Abstract
Fluorescence nanoscopy represented a breakthrough for the life sciences as it delivers 20–30 nm resolution using far-field fluorescence microscopes. This resolution limit is not fundamental but imposed by the limited photostability of fluorophores under ambient conditions. This has motivated the development of a second generation of fluorescence nanoscopy methods that aim to deliver sub-10 nm resolution, reaching the typical size of structural proteins and thus providing true molecular resolution. In this review, we present common fundamental aspects of these nanoscopies, discuss the key experimental factors that are necessary to fully exploit their capabilities, and discuss their current and future challenges.
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References
Hell SW et al (2007) Far-field optical nanoscopy. Science 316:1153–1158. https://doi.org/10.1126/science.1137395
Hell SW (2015) Nanoscopy with Focused Light (Nobel Lecture). Angew Chemie Int Ed 54:8054–8066. https://doi.org/10.1002/anie.201504181
Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci 97:8206–8210. https://doi.org/10.1073/pnas.97.15.8206
Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782. https://doi.org/10.1364/OL.19.000780
Hofmann M, Eggeling C, Jakobs S, Hell SW (2005) Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci 102:17565–17569. https://doi.org/10.1073/pnas.0506010102
Hell SW, Jakobs S, Kastrup L (2003) Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl Phys A Mater Sci Process 77:859–860. https://doi.org/10.1007/s00339-003-2292-4
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–796. https://doi.org/10.1038/nmeth929
Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645. https://doi.org/10.1126/science.1127344
Huang B, Bates M, Zhuang X (2009) Super-resolution fluorescence microscopy. Annu Rev Biochem 78:993–1016. https://doi.org/10.1146/annurev.biochem.77.061906.092014
Sahl SJ, Hell SW, Jakobs S (2017) Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18:685–701. https://doi.org/10.1038/nrm.2017.71
Schnitzbauer J, Strauss MT, Schlichthaerle T, Schueder F, Jungmann R (2017) Super-resolution microscopy with DNA-PAINT. Nat Protoc 12:1198–1228. https://doi.org/10.1038/nprot.2017.024
Jungmann R et al (2010) Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett 10:4756–4761. https://doi.org/10.1021/nl103427w
Dai M, Jungmann R, Yin P (2016) Optical imaging of individual biomolecules in densely packed clusters. Nat Nanotechnol 11:798–807. https://doi.org/10.1038/nnano.2016.95
Strauss S et al (2018) Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging. Nat Methods 15:685–688. https://doi.org/10.1038/s41592-018-0105-0
Strauss S, Jungmann R (2020) Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT. Nat Methods 17:789–791. https://doi.org/10.1038/s41592-020-0869-x
Balzarotti F et al (2017) Nanometer resolution imaging and tracking with minimal photon fluxes. Science 355:606–612. https://doi.org/10.1126/science.aak9913
Masullo LA et al (2021) Pulsed Interleaved MINFLUX. Nano Lett 21:840–846. https://doi.org/10.1021/acs.nanolett.0c04600
Jouchet P et al (2021) Nanometric axial localization of single fluorescent molecules with modulated excitation. Nat Photonics 15:297–304. https://doi.org/10.1038/s41566-020-00749-9
Gu L et al (2019) Molecular resolution imaging by repetitive optical selective exposure. Nat Methods 16:1114–1118. https://doi.org/10.1038/s41592-019-0544-2
Cnossen J et al (2020) Localization microscopy at doubled precision with patterned illumination. Nat Methods 17:59–63. https://doi.org/10.1038/s41592-019-0657-7
Reymond L et al (2019) SIMPLE: Structured illumination based point localization estimator with enhanced precision. Opt Express 27:24578. https://doi.org/10.1364/OE.27.024578
Reymond L, Huser T, Ruprecht V, Wieser S (2020) Modulation-enhanced localization microscopy. J Phys Photonics 2:041001. https://doi.org/10.1088/2515-7647/ab9eac
Masullo LA, Lopez LF, Stefani FD (2021) A common framework for single-molecule localization using sequential structured illumination. Biophysical Reports, accepted. https://doi.org/10.1016/j.bpr.2021.100036
Huang F et al (2016) Ultra-high resolution 3D imaging of whole cells. Cell 166:1028–1040. https://doi.org/10.1016/j.cell.2016.06.016
Shtengel G et al (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci 106:3125–3130. https://doi.org/10.1073/pnas.0813131106
Aquino D et al (2011) Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat Methods 8:353–359. https://doi.org/10.1038/nmeth.1583
Szalai AM et al (2021) Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules. Nat Commun 12:517. https://doi.org/10.1038/s41467-020-20863-0
Bourg N et al (2015) Direct optical nanoscopy with axially localized detection. Nat Photonics 9:587–593. https://doi.org/10.1038/nphoton.2015.132
Dasgupta A et al (2021) Direct supercritical angle localization microscopy for nanometer 3D superresolution. Nat Commun 12:1180. https://doi.org/10.1038/s41467-021-21333-
Chizhik AI, Rother J, Gregor I, Janshoff A, Enderlein J (2014) Metal-induced energy transfer for live cell nanoscopy. Nat Photonics 8:124–127. https://doi.org/10.1038/nphoton.2013.345
Ghosh A et al (2019) Graphene-based metal-induced energy transfer for sub-nanometre optical localization. Nat Photonics 13:860–865. https://doi.org/10.1038/s41566-019-0510-7
Kaminska I et al (2019) Distance dependence of single-molecule energy transfer to graphene measured with DNA origami nanopositioners. Nano Lett 19:4257–4262. https://doi.org/10.1021/acs.nanolett.9b00172
Kamińska I et al (2021) Graphene energy transfer for single-molecule biophysics, biosensing, and super-resolution microscopy. Adv Mater 33:2101099. https://doi.org/10.1002/adma.202101099
Gwosch KC et al (2020) MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat Methods 17:217–224. https://doi.org/10.1038/s41592-019-0688-
Gu L et al (2021) Molecular-scale axial localization by repetitive optical selective exposure. Nat Methods 18:369–373. https://doi.org/10.1038/s41592-021-01099-2
Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813. https://doi.org/10.1126/science.1153529
Willig KI, Harke B, Medda R, Hell SW (2007) STED microscopy with continuous wave beams. Nat Methods 4:915–918. https://doi.org/10.1038/nmeth1108
Rittweger E, Han KY, Irvine SSE, Eggeling C, Hell SW (2009) STED microscopy reveals crystal colour centres with nanometric resolution. Nat Photonics 3:1–4. https://doi.org/10.1038/nphoton.2009.2
Heilemann M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chemie Int Ed 47:6172–6176. https://doi.org/10.1002/anie.200802376
Nieuwenhuizen RPJJ et al (2013) Measuring image resolution in optical nanoscopy. Nat Methods 10:557–562. https://doi.org/10.1038/nmeth.2448
Descloux A, Grußmayer KS, Radenovic A (2019) Parameter-free image resolution estimation based on decorrelation analysis. Nat Methods 16:918–924. https://doi.org/10.1038/s41592-019-0515-7
Descloux AC, Grußmayer KS, Radenovic A (2021) Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation. Commun Biol 4:550. https://doi.org/10.1038/s42003-021-02086-1
Schmied JJ et al (2014) DNA origami–based standards for quantitative fluorescence microscopy. Nat Protoc 9:1367–1391. https://doi.org/10.1038/nprot.2014.079
Scheckenbach M, Bauer J, Zähringer J, Selbach F, Tinnefeld P (2020) DNA origami nanorulers and emerging reference structures. APL Mater 8:110902. https://doi.org/10.1063/5.0022885
Schlichthaerle T et al (2019) Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT. Angew Chemie Int Ed 58:13004–13008. https://doi.org/10.1002/anie.201905685
Thevathasan JV et al (2019) Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat Methods 16:1045–1053. https://doi.org/10.1038/s41592-019-0574-9
von Appen A et al (2015) In situ structural analysis of the human nuclear pore complex. Nature 526:140–143. https://doi.org/10.1038/nature15381
Loïodice I et al (2004) The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. Mol Biol Cell 15:3333–3344. https://doi.org/10.1091/mbc.e03-12-0878
Heydarian H et al (2021) 3D particle averaging and detection of macromolecular symmetry in localization microscopy. Nat Commun 12:2847. https://doi.org/10.1038/s41467-021-22006-5
Schmidt R et al (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat Commun 12:1478. https://doi.org/10.1038/s41467-021-21652-z
Li Y et al (2018) Real-time 3D single-molecule localization using experimental point spread functions. Nat Methods 15:367–369. https://doi.org/10.1038/nmeth.4661
Wang Y et al (2014) Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm. Opt Express 22:15982–15991. https://doi.org/10.1364/OE.22.015982
Balinovic A, Albrecht D, Endesfelder U (2019) Spectrally red-shifted fluorescent fiducial markers for optimal drift correction in localization microscopy. J Phys D Appl Phys 52:204002. https://doi.org/10.1088/1361-6463/ab0862
Carter AR et al (2007) Stabilization of an optical microscope to 0.1 nm in three dimensions. Appl Opt 46:421–427. https://doi.org/10.1364/AO.46.000421
Pertsinidis A, Zhang Y, Chu S (2010) Subnanometre single-molecule localization, registration and distance measurements. Nature 466:647–651. https://doi.org/10.1038/nature09163
Coelho S et al (2020) Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells. Sci Adv 6:eaay8271. https://doi.org/10.1126/sciadv.aay8271
Ganji M, Schlichthaerle T, Eklund AS, Strauss S, Jungmann R (2021) Quantitative assessment of labeling probes for super-resolution microscopy using designer DNA nanostructures. ChemPhysChem 22:911–914. https://doi.org/10.1002/cphc.202100185
Früh SM et al (2021) Site-specifically-labeled antibodies for super-resolution microscopy reveal in situ linkage errors. ACS Nano 15:12161–12170. https://doi.org/10.1021/acsnano.1c03677
Ries J, Kaplan C, Platonova E, Eghlidi H, Ewers H (2012) A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9:582–584. https://doi.org/10.1038/nmeth.1991
Pleiner T et al (2015) Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. Elife 4:e11349. https://doi.org/10.7554/eLife.11349
Schlichthaerle T et al (2018) Site-Specific Labeling of Affimers for DNA-PAINT Microscopy. Angew Chemie Int Ed 57:11060–11063. https://doi.org/10.1002/anie.201804020
Wang L, Frei MS, Salim A, Johnsson K (2019) Small-molecule fluorescent probes for live-cell super-resolution microscopy. J Am Chem Soc 141:2770–2781. https://doi.org/10.1021/jacs.8b11134
Keppler A et al (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89. https://doi.org/10.1038/nbt765
Los GV et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382. https://doi.org/10.1021/cb800025k
Tiede C et al (2017) Affimer proteins are versatile and renewable affinity reagents. Elife 6:e24903. https://doi.org/10.7554/eLife.24903
Opazo F et al (2012) Aptamers as potential tools for super-resolution microscopy. Nat Methods 9:938–939. https://doi.org/10.1038/nmeth.2179
Bedford R et al (2017) Alternative reagents to antibodies in imaging applications. Biophys Rev 9:299–308. https://doi.org/10.1007/s12551-017-0278-2
Lukinavičius G et al (2014) Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11:731–733. https://doi.org/10.1038/nmeth.2972
Lukinavičius G et al (2013) A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem 5:132–139. https://doi.org/10.1038/nchem.1546
D’Este E et al (2015) STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. Cell Rep 10:1246–1251. https://doi.org/10.1016/j.celrep.2015.02.007
Dancker P, Löw I, Hasselbach W, Wieland T (1975) Interaction of actin with phalloidin: polymerization and stabilization of F-actin. Biochim Biophys Acta 400:407–414. https://doi.org/10.1016/0005-2795(75)90196-8
Hern J, a, et al (2010) Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc Natl Acad Sci U S A 107:2693–2698. https://doi.org/10.1073/pnas.0907915107
Cai M et al (2004) Real time differentiation of G-Protein Coupled Receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy. J Am Chem Soc 126:7160–7161. https://doi.org/10.1021/ja049473m
Szalai AM et al (2018) A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and: in situ affinity constant in cells. Phys Chem Chem Phys 20:29212–29220. https://doi.org/10.1039/C8CP06196C
Ha T, Tinnefeld P (2012) Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 63:595–617. https://doi.org/10.1146/annurev-physchem-032210-103340
Vogelsang J et al (2010) Make them blink: probes for super-resolution microscopy. ChemPhysChem 11:2475–2490. https://doi.org/10.1002/cphc.201000189
Dempsey GTGGT, Vaughan JCJJC, Chen KHK, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8:1027–1036. https://doi.org/10.1038/nmeth.1768
van de Linde S, Heilemann M, Sauer M (2012) Live-cell super-resolution imaging with synthetic fluorophores. Annu Rev Phys Chem 63:519–540. https://doi.org/10.1146/annurev-physchem-032811-112012
Vogelsang J et al (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chemie 47:5465–5469. https://doi.org/10.1002/anie.200801518
Cordes T, Vogelsang J, Tinnefeld P (2009) On the mechanism of Trolox as antiblinking and antibleaching reagent. J Am Chem Soc 131:5018–5019. https://doi.org/10.1021/ja809117z
Harada Y, Sakurada K, Aoki T, Thomas DD, Yanagida T (1990) Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. J Mol Biol 216:49–68. https://doi.org/10.1016/S0022-2836(05)80060-9
Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94:1826–1835. https://doi.org/10.1529/biophysj.107.117689
Szalai AM et al (2021) Super-resolution imaging of energy transfer by intensity-based STED-FRET. Nano Lett 21:2296–2303. https://doi.org/10.1021/acs.nanolett.1c00158
Kasper R et al (2010) Single-molecule STED microscopy with photostable organic fluorophores. Small 6:1379–1384. https://doi.org/10.1002/smll.201000203
Blumhardt P et al (2018) Photo-induced depletion of binding sites in DNA-PAINT microscopy. Molecules 23:3165. https://doi.org/10.3390/molecules23123165
Olivier N, Keller D, Gönczy P, Manley S (2013) Resolution doubling in 3D-STORM imaging through improved buffers. PLoS ONE 8:e69004. https://doi.org/10.1371/journal.pone.0069004
Schueder F et al (2019) An order of magnitude faster DNA-PAINT imaging by optimized sequence design and buffer conditions. Nat Methods 16:1101–1104. https://doi.org/10.1038/s41592-019-0584-7
Civitci F et al (2020) Fast and multiplexed superresolution imaging with DNA-PAINT-ERS. Nat Commun 11:4339. https://doi.org/10.1038/s41467-020-18181-6
Chung KKH et al (2020) Fluorogenic probe for fast 3D whole-cell DNA-PAINT. bioRxiv 2020.04.29.066886. https://doi.org/10.1101/2020.04.29.066886
Lee J, Park S, Kang W, Hohng S (2017) Accelerated super-resolution imaging with FRET-PAINT. Mol Brain 10:63. https://doi.org/10.1186/s13041-017-0344-5
Auer A, Strauss MT, Schlichthaerle T, Jungmann R (2017) Fast, background-free DNA-PAINT imaging using FRET-based probes. Nano Lett 17:6428–6434. https://doi.org/10.1021/acs.nanolett.7b03425
Mortensen KI, Churchman LS, Spudich JA, Flyvbjerg H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods 7:377–381. https://doi.org/10.1038/nmeth.1447
Huang F et al (2013) Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms. Nat Methods 10:653–658. https://doi.org/10.1038/nmeth.2488
Westphal V et al (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320:246–249. https://doi.org/10.1126/science.1154228
Bergermann F, Alber L, Sahl SJ, Engelhardt J, Hell SW (2015) 2000-fold parallelized dual-color STED fluorescence nanoscopy. Opt Express 23:211. https://doi.org/10.1364/OE.23.000211
Masullo LA et al (2018) Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems. Nat Commun 9:3281. https://doi.org/10.1038/s41467-018-05799-w
Bodén A et al (2021) Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy. Nat Biotechnol 39:609–618. https://doi.org/10.1038/s41587-020-00779-2
Ouyang W, Aristov A, Lelek M, Hao X, Zimmer C (2018) Deep learning massively accelerates super-resolution localization microscopy. Nat Biotechnol 36:460–468. https://doi.org/10.1038/nbt.4106
Filius M et al (2020) High-speed super-resolution imaging using protein-assisted DNA-PAINT. Nano Lett 20:2264–2270. https://doi.org/10.1021/acs.nanolett.9b04277
Enderlein J (2000) Tracking of fluorescent molecules diffusing within membranes. Appl Phys B Lasers Opt 71:773–777. https://doi.org/10.1007/s003400000409
Weigel AV, Simon B, Tamkun MM, Krapf D (2011) Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking. Proc Natl Acad Sci 108:6438–6443. https://doi.org/10.1073/pnas.1016325108
Sako Y, Minoghchi S, Yanagida T (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2:168–172. https://doi.org/10.1038/35004044
Eilers Y, Ta H, Gwosch KC, Balzarotti F, Hell SW (2018) MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution. Proc Natl Acad Sci 115:6117–6122. https://doi.org/10.1073/pnas.1801672115
Piston DW, Kremers G-J (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32:407–414. https://doi.org/10.1016/j.tibs.2007.08.003
Lerner E et al (2018) Toward dynamic structural biology: two decades of single-molecule Förster resonance energy transfer. Science 359:eaan1133. https://doi.org/10.1126/science.aan1133
Deußner-Helfmann NS et al (2018) Correlative single-molecule FRET and DNA-PAINT imaging. Nano Lett 18:4626–4630. https://doi.org/10.1021/acs.nanolett.8b02185
Fernández-Suárez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9:929–943. https://doi.org/10.1038/nrm2531
Grimm JB et al (2020) A general method to optimize and functionalize red-shifted rhodamine dyes. Nat Methods 17:815–821. https://doi.org/10.1038/s41592-020-0909-6
Chen F, Tillberg PW, Boyden ES (2015) Expansion microscopy. Science 347:543–548. https://doi.org/10.1126/science.1260088
Mau A, Friedl K, Leterrier C, Bourg N, Lévêque-Fort S (2021) Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields. Nat Commun 12:3077. https://doi.org/10.1038/s41467-021-23405-4
Stehr F, Stein J, Schueder F, Schwille P, Jungmann R (2019) Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification. Nat Commun 10:1268. https://doi.org/10.1038/s41467-019-09064-6
Pape JK et al (2020) Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins. Proc Natl Acad Sci 117:20607–20614. https://doi.org/10.1073/pnas.2009364117
Tardif C et al (2019) Fluorescence lifetime imaging nanoscopy for measuring Förster resonance energy transfer in celular nanodomains. Neurophotonics 6: 015002. https://doi.org/10.1117/1.NPh.6.1.015002
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This work has been funded by CONICET, ANPCYT Projects PICT-2017–0870, and PICT-2014–0739. F.D.S. received support from the Max-Planck-Society and the Alexander von Humboldt Foundation.
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Luciano A. Masullo and Alan M. Szalai contributed equally to this work.
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Masullo, L.A., Szalai, A.M., Lopez, L.F. et al. Fluorescence nanoscopy at the sub-10 nm scale. Biophys Rev 13, 1101–1112 (2021). https://doi.org/10.1007/s12551-021-00864-z
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DOI: https://doi.org/10.1007/s12551-021-00864-z