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Imaging biological structures with fluorescence photoactivation localization microscopy

Abstract

Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1–30 min and analyzed in approximately 0.5–4 h.

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Figure 1: Concept of FPALM.
Figure 2: FPALM experimental setup.
Figure 3: Identifying single molecules.
Figure 4: Example timing sequences for FPALM acquisitions.
Figure 5: Typical FPALM image of Dendra2-actin expressed in a fixed mouse fibroblast.
Figure 6: Illustration of troubleshooting of pixelization artifact in FPALM image of Dendra2-actin expressed in a fixed fibroblast.

References

  1. 1

    Born, M. & Wolf, E. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge University Press, New York, 1997).

    Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

    Hess, S.T., Girirajan, T.P. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy (FPALM). Biophys. J. 91, 4258–4272 (2006).

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Google Scholar 

  6. 6

    Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Hess, S.T. et al. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Geisler, C. et al. Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching. Appl. Phys. A 88, 223–226 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Flors, C. et al. A stroboscopic approach for fast photoactivation-localization microscopy with dronpa mutants. J. Am. Chem. Soc. 129, 13970–13977 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5, 417–423 (2008).

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Shroff, H. et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc. Natl. Acad. Sci. USA 104, 20308–20313 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Bock, H. et al. Two-color far-field fluorescence nanoscopy based on photo-switchable emitters. Appl. Phys. B 88, 161–165 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Juette, M.F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047–1052 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Gould, T.J. et al. Nanoscale imaging of molecular positions and anisotropies. Nat. Methods 5, 1027–1030 (2008).

    CAS  Article  Google Scholar 

  19. 19

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

    Article  Google Scholar 

  20. 20

    Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Innovation: photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6, 885–891 (2005).

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Chudakov, D.M. et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22, 1435–1439 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Verkhusha, V.V. & Sorkin, A. Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem. Biol. 12, 279–85 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Subach, F.V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods (in press).

  25. 25

    van Thor, J.J., Gensch, T., Hellingwerf, K.J. & Johnson, L.N. Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat. Struct. Biol. 9, 37–41 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Wiedenmann, J. et al. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N. & Miyawaki, A. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6, 233–238 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Gurskaya, N.G. et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24, 461–465 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and dendra2. Nat. Protoc. 2, 2024–2032 (2007).

    CAS  Article  Google Scholar 

  32. 32

    Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).

    CAS  Article  Google Scholar 

  33. 33

    Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Stiel, A.C. et al. 1.8 a bright-state structure of the reversibly switchable fluorescent protein dronpa guides the generation of fast switching variants. Biochem. J. 402, 35–42 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Ando, R., Flors, C., Mizuno, H., Hofkens, J. & Miyawaki, A. Highlighted generation of fluorescence signals using simultaneous two-color irradiation on dronpa mutants. Biophys. J. 92, L97–L99 (2007).

    CAS  Article  Google Scholar 

  36. 36

    Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    CAS  Article  Google Scholar 

  37. 37

    Egner, A. et al. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Haupts, U., Maiti, S., Schwille, P. & Webb, W.W. Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).

    CAS  Article  Google Scholar 

  39. 39

    Schwille, P., Kummer, S., Heikal, A.A., Moerner, W.E. & Webb, W.W. Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 97, 151–156 (2000).

    CAS  Article  Google Scholar 

  40. 40

    Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y. & Webb, W.W. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsred) and yellow (citrine). Proc. Natl. Acad. Sci. USA 97, 11996–12001 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Dickson, R.M., Cubitt, A.B., Tsien, R.Y. & Moerner, W.E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

    CAS  Article  Google Scholar 

  42. 42

    Enderlein, J., Toprak, E. & Selvin, P.R. Polarization effect on position accuracy of fluorophore localization. Opt. Expr. 14, 8111–8120 (2006).

    CAS  Article  Google Scholar 

  43. 43

    Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    CAS  Article  Google Scholar 

  44. 44

    Shaner, N.C., Patterson, G.H. & Davidson, M.W. Advances in fluorescent protein technology. J. Cell Sci. 120, 4247–4260 (2007).

    CAS  Article  Google Scholar 

  45. 45

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

    CAS  Article  Google Scholar 

  46. 46

    Hess, S.T., Gould, T.J., Gunewardene, M., Bewersdorf, J. & Mason, M.D. Ultra-high resolution imaging of biomolecules by fluorescence photoactivation localization microscopy (FPALM). In Methods in Molecular Biology (eds. Foote, R.S. & Lee, J.W.) in press (Humana Press, Totowa, NJ).

  47. 47

    Self, S.A. Focusing of spherical Gaussian beams. Appl. Opt. 22, 658 (1983).

    CAS  Article  Google Scholar 

  48. 48

    Currie, L.A. Limits for qualitative detection and quantitative determination. Anal. Chem. 40, 586–593 (1968).

    CAS  Article  Google Scholar 

  49. 49

    Gordon, M.P., Ha, T. & Selvin, P.R. Single-molecule high-resolution imaging with photobleaching. Proc. Natl. Acad. Sci. USA 101, 6462–6465 (2004).

    CAS  Article  Google Scholar 

  50. 50

    Qu, X., Wu, D., Mets, L. & Scherer, N.F. Nanometer-localized multiple single-molecule fluorescence microscopy. Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).

    CAS  Article  Google Scholar 

  51. 51

    Cheezum, M.K., Walker, W.F. & Guilford, W.H. Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys. J. 81, 2378–2388 (2001).

    CAS  Article  Google Scholar 

  52. 52

    Pawley, J.B. Handbook of Biological Confocal Microscopy (Plenum Press, New York, 1995).

    Book  Google Scholar 

  53. 53

    Ripley, B.D. Tests of randomness for spatial point patterns. J. R. Stat. Soc. Ser. B 41, 368–374 (1979).

    Google Scholar 

  54. 54

    Pathria, R.K. Statistical Mechanics (Butterworth-Heinemann, Oxford, Boston, 1996).

    Google Scholar 

  55. 55

    Lakowicz, J.R. Principles of Fluorescence Spectroscopy (Springer Science, New York, 2006).

    Book  Google Scholar 

  56. 56

    Sternberg, S.R. Biomedical image processing. IEEE Comput. 16, 22–34 (1983).

    Article  Google Scholar 

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Acknowledgements

We thank Christopher Fang-Yen, Paul Blank, Joerg Bewersdorf, Joshua Zimmerberg, George Patterson, Julie Gosse and Michael Mason for useful discussions, Paul Millard and Carol Kim for use of equipment and reagents, Ed Allgeyer, Manasa Gudheti and Siyath Gunewardene for laboratory assistance, Matthew Parent for programming assistance and Thomas Tripp, Tony McGinn and Kyle Jensen for machining services. This work was supported by grants K25-AI-65459 from the National Institute of Allergy and Infectious Diseases (S.T.H.), CHE-0722759 from the National Science Foundation (S.T.H.), start-up funds from the University of Maine (S.T.H.) and by grants GM070358 and GM073913 from the National Institute of General Medical Sciences (V.V.V.).

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Correspondence to Samuel T Hess.

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Gould, T., Verkhusha, V. & Hess, S. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat Protoc 4, 291–308 (2009). https://doi.org/10.1038/nprot.2008.246

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