for External Collaborative Research. M.S. acknowledges support by the Deutsche Forschungsgemeinschaft (DFG) within the Collaborative Research Center 166 ReceptorLight (projects A04 and B04). C.E. acknowledges support by the Medical Research Council (grant number MC_UU_12010/unit programs G0902418 and MC_UU_12025, grant MR/K01577X/1), Wellcome Trust (grant 104924/14/Z/14 and Strategic Award 107457), DFG (Research unit FOR 1905) and Oxford internal funds (EPA Cephalosporin Fund and John Fell Fund).
G.D. is partially exempted from his duties at BNS to pursue fundamental scientific research. All other authors declare no competing interest.
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Schermelleh, L., Ferrand, A., Huser, T. et al. Super-resolution microscopy demystified. Nat Cell Biol 21, 72–84 (2019). https://doi.org/10.1038/s41556-018-0251-8
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