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  • Perspective
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Opportunities and challenges in single-molecule and single-particle fluorescence microscopy for mechanistic studies of chemical reactions

Nature Chemistry volume 5, pages 993–999 (2013)Cite this article

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Abstract

In recent years, single-molecule and single-particle fluorescence microscopy has emerged as a tool to investigate chemical systems. After an initial lag of over a decade with respect to biophysical studies, this powerful imaging technique is now revealing mechanisms of 'classical' organic reactions, spatial distribution of chemical reactivity on surfaces and the phase of active catalysts. The recent advance into commercial imaging systems obviates the need for home-built laser systems and thus opens this technique to traditionally trained synthetic chemists. We discuss the requisite photophysical and chemical properties of fluorescent reporters and highlight the main challenges in applying single-molecule techniques to chemical questions. The goal of this Perspective is to provide a snapshot of an emerging multidisciplinary field and to encourage broader use of this young experimental approach that aids the observation of chemical reactions as depicted in many textbooks: molecule by molecule.

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Figure 1: Types of fluorophore used in chemical single-molecule studies.
Figure 2: Examples of single-molecule fluorescence microscopy studies of stochiometric chemical reactions.
Figure 3: Examples of single-molecule fluorescence microscopy studies of catalytic chemical reactions.
Figure 4: Comprehensive list of all fluorophores used to study chemical reactions on the level of single-molecules.
Figure 5: Classes of fluorophores, their photophysical behaviour in solution, and microscopy cells suited to studying chemical reactions.

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References

  1. Gladysz, J. A. The Experimental Assay of Catalyst Recovery: General Concepts (Wiley, 2009).

    Google Scholar 

  2. Whitesides, G. M. et al. Suppression of unwanted heterogeneous platinum(0)-catalyzed reactions by poisoning with mercury(0) in systems involving competing homogeneous reactions of soluble organoplatinum compounds: thermal decomposition of bis(triethylphosphine)-3,3,4,4-tetramethylplatinacyclopentane. Organometallics 4, 1819–1830 (1985).

    Article  CAS  Google Scholar 

  3. Crabtree, R. H. The Organometallic Chemistry of the Transition Metals (Wiley, 2009).

    Google Scholar 

  4. Hartwig, J. F. Organotransition Metal Chemistry: From Bonding to Catalysis (University Science Books, 2010).

    Google Scholar 

  5. Roeffaers, M. B. J. et al. Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting. Nature 439, 572–575 (2006).

    Article  CAS  Google Scholar 

  6. Kiel, A. et al. Direct monitoring of formation and dissociation of individual metal complexes by single-molecule fluorescence spectroscopy. Angew. Chem. Int. Ed. 46, 5049–5049 (2007).

    Article  CAS  Google Scholar 

  7. Jung, G., Schmitt, A., Jacob, M. & Hinkeldey, B. Fluorescent probes for chemical transformations on the single-molecule level. Ann. NY Acad. Sci. 1130, 131–137 (2008).

    Article  CAS  Google Scholar 

  8. Esfandiari, N. M. et al. Single-molecule imaging of platinum ligand exchange reaction reveals reactivity distribution. J. Am. Chem. Soc. 132, 15167–15169 (2010).

    Article  CAS  Google Scholar 

  9. Esfandiari, N. M. & Blum, S. A. Homogeneous vs heterogeneous polymerization catalysis revealed by single-particle fluorescence microscopy. J. Am. Chem. Soc. 133, 18145–18147 (2011).

    Article  CAS  Google Scholar 

  10. Esfandiari, N. M., Wang, Y., McIntire, T. M. & Blum, S. A. Real-time imaging of platinum–sulfur ligand exchange reactions at the single-molecule level via a general chemical technique. Organometallics 30, 2901–2907 (2010).

    Article  Google Scholar 

  11. Rybina, A. et al. Distinguishing alternative reaction pathways by single-molecule fluorescence spectroscopy. Angew. Chem. Int. Ed. 52, 6322–6325 (2013).

    Article  CAS  Google Scholar 

  12. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996).

    Article  CAS  Google Scholar 

  13. van Oijen, A. M. et al. Unraveling the electronic structure of individual photosynthetic pigment–protein complexes. Science 285, 400–402 (1999).

    Article  CAS  Google Scholar 

  14. Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001).

    Article  CAS  Google Scholar 

  15. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

    Article  CAS  Google Scholar 

  16. Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).

    Article  Google Scholar 

  17. English, B. P. et al. Ever-fluctuating single enzyme molecules: Michaelis–Menten equation revisited. Nature Chem. Biol. 2, 87–94 (2006).

    Article  CAS  Google Scholar 

  18. Hohlbein, J., Gryte, K., Heilemann, M. & Kapanidis, A. N. Surfing on a new wave of single-molecule fluorescence methods. Phys. Biol. 7, 031001 (2010).

    Article  Google Scholar 

  19. Ha, T. & Tinnefeld, P. Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu. Rev. Phys. Chem. 63, 595–617 (2012).

    Article  CAS  Google Scholar 

  20. Aitken, C. E., Marshall, R. A. & Pulglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

    Article  CAS  Google Scholar 

  21. Xu, W., Kong, J. S., Yeh, Y. T. & Chen, P. Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics. Nature Mater. 7, 992–996 (2008).

    Article  CAS  Google Scholar 

  22. Xu, W., Kong, J. S. & Chen, P. Probing the catalytic activity and heterogeneity of Au-nanoparticles at the single-molecule level. Phys. Chem. Chem. Phys. 11, 2767–2778 (2009).

    Article  CAS  Google Scholar 

  23. Zhou, X. et al. Size-dependent catalytic activity and dynamics of gold nanoparticles at the single-molecule level. J. Am. Chem. Soc. 132, 138–146 (2010).

    Article  CAS  Google Scholar 

  24. Ochoa, M. A., Zhou, X., Chen, P. & Loring, R. F. Interpreting single turnover catalysis measurements with constrained mean dwell times. J. Chem. Phys. 135, 174509 (2011).

    Article  Google Scholar 

  25. Han, K. S. et al. How does a single Pt nanocatalyst behave in two different reactions? A single-molecule study. Nano Lett. 12, 1253–1259 (2012).

    Article  CAS  Google Scholar 

  26. Esfandiari, N. M., Wang, Y., Bass, J. Y. & Blum, S. A. Deconvoluting subensemble chemical reaction kinetics of platinum–sulfur ligand exchange detected with single-molecule fluorescence microscopy. Inorg. Chem. 50, 9201–9203 (2011).

    Article  CAS  Google Scholar 

  27. Smith, J. G. Organic Chemistry (McGraw-Hill, 2001).

    Google Scholar 

  28. Canham, S. M. et al. Toward the single-molecule investigation of organometallic reaction mechanisms: Single-molecule imaging of fluorophore-tagged palladium(II) complexes. Organometallics 27, 2172–2175 (2008).

    Article  CAS  Google Scholar 

  29. Ameloot, R. et al. Towards direct monitoring of discrete events in a catalytic cycle at the single molecule level. Photochem. Photobiol. Sci. 8, 453–456 (2009).

    Article  CAS  Google Scholar 

  30. Hensle, E. M. & Blum, S. A. Phase separation polymerization of dicyclopentadiene characterized by in operando fluorescence microscopy. J. Am. Chem. Soc. 135, 12324–12328 (2013).

    Article  CAS  Google Scholar 

  31. Andoy, N. M. et al. Single-molecule catalysis mapping quantifies site-specific activity and uncovers radial activity gradient on single 2D nanocrystals. J. Am. Chem. Soc. 135, 1845–1852 (2013).

    Article  CAS  Google Scholar 

  32. Chen, P. et al. Single-molecule fluorescence imaging of nanocatalytic processes. Chem. Soc. Rev. 39, 4560–4570 (2010).

    Article  CAS  Google Scholar 

  33. Buurmans, I. L. C. & Weckhuysen, B. M. Heterogeneities of individual catalyst particles in space and time as monitored by spectroscopy. Nature Chem. 4, 873–886 (2012).

    Article  CAS  Google Scholar 

  34. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  35. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  36. Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

    Article  CAS  Google Scholar 

  37. Vogelsang, J. et al. Make them blink: probes for super-resolution microscopy. ChemPhysChem 11, 2475–2490 (2010).

    Article  CAS  Google Scholar 

  38. Tinnefeld, P. & Sauer, M. Branching out of single-molecule fluorescence spectroscopy: challenges for chemistry and influence on biology. Angew. Chem. Int. Ed. 44, 2642–2671 (2005).

    Article  CAS  Google Scholar 

  39. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nature Methods 5, 507–516 (2008).

    Article  CAS  Google Scholar 

  40. Roeffaers, M. B. J. et al. Super-resolution reactivity mapping of nanostructured catalyst particles. Angew. Chem. Int. Ed. 48, 9285–9289 (2009).

    Article  CAS  Google Scholar 

  41. Wöll, D. et al. Radical polymerization tracked by single molecule spectroscopy. Angew. Chem. Int. Ed. 47, 783–787 (2008).

    Article  Google Scholar 

  42. Kim, K. H. et al. Quantitative catalyst–substrate association relationships between metathesis molybdenum or ruthenium carbene complexes and their substrates. J. Am. Chem. Soc. 132, 12027–12033 (2010).

    Article  CAS  Google Scholar 

  43. Lim, S. G. & Blum, S. A. A General fluorescence resonance energy transfer (FRET) method for observation and quantification of organometallic complexes under reaction conditions. Organometallics 28, 4643–4645 (2009).

    Article  CAS  Google Scholar 

  44. Cordes, T. et al. Single-molecule redox blinking of perylene diimide derivatives in water. J. Am. Chem. Soc. 132, 2404–2409 (2010).

    Article  CAS  Google Scholar 

  45. Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and longlasting single-molecule fluorescence imaging. Nature Methods 3, 891–893 (2006).

    Article  CAS  Google Scholar 

  46. Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Ed. 47, 5465–5469 (2008).

    Article  CAS  Google Scholar 

  47. Cordes, T., Vogelsang, J. & Tinnefeld, P. On the mechanism of Trolox as antiblinking and antibleaching reagent. J. Am. Chem. Soc. 131, 5018–5019 (2009).

    Article  CAS  Google Scholar 

  48. Altman, R. B. et al. Cyanine fluorophore derivatives with enhanced photostability. Nature Methods 9, 68–71 (2012).

    Article  CAS  Google Scholar 

  49. Fast, A. & Blum, S. A. Small number of active sites and single-locus kinetics revealed in (salph)co-catalyzed ethylene oxide polymerization. ACS Catal. 3, 2150–2153 (2013).

    Article  CAS  Google Scholar 

  50. Wöll, D. et al. Polymers and single molecule fluorescence spectroscopy, what can we learn? Chem. Soc. Rev. 38, 313–328 (2009).

    Article  Google Scholar 

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Acknowledgements

T.C. was supported by the Zernike Institute for Advanced Materials and the Centre for Synthetic Biology (University of Groningen). S.A.B. thanks the US Department of Energy, Office of Basic Energy Sciences for support (DE-FG02-08ER15994).

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Authors and Affiliations

  1. Molecular Microscopy Research Group and Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, The Netherlands

    Thorben Cordes

  2. Department of Chemistry, University of California, Irvine, 92697-2025, California, USA

    Suzanne A. Blum

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Correspondence to Thorben Cordes or Suzanne A. Blum.

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Cordes, T., Blum, S. Opportunities and challenges in single-molecule and single-particle fluorescence microscopy for mechanistic studies of chemical reactions. Nature Chem 5, 993–999 (2013). https://doi.org/10.1038/nchem.1800

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