Skip to main content
SpringerLink
Log in

Photoswitchable Fluorophores for Single-Molecule Localization Microscopy

  • Protocol
  • First Online:
Book cover Nanoimaging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 950))

Abstract

Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods—collectively referred to as “single-molecule localization microscopy” (SMLM)—are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.

Key to this strategy is the use of fluorophores that can be switched “on” and “off” in a controllable manner. Here we review the strengths and weaknesses of the wide variety of SMLM-compatible photoswitchable fluorophores and labeling strategies currently available. We also discuss their suitability for live-cell and multicolor imaging, as well as molecular counting.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783

    Article  PubMed  CAS  Google Scholar 

  2. Abbe E (1873) Beitrage zur theorie des mikroskops und der mikroskopischen wahrnehmung. Archive Mikroskop Anat 9:413–420

    Article  Google Scholar 

  3. Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149:243–251

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  5. Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158

    Article  PubMed  CAS  Google Scholar 

  6. McEvoy AL, Greenfield D, Bates M, Liphardt J (2010) Q&A: single-molecule localization microscopy for biological imaging. BMC Biol 8:106

    Article  PubMed  Google Scholar 

  7. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  PubMed  CAS  Google Scholar 

  8. Rust MJ, Bates M, Zhuang XW (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795

    Article  PubMed  CAS  Google Scholar 

  9. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47:6172–6176

    Article  CAS  Google Scholar 

  10. Folling J, Belov V, Kunetsky R, Medda R, Schonle A et al (2007) Photochromic rhodamines provide nanoscopy with optical sectioning. Angew Chem Int Ed 46:6266–6270

    Article  CAS  Google Scholar 

  11. Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272

    Article  PubMed  CAS  Google Scholar 

  12. Folling J, Bossi M, Bock H, Medda R, Wurm CA et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5:943–945

    Article  PubMed  Google Scholar 

  13. Lemmer P, Gunkel M, Baddeley D, Kaufmann R, Urich A et al (2008) SPDM: light microscopy with single-molecule resolution at the nanoscale. Appl Phys B 93:1–12

    Article  CAS  Google Scholar 

  14. Steinhauer C, Forthmann C, Vogelsang J, Tinnefeld P (2008) Superresolution microscopy on the basis of engineered dark states. J Am Chem Soc 130:16840

    Article  PubMed  CAS  Google Scholar 

  15. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877

    Article  PubMed  CAS  Google Scholar 

  16. van de Linde S, Wolter S, Heilemann M, Sauer M (2010) The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. J Biotechnol 149:260–266

    Article  PubMed  Google Scholar 

  17. Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J et al (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci USA 104:20308–20313

    Article  PubMed  CAS  Google Scholar 

  18. Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565

    Article  PubMed  CAS  Google Scholar 

  19. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Phys Rev 90:1103–1163

    Article  CAS  Google Scholar 

  20. Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388:355–358

    Article  PubMed  CAS  Google Scholar 

  21. Fuchs J, Bohme S, Oswald F, Hedde PN, Krause M et al (2010) A photoactivatable marker protein for pulse-chase imaging with superresolution. Nat Methods 7:627–630

    Article  PubMed  CAS  Google Scholar 

  22. Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S et al (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22:1435–1439

    Article  PubMed  CAS  Google Scholar 

  23. McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133

    Article  PubMed  CAS  Google Scholar 

  24. Habuchi S, Ando R, Dedecker P, Verheijen W, Mizuno H et al (2005) Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci USA 102:9511–9516

    Article  PubMed  CAS  Google Scholar 

  25. Andresen M, Stiel AC, Folling J, Wenzel D, Schonle A et al (2008) Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26:1035–1040

    Article  PubMed  CAS  Google Scholar 

  26. Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C et al (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101:15905–15910

    Article  PubMed  CAS  Google Scholar 

  27. Annibale P, Scarselli M, Kodiyan A, Radenovic A (2010) Photoactivatable fluorescent protein mEos2 displays repeated photoactivation after a long-lived dark state in the red photoconverted form. J Phys Chem Lett 1:1506–1510

    Article  CAS  Google Scholar 

  28. Endesfelder U, Malkusch S, Flottmann B, Mondry J, Liguzinski P et al (2011) Chemically induced photoswitching of fluorescent probes-a general concept for super-resolution microscopy. Molecules 16:3106–3118

    Article  PubMed  CAS  Google Scholar 

  29. Chudakov DM, Lukyanov S, Lukyanov KA (2007) Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc 2:2024–2032

    Article  PubMed  CAS  Google Scholar 

  30. Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008) mKikGR, a monomeric photoswitchable fluorescent protein. PLoS One 3:e3944

    Article  PubMed  Google Scholar 

  31. Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367

    Article  PubMed  CAS  Google Scholar 

  32. Subach FV, Patterson GH, Manley S, Gillette JM, Lippincott-Schwartz J et al (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6:153–159

    Article  PubMed  CAS  Google Scholar 

  33. Subach FV, Patterson GH, Renz M, Lippincott-Schwartz J, Verkhusha VV (2010) Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J Am Chem Soc 132:6481–6491

    Article  PubMed  CAS  Google Scholar 

  34. Subach OM, Patterson GH, Ting LM, Wang Y, Condeelis JS et al (2011) A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat Methods 8:771–777

    Article  PubMed  CAS  Google Scholar 

  35. Kim SY, Gitai Z, Kinkhabwala A, Shapiro L, Moerner WE (2006) Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc Natl Acad Sci USA 103:10929–10934

    Article  PubMed  CAS  Google Scholar 

  36. Matsuda A, Shao L, Boulanger J, Kervrann C, Carlton PM et al (2010) Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones. PLoS One 5:e12768

    Article  PubMed  Google Scholar 

  37. Li S, Kimura E, Ng R, Fall BM, Meuse L et al (2006) A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of duchenne muscular dystrophy. Hum Mol Genet 15:1610–1622

    Article  PubMed  CAS  Google Scholar 

  38. Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS et al (2009) Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7:e1000137

    Article  PubMed  Google Scholar 

  39. Belov VN, Wurm CA, Boyarskiy VP, Jakobs S, Hell SW (2010) Rhodamines NN: a novel class of caged fluorescent dyes. Angew Chem Int Ed Engl 49:3520–3523

    Article  PubMed  CAS  Google Scholar 

  40. Heilemann M, Sauer M, Margeat E, Kasper R, Tinnefeld P (2005) Carbocyanine dyes as efficient reversible single-molecule optical switch. J Am Chem Soc 127:3801–3806

    Article  PubMed  CAS  Google Scholar 

  41. Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317:1749–1753

    Article  PubMed  CAS  Google Scholar 

  42. Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angew Chem Int Ed 48:6903–6908

    Article  CAS  Google Scholar 

  43. van de Linde S, Krstic I, Prisner T, Doose S, Heilemann M et al (2011) Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging. Photochem Photobiol Sci 10:499–506

    Article  PubMed  Google Scholar 

  44. Kottke T, van de Linde S, Sauer M, Kakorin S, Heilemann M (2010) Identification of the product of photoswitching of an oxazine fluorophore using fourier transform infrared difference spectroscopy. J Phys Chem Lett 1:3156–3159

    Article  CAS  Google Scholar 

  45. Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M et al (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed 47:5465–5469

    Article  CAS  Google Scholar 

  46. Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D et al (2007) Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J 93:3285–3290

    Article  PubMed  CAS  Google Scholar 

  47. Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C et al (2010) Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J 99:2686–2694

    Article  PubMed  CAS  Google Scholar 

  48. Lemmer P, Gunkel M, Weiland Y, Muller P, Baddeley D et al (2009) Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range. J Microsc 235:163–171

    Article  PubMed  CAS  Google Scholar 

  49. Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:108101

    Article  PubMed  Google Scholar 

  50. Belov VN, Bossi ML, Folling J, Boyarskiy VP, Hell SW (2009) Rhodamine spiroamides for multi­color single-molecule switching fluorescent nanoscopy. Chemistry 15:10762–10776

    Article  PubMed  CAS  Google Scholar 

  51. Lidke KA, Rieger B, Jovin TM, Heintzmann R (2005) Superresolution by localization of quantum dots using blinking statistics. Opt Express 13:7052–7062

    Article  PubMed  Google Scholar 

  52. Goldstein M, Watkins S (2008) Immunohisto­chemistry. Curr Protoc Mol Biol 14:Unit 14.6.

    Google Scholar 

  53. Puchtler H, Meloan SN (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201–204

    Article  PubMed  CAS  Google Scholar 

  54. Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H et al (2007) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 1:Unit 1.16

    Google Scholar 

  55. Mortensen RM, Kingston RE (2009) Selection of transfected mammalian cells. Curr Protoc Mol Biol 9:Unit 9.5.

    Google Scholar 

  56. Subach FV, Malashkevich VN, Zencheck WD, Xiao H, Filonov GS et al (2009) Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc Natl Acad Sci USA 106:21097–21102

    Article  PubMed  CAS  Google Scholar 

  57. You X, Nguyen AW, Jabaiah A, Sheff MA, Thorn KS et al (2006) Intracellular protein interaction mapping with FRET hybrids. Proc Natl Acad Sci USA 103:18458–18463

    Article  PubMed  CAS  Google Scholar 

  58. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H et al (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89

    Article  PubMed  CAS  Google Scholar 

  59. Sun X, Zhang A, Baker B, Sun L, Howard A et al (2011) Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. Chembiochem 12:2217–2226

    Article  PubMed  CAS  Google Scholar 

  60. Gautier A, Juillerat A, Heinis C, Correa IR Jr, Kindermann M et al (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136

    Article  PubMed  CAS  Google Scholar 

  61. Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382

    Article  PubMed  CAS  Google Scholar 

  62. Miller LW, Cai Y, Sheetz MP, Cornish VW (2005) In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag. Nat Methods 2:255–257

    Article  PubMed  CAS  Google Scholar 

  63. Klein T, Loschberger A, Proppert S, Wolter S, van de Linde S et al (2011) Live-cell dSTORM with SNAP-tag fusion proteins. Nat Methods 8:7–9

    Article  PubMed  CAS  Google Scholar 

  64. Wombacher R, Heidbreder M, van de Linde S, Sheetz MP, Heilemann M et al (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7:717–719

    Article  PubMed  CAS  Google Scholar 

  65. Lee HLD, Lord SJ, Iwanaga S, Zhan K, Xie HX et al (2010) Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores. J Am Chem Soc 132:15099–15101

    Article  PubMed  CAS  Google Scholar 

  66. Wombacher R, Cornish VW (2011) Chemical tags: applications in live cell fluorescence imaging. J Biophotonics 4:391–402

    Article  PubMed  CAS  Google Scholar 

  67. Henriques R, Griffiths C, Hesper RE, Mhlanga MM (2011) PALM and STORM: unlocking live-cell super-resolution. Biopolymers 95:322–331

    Article  PubMed  CAS  Google Scholar 

  68. Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S et al (2009) Cell culture medium affects GFP photostability: a solution. Nat Methods 6:859–860

    Article  PubMed  CAS  Google Scholar 

  69. Dani A, Huang B, Bergan J, Dulac C, Zhuang X (2010) Superresolution imaging of chemical synapses in the brain. Neuron 68:843–856

    Article  PubMed  CAS  Google Scholar 

  70. van de Linde S, Endesfelder U, Mukherjee A, Schuttpelz M, Wiebusch G et al (2009) Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging. Photochem Photobiol Sci 8:465–469

    Article  PubMed  Google Scholar 

  71. Löschberger A, van de Linde S, Dabauvalle MC, Rieger B, Heilemann M et al (2011) Super-resolution imaging reveals eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J Cell Sci 125: 570–575

    Google Scholar 

  72. Baddeley D, Crossman D, Rossberger S, Cheyne JE, Montgomery JM et al (2011) 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS One 6:e20645

    Article  PubMed  CAS  Google Scholar 

  73. Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011) Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8:527–528

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Sebastian Malkusch and Patrick Zessin for critical reading of the manuscript and to Prof. G.U. Nienhaus for providing spectral data for the fluorescent protein mEosFPthermo. This work was supported by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315262), the German Science Foundation (DFG, grant nr. HE 6166/2-1), and by contract research “Methoden für die Lebenswissenschaften” of the Baden-Württemberg Stiftung (grant nr. P-LS-SPII/11).

Author information

Authors and Affiliations

  1. Department of Biotechnology and Biophysics, Julius-Maximilians University Würzburg, Würzburg, Germany

    Kieran Finan & Mike Heilemann

  2. Heidelberg Collaboratory for Image Processing, University of Heidelberg, Heidelberg, Germany

    Benjamin Flottmann & Mike Heilemann

Authors
  1. Kieran Finan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benjamin Flottmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mike Heilemann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mike Heilemann .

Editor information

Editors and Affiliations

  1. Nat. Inst. Biomedical Imaging & Bioengin, National Institutes of Health, South Drive 13, Bethesda, 20892, Maryland, USA

    Alioscka A. Sousa

  2. , National Cancer Institute, National Institutes of Health, Center Drive 10, Bethesda, 20892, USA

    Michael J. Kruhlak

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Finan, K., Flottmann, B., Heilemann, M. (2013). Photoswitchable Fluorophores for Single-Molecule Localization Microscopy. In: Sousa, A., Kruhlak, M. (eds) Nanoimaging. Methods in Molecular Biology, vol 950. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-137-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-137-0_9

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-136-3

  • Online ISBN: 978-1-62703-137-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation