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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References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

    , , & Lens-based fluorescence nanoscopy. Q. Rev. Biophys. 48, 178–243 (2015).

  5. 5.

    Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

  6. 6.

    , & Eight years of single-molecule localization microscopy. Histochem. Cell Biol. 141, 561–575 (2014).

  7. 7.

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

  8. 8.

    & Principles of Optics (Cambridge University Press, 1999).

  9. 9.

    , , , & Evaluating image resolution in STED microscopy. Optica 5, 32–35 (2018).

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 13.

    , , , & A STED microscope aligned by design. Opt. Express 17, 16100–16110 (2009).

  14. 14.

    , & Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation. Opt. Express 18, 1049–1058 (2010).

  15. 15.

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

  16. 16.

    , , , & Auto-aligning stimulated emission depletion microscope using adaptive optics. Opt. Lett. 38, 1860–1862 (2013).

  17. 17.

    , , , & Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).

  18. 18.

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

  19. 19.

    , , , & Laser-diode-stimulated emission depletion microscopy. Appl. Phys. Lett. 82, 3125 (2003).

  20. 20.

    , & Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source. Opt. Lett. 33, 2491–2493 (2008).

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 24.

    , , & STED microscopy with a supercontinuum laser source. Opt. Express 16, 9614–9621 (2008).

  25. 25.

    , , , & Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses. Opt. Express 19, 3130–3143 (2011).

  26. 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. 27.

    , , , & Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging. Proc. Natl. Acad. Sci. USA 109, 6390–6393 (2012).

  28. 28.

    et al. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization. Nat. Commun. 7, 10144 (2016).

  29. 29.

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

  30. 30.

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

  31. 31.

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

  32. 32.

    , , , & Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers. Phys. Chem. Chem. Phys. 16, 6990–6996 (2014).

  33. 33.

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

  34. 34.

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

  35. 35.

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

  36. 36.

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

  37. 37.

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

  38. 38.

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

  39. 39.

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

  40. 40.

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

  41. 41.

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

  42. 42.

    , , , & Light-induced cell damage in live-cell super-resolution microscopy. Sci. Rep. 5, 15348 (2015).

  43. 43.

    , , , & Assessing phototoxicity in live fluorescence imaging. Nat. Methods 14, 657–661 (2017).

  44. 44.

    , & Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc. Natl. Acad. Sci. USA 105, 14271–14276 (2008).

  45. 45.

    , & A guide to super-resolution fluorescence microscopy. J. Cell Biol. 190, 165–175 (2010).

  46. 46.

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

  47. 47.

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

  48. 48.

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

  49. 49.

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

  50. 50.

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

  51. 51.

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

  52. 52.

    & Chromophore chemistry of fluorescent proteins controlled by light. Curr. Opin. Chem. Biol. 20, 60–68 (2014).

  53. 53.

    Toward fluorescence nanoscopy. Nat. Biotechnol. 21, 1347–1355 (2003).

  54. 54.

    , , & STED-SPIM: stimulated emission depletion improves sheet illumination microscopy resolution. Biophys. J. 100, L43–L45 (2011).

  55. 55.

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

  56. 56.

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

  57. 57.

    , , & STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J. 101, 1277–1284 (2011).

  58. 58.

    , , & Is phase-mask alignment aberrating your STED microscope? Methods Appl. Fluoresc. 3, 024002 (2015).

  59. 59.

    & Superresolving dendritic spines. Biophys. J. 104, 741–743 (2013).

  60. 60.

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

  61. 61.

    , , & Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue. Kidney Int. 89, 243–247 (2016).

  62. 62.

    , , & Parallelized STED fluorescence nanoscopy. Opt. Express 19, 23716–23726 (2011).

  63. 63.

    , , , & Large parallelization of STED nanoscopy using optical lattices. Opt. Express 22, 5581–5589 (2014).

  64. 64.

    , , , & 2000-fold parallelized dual-color STED fluorescence nanoscopy. Opt. Express 23, 211–223 (2015).

  65. 65.

    , , & Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Phys. Rev. Lett. 94, 178104 (2005).

  66. 66.

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

  67. 67.

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

  68. 68.

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

  69. 69.

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

  70. 70.

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

  71. 71.

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

  72. 72.

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

  73. 73.

    , & Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review. Methods Appl. Fluoresc. 3, 042004 (2015).

  74. 74.

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

  75. 75.

    , , , & Two-color STED microscopy of living synapses using a single laser-beam pair. Biophys. J. 101, 2545–2552 (2011).

  76. 76.

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

  77. 77.

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

  78. 78.

    , , , & A novel nanoscopic tool by combining AFM with STED microscopy. Opt. Nanoscopy 1, 3 (2012).

  79. 79.

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

  80. 80.

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

  81. 81.

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

  82. 82.

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

  83. 83.

    , , & Influence of laser intensity noise on gated CW-STED microscopy. Laser Phys. Lett. 11, 095603 (2014).

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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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Affiliations

  1. Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy.

    • Giuseppe Vicidomini
  2. Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy.

    • Paolo Bianchini
    •  & Alberto Diaspro
  3. Department of Physics, University of Genoa, Genoa, Italy.

    • Alberto Diaspro

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Giuseppe Vicidomini.

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https://doi.org/10.1038/nmeth.4593