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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 11 — Nov. 1, 2011
  • pp: 2934–2949

Model of bleaching and acquisition for superresolution microscopy controlled by a single wavelength

Alex Small  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 11, pp. 2934-2949 (2011)

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We consider acquisition schemes that maximize the fraction of images that contain only a single activated molecule (as opposed to multiple activated molecules) in superresolution localization microscopy of fluorescent probes. During a superresolution localization microscopy experiment, irreversible photobleaching destroys fluorescent molecules, limiting the ability to monitor the dynamics of long-lived processes. Here we consider experiments controlled by a single wavelength, so that the bleaching and activation rates are coupled variables. We use variational techniques and kinetic models to demonstrate that this coupling of bleaching and activation leads to very different optimal control schemes, depending on the detailed kinetics of fluorophore activation and bleaching. Likewise, we show that the robustness of the acquisition scheme is strongly dependent on the detailed kinetics of activation and bleaching.

© 2011 OSA

OCIS Codes
(100.6640) Image processing : Superresolution
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: August 8, 2011
Revised Manuscript: August 28, 2011
Manuscript Accepted: August 31, 2011
Published: September 30, 2011

Alex Small, "Model of bleaching and acquisition for superresolution microscopy controlled by a single wavelength," Biomed. Opt. Express 2, 2934-2949 (2011)

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  1. K. Lidke, B. Rieger, T. Jovin, and R. Heintzmann, “Superresolution by localization of quantum dots using blinking statistics,” Opt. Express13, 7052–7062 (2005). [CrossRef] [PubMed]
  2. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313, 1642–1645 (2006). [CrossRef] [PubMed]
  3. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (storm),” Nat. Methods3, 793–795 (2006). [CrossRef] [PubMed]
  4. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91, 4258–4272 (2006). [CrossRef] [PubMed]
  5. R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J.82, 2775–2783 (2002). [CrossRef] [PubMed]
  6. R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophysical Journal86, 1185–1200 (2004). [CrossRef] [PubMed]
  7. H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5, 417–423 (2008). [CrossRef] [PubMed]
  8. S. A. Ribeiro, P. Vagnarelli, Y. Dong, T. Hori, B. F. McEwen, T. Fukagawa, C. Flors, and W. C. Earnshaw, “A super-resolution map of the vertebrate kinetochore,” Proc. Natl Acad. Sci. U.S.A.107, 10484–10489 (2010). [CrossRef] [PubMed]
  9. D. Greenfield, A. L. McEvoy, H. Shroff, G. E. Crooks, N. S. Wingreen, E. Betzig, and J. Liphardt, “Self-organization of the escherichia coli chemotaxis network imaged with super-resolution light microscopy,” PLoS Biol.7, e1000137 (2009). [CrossRef] [PubMed]
  10. N. A. Frost, H. Shroff, H. Kong, E. Betzig, and T. A. Blanpied, “Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines,” Neuron67, 86–99 (2010). [CrossRef] [PubMed]
  11. S. T. Hess, T. J. Gould, M. V. Gudheti, S. A. Maas, K. D. Mills, and J. Zimmerberg, “Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories,” Proc. Natl. Acad. Sci. U.S.A.104, 17370 (2007). [CrossRef] [PubMed]
  12. T. A. Brown, R. D. Fetter, A. N. Tkachuk, and D. A. Clayton, “Approaches toward super-resolution fluorescence imaging of mitochondrial proteins using palm,” Methods51, 458–463 (2010). [CrossRef] [PubMed]
  13. B. Huang, S. A. Jones, B. Brandenburg, and X. Zhuang, “Whole-cell 3d storm reveals interactions between cellular structures with nanometer-scale resolution,” Nat. Methods5, 1047 (2008). [CrossRef] [PubMed]
  14. J. Folling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods5, 943–945 (2008). [CrossRef] [PubMed]
  15. C. Steinhauer, C. Forthmann, J. Vogelsang, and P. Tinnefeld, “Superresolution microscopy on the basis of engineered dark states,” J. Am. Chem. Soc130, 16840–16841 (2008). [CrossRef] [PubMed]
  16. D. Baddeley, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “Light-induced dark states of organic fluochromes enable 30 nm resolution imaging in standard media,” Biophys. J.96, L22–L24 (2009). [CrossRef] [PubMed]
  17. M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed.48, 6903–6908 (2009). [CrossRef]
  18. I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. Von Middendorf, J. Flling, S. Jakobs, A. Schnle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J.99, 2686–2694 (2010). [CrossRef] [PubMed]
  19. S. Lee, M. Thompson, M. A. Schwartz, L. Shapiro, and W. E. Moerner, “Super-resolution imaging of the nucleoid-associated protein hu in caulobacter crescentus,” Biophys. J.100, L31–L33 (2011). [CrossRef] [PubMed]
  20. J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106, 8107–8112 (2009). [CrossRef] [PubMed]
  21. E. Shore and A. Small, “Optimal acquisition scheme for subwavelength localization microscopy of bleachable fluorophores,” Opt. Lett.36, 289–291 (2011). [CrossRef] [PubMed]
  22. A. Small, “Theoretical limits on errors and acquisition rates in localizing switchable fluorophores,” Biophys. J.96, L16–L18 (2009). [CrossRef] [PubMed]
  23. F. Huang, S. L. Schwartz, J. M. Byars, and K. A. Lidke, “Simultaneous multiple-emitter fitting for single molecule super-resolution imaging,” Biomed. Opt. Express2, 1377–1393 (2011). [CrossRef] [PubMed]
  24. H. Goldstein, Classical mechanics (Addison-Wesley Pub. Co., 1980).
  25. B. Chachuat, Nonlinear and Dynamic Optimization: From Theory to Practice (Laboratoire dAutomatique, Ecole Polytechnique Federale de Lausanne, Lecture Notes for Winter Semester, 2007).
  26. M. Bates, B. Huang, and X. Zhuang, “Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes,” Curr. Opinion Chem. Biol.12, 505–514 (2008). [CrossRef]
  27. T. Gould and S. Hess, “Nanoscale biological fluorescence imaging: Breaking the diffraction barrier,” Methods Cell Biol.89, 329–358 (2008). [CrossRef]
  28. C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods7, 373–375 (2010). [CrossRef] [PubMed]
  29. C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem70, 2651–2659 (1998). [CrossRef] [PubMed]
  30. G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods4, 81–86 (2007). [CrossRef]

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