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STED super-resolved microscopy

Abstract

Stimulated emission depletion (STED) microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. However, sophisticated microscopy architectures and high illumination intensities have limited STED microscopy's widespread use in the past. Here we summarize the progress that is mitigating these problems and giving substantial momentum to STED microscopy applications. We discuss the future of this method in regard to spatiotemporal limits, live-cell imaging and combination with spectroscopy. Advances in these areas may elevate STED microscopy to a standard method for imaging in the life sciences.

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Figure 1: STED microscopy principles.
Figure 2: STED microscopy architectures.
Figure 3: RESCue- and protected-STED microscopy.
Figure 4: Comparison of confocal and STED microscopy images.

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References

  1. Hell, S.W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  PubMed  Google Scholar 

  2. Hell, S.W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Diaspro, A. ed. Nanoscopy and Multidimensional Optical Fluorescence Microscopy (Chapman and Hall/CRC, 2010).

  4. Eggeling, C., Willig, K.I., Sahl, S.J. & Hell, S.W. Lens-based fluorescence nanoscopy. Q. Rev. Biophys. 48, 178–243 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Klein, T., Proppert, S. & Sauer, M. Eight years of single-molecule localization microscopy. Histochem. Cell Biol. 141, 561–575 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bianchini, P. et al. STED nanoscopy: a glimpse into the future. Cell Tissue Res. 360, 143–150 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Born, M. & Wolf, E. Principles of Optics (Cambridge University Press, 1999).

  9. Tortarolo, G., Castello, M., Diaspro, A., Koho, S. & Vicidomini, G. Evaluating image resolution in STED microscopy. Optica 5, 32–35 (2018).

    Article  CAS  Google Scholar 

  10. Galiani, S. et al. Strategies to maximize the performance of a STED microscope. Opt. Express 20, 7362–7374 (2012).

    Article  PubMed  Google Scholar 

  11. Vicidomini, G. et al. STED nanoscopy with time-gated detection: theoretical and experimental aspects. PLoS One 8, e54421 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hotta, J. et al. Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores. J. Am. Chem. Soc. 132, 5021–5023 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Wildanger, D., Bückers, J., Westphal, V., Hell, S.W. & Kastrup, L. A STED microscope aligned by design. Opt. Express 17, 16100–16110 (2009).

    Article  PubMed  Google Scholar 

  14. Reuss, M., Engelhardt, J. & Hell, S.W. Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation. Opt. Express 18, 1049–1058 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Yan, L. et al. Q-plate enabled spectrally diverse orbital-angular-momentum conversion for stimulated emission depletion microscopy. Optica 2, 900–903 (2015).

    Article  Google Scholar 

  16. Gould, T.J., Kromann, E.B., Burke, D., Booth, M.J. & Bewersdorf, J. Auto-aligning stimulated emission depletion microscope using adaptive optics. Opt. Lett. 38, 1860–1862 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Willig, K.I. et al. Nanoscale resolution in GFP-based microscopy. Nat. Methods 3, 721–723 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Westphal, V., Blanca, C.M., Dyba, M., Kastrup, L. & Hell, S.W. Laser-diode-stimulated emission depletion microscopy. Appl. Phys. Lett. 82, 3125 (2003).

    Article  CAS  Google Scholar 

  20. Rankin, B.R., Kellner, R.R. & Hell, S.W. Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source. Opt. Lett. 33, 2491–2493 (2008).

    Article  PubMed  Google Scholar 

  21. Göttfert, F. et al. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys. J. 105, L01–L03 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bottanelli, F. et al. Two-colour live-cell nanoscale imaging of intracellular targets. Nat. Commun. 7, 10778 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vicidomini, G. et al. Sharper low-power STED nanoscopy by time gating. Nat. Methods 8, 571–573 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Wildanger, D., Rittweger, E., Kastrup, L. & Hell, S.W. STED microscopy with a supercontinuum laser source. Opt. Express 16, 9614–9621 (2008).

    Article  PubMed  Google Scholar 

  25. Bückers, J., Wildanger, D., Vicidomini, G., Kastrup, L. & Hell, S.W. Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses. Opt. Express 19, 3130–3143 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Winter, F.R. et al. Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection. Sci. Rep. 7, 46492 (2017).

  27. Bianchini, P., Harke, B., Galiani, S., Vicidomini, G. & Diaspro, A. Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging. Proc. Natl. Acad. Sci. USA 109, 6390–6393 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  28. van der Velde, J.H.M. et al. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization. Nat. Commun. 7, 10144 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Donnert, G. et al. Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl. Acad. Sci. USA 103, 11440–11445 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Moneron, G. et al. Fast STED microscopy with continuous wave fiber lasers. Opt. Express 18, 1302–1309 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Schneider, J. et al. Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics. Nat. Methods 12, 827–830 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. Beater, S., Holzmeister, P., Pibiri, E., Lalkens, B. & Tinnefeld, P. Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers. Phys. Chem. Chem. Phys. 16, 6990–6996 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Han, K.Y. et al. Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light. Nano Lett. 9, 3323–3329 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Lesoine, M.D. et al. Subdiffraction, luminescence-depletion imaging of isolated, giant, CdSe/CdS nanocrystal quantum dots. J. Phys. Chem. C 117, 3662–3667 (2013).

    Article  CAS  Google Scholar 

  35. Liu, Y. et al. Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy. Nature 543, 229–233 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Staudt, T. et al. Far-field optical nanoscopy with reduced number of state transition cycles. Opt. Express 19, 5644–5657 (2011).

    Article  PubMed  Google Scholar 

  37. Göttfert, F. et al. Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent. Proc. Natl. Acad. Sci. USA 114, 2125–2130 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Heine, J. et al. Adaptive-illumination STED nanoscopy. Proc. Natl. Acad. Sci. USA 114, 9797–9802 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Danzl, J.G. et al. Coordinate-targeted fluorescence nanoscopy with multiple off states. Nat. Photonics 10, 122–128 (2016).

    Article  CAS  Google Scholar 

  40. Castello, M. et al. Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching. Microsc. Res. Tech. 79, 785–791 (2016).

  41. Strack, R. Death by super-resolution imaging. Nat. Methods 12, 1111 (2015).

    Article  CAS  PubMed  Google Scholar 

  42. Wäldchen, 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Laissue, P.P., Alghamdi, R.A., Tomancak, P., Reynaud, E.G. & Shroff, H. Assessing phototoxicity in live fluorescence imaging. Nat. Methods 14, 657–661 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Hein, B., Willig, K.I. & Hell, S.W. Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc. Natl. Acad. Sci. USA 105, 14271–14276 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Schermelleh, L., Heintzmann, R. & Leonhardt, H. A guide to super-resolution fluorescence microscopy. J. Cell Biol. 190, 165–175 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lukinavičius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Lukinavičius, G. et al. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 6, 8497 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Butkevich, A.N. et al. Fluorescent rhodamines and fluorogenic carbopyronines for super-resolution STED microscopy in living cells. Angew. Chem. Int. Ed. Engl. 55, 3290–3294 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Matela, G. et al. A far-red emitting fluorescent marker protein, mGarnet2, for microscopy and STED nanoscopy. Chem. Commun. (Camb.) 53, 979–982 (2017).

    Article  CAS  Google Scholar 

  51. Opazo, F. et al. Aptamers as potential tools for super-resolution microscopy. Nat. Methods 9, 938–939 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Shcherbakova, D.M. & Verkhusha, V.V. Chromophore chemistry of fluorescent proteins controlled by light. Curr. Opin. Chem. Biol. 20, 60–68 (2014).

    Article  CAS  PubMed  Google Scholar 

  53. Hell, S.W. Toward fluorescence nanoscopy. Nat. Biotechnol. 21, 1347–1355 (2003).

    Article  CAS  PubMed  Google Scholar 

  54. Friedrich, M., Gan, Q., Ermolayev, V. & Harms, G.S. STED-SPIM: stimulated emission depletion improves sheet illumination microscopy resolution. Biophys. J. 100, L43–L45 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Gohn-Kreuz, C. & Rohrbach, A. Light-sheet generation in inhomogeneous media using self-reconstructing beams and the STED-principle. Opt. Express 24, 5855–5865 (2016).

    Article  PubMed  Google Scholar 

  56. Chen, B.-C. et al.Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Urban, N.T., Willig, K.I., Hell, S.W. & Nägerl, U.V. STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J. 101, 1277–1284 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Patton, B.R., Burke, D., Vrees, R. & Booth, M.J. Is phase-mask alignment aberrating your STED microscope? Methods Appl. Fluoresc. 3, 024002 (2015).

    Article  PubMed  Google Scholar 

  59. Loew, L.M. & Hell, S.W. Superresolving dendritic spines. Biophys. J. 104, 741–743 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ke, M.-T. et al. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Cell Rep. 14, 2718–2732 (2016).

    Article  CAS  PubMed  Google Scholar 

  61. Unnersjö-Jess, D., Scott, L., Blom, H. & Brismar, H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue. Kidney Int. 89, 243–247 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Bingen, P., Reuss, M., Engelhardt, J. & Hell, S.W. Parallelized STED fluorescence nanoscopy. Opt. Express 19, 23716–23726 (2011).

    Article  CAS  PubMed  Google Scholar 

  63. Yang, B., Przybilla, F., Mestre, M., Trebbia, J.-B. & Lounis, B. Large parallelization of STED nanoscopy using optical lattices. Opt. Express 22, 5581–5589 (2014).

    Article  PubMed  Google Scholar 

  64. Bergermann, F., Alber, L., Sahl, S.J., Engelhardt, J. & Hell, S.W. 2000-fold parallelized dual-color STED fluorescence nanoscopy. Opt. Express 23, 211–223 (2015).

    Article  PubMed  Google Scholar 

  65. Kastrup, L., Blom, H., Eggeling, C. & Hell, S.W. Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Phys. Rev. Lett. 94, 178104 (2005).

    Article  CAS  PubMed  Google Scholar 

  66. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

    Article  CAS  PubMed  Google Scholar 

  67. Lanzanò, L. et al. Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS. Nat. Commun. 8, 65 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Niehörster, T. et al. Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Nat. Methods 13, 257–262 (2016).

    Article  CAS  PubMed  Google Scholar 

  69. Donnert, G. et al. Two-color far-field fluorescence nanoscopy. Biophys. J. 92, L67–L69 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

    Article  CAS  PubMed  Google Scholar 

  71. Sidenstein, S.C. et al. Multicolour multilevel STED nanoscopy of actin/spectrin organization at synapses. Sci. Rep. 6, 26725 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Galiani, S. et al. Super-resolution microscopy reveals compartmentalization of peroxisomal membrane proteins. J. Biol. Chem. 291, 16948–16962 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Sednev, M.V., Belov, V.N. & Hell, S.W. Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review. Methods Appl. Fluoresc. 3, 042004 (2015).

    Article  CAS  PubMed  Google Scholar 

  74. Lukinavičius, G. et al. Fluorogenic probes for multicolor imaging in living cells. J. Am. Chem. Soc. 138, 9365–9368 (2016).

    Article  CAS  PubMed  Google Scholar 

  75. Tønnesen, J., Nadrigny, F., Willig, K.I., Wedlich-Söldner, R. & Nägerl, U.V. Two-color STED microscopy of living synapses using a single laser-beam pair. Biophys. J. 101, 2545–2552 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Ringemann, C. et al. Exploring single-molecule dynamics with fluorescence nanoscopy. New J. Phys. 11, 103054 (2009).

    Article  CAS  Google Scholar 

  77. Ta, H. et al. Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6, 7977 (2015).

    Article  CAS  PubMed  Google Scholar 

  78. Harke, B., Chacko, J., Haschke, H., Canale, C. & Diaspro, A. A novel nanoscopic tool by combining AFM with STED microscopy. Opt. Nanoscopy 1, 3 (2012).

    Article  Google Scholar 

  79. Zanella, R. et al. Towards real-time image deconvolution: application to confocal and STED microscopy. Sci. Rep. 3, 2523 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Hell, S.W. et al. The 2015 super-resolution microscopy roadmap. J. Phys. D Appl. Phys. 48, 443001 (2015).

    Article  CAS  Google Scholar 

  81. Rankin, B.R. et al. Nanoscopy in a living multicellular organism expressing GFP. Biophys. J. 100, L63–L65 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Meyer, L. et al. Dual-color STED microscopy at 30-nm focal-plane resolution. Small 4, 1095–1100 (2008).

    Article  CAS  PubMed  Google Scholar 

  83. Coto Hernàndez, I., d'Amora, M., Diaspro, A. & Vicidomini, G. Influence of laser intensity noise on gated CW-STED microscopy. Laser Phys. Lett. 11, 095603 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

We equally acknowledge S. Koho, M. Castello and G. Tortarolo (Istituto Italiano di Tecnologia) for fruitful discussions.

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Correspondence to Giuseppe Vicidomini.

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Integrated supplementary information

Supplementary Figure 1 Milestones of the laser technology developments on STED microscopy.1–23

The colour of the milestone year denotes if the associated STED implementation is: (i) obsolete (black); (ii) commercial available (green); routinely used but custom-made (red). The dismissed and current commercial STED microscopes are: Leica TCS-STED (LeTCS, dismissed); Leica TCS-STED-CW (LeTCSCW, dismissed); Leica TCS-STED-3X (LeTCS3X); Abberior STED QUAD Scan (AbQS); Abberior STEDYCON (AbSYC); PicoQuant MicroTime 200 STED (PqMT200); ISS Alba-STED (Alba)

Supplementary Figure 2 Multi-colour STED microscopy.15,21,22,24–47

For each dyes combination, the STED implementation used is reported: custom made (CM) or commercial. Compatible commercial implementations have been also reported. AbQS@775 and AbQS@595 indicated the STED beam's wavelength configuration (775 nm or 595 nm, respectively) for the AbQS commercial systems.

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Supplementary Figures 1–2 and Supplementary Notes 1–4 (PDF 1664 kb)

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Vicidomini, G., Bianchini, P. & Diaspro, A. STED super-resolved microscopy. Nat Methods 15, 173–182 (2018). https://doi.org/10.1038/nmeth.4593

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