Abstract
We demonstrate two-color far-field fluorescence microscopy with nanoscale spatial resolution based on the photoswitching of individual fluorescent markers. By enabling, recording, and disabling the emission of the reversibly switchable fluorescent protein rsFastLime and of the organic fluorophore cyanine5, we recorded two-color nanoscale images inside whole cells. The position of individual emitters was determined with a typical accuracy of 20 nm, which largely constitutes the lateral resolution of the system. Photoswitching in two-color colocalization experiments represents a major step towards the application of far-field fluorescence nanoscopy to the study of (biological) samples on the macromolecular level.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
E. Abbe, Arch. Mikrosk. Anat. 9, 413 (1873)
S.W. Hell, Science 316, 1153 (2007)
S.W. Hell, Opt. Commun. 106, 19 (1994)
S.W. Hell, J. Wichmann, Opt. Lett. 19, 780 (1994)
S.W. Hell, S. Jakobs, L. Kastrup, Appl. Phys. A 77, 859 (2003)
M. Hofmann, C. Eggeling, S. Jakobs, S.W. Hell, Proc. Nat. Acad. Sci. 102, 17565 (2005)
E. Betzig, G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, H.F. Hess, Science 313, 1642 (2006)
S.T. Hess, T.P.K. Girirajan, M.D. Mason, Biophys. J. 91, 4258 (2006)
M.J. Rust, M. Bates, X. Zhuang, Nature Methods 3, 793 (2006)
E. Betzig, Opt. Lett. 20, 237 (1995)
G.J. Schütz, H. Schindler, T. Schmidt, Biophys. J. 73, 1073 (1997)
T.D. Lacoste, X. Michalet, F. Pinaud, D.S. Chemla, A.P. Alivisatos, S. Weiss, Proc. Nat. Acad. Sci. 97, 9461 (2000)
R.E. Thompson, D.R. Larson, W.W. Webb, Biophys. J. 82, 2775 (2002)
S.W. Hell, J. Soukka, P.E. Hänninen, Bioimaging 3, 65 (1995)
C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, S.W. Hell, Appl. Phys. A 88, 223 (2007)
E.A. Jares-Erijman, T.M. Jovin, Nature Biotechnol. 21, 1387 (2003)
G. Donnert, J. Keller, C.A. Wurm, S.O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, S.W. Hell, Biophys. J. 92, L67 (2007)
A.C. Stiel, S. Trowitzsch, G. Weber, M. Andresen, C. Eggeling, S.W. Hell, S. Jakobs, M.C. Wahl, Biochem. J. 402, 35 (2007)
R. Ando, H. Mizuno, A. Miyawaki, Science 306, 1370 (2004)
S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, J. Hofkens, Proc. Nat. Acad. Sci. 102, 9511 (2005)
M. Bates, T.R. Blosser, X. Zhuang, Phys. Rev. Lett. 94, 108101 (2005)
M. Heilemann, E. Margeat, R. Kasper, M. Sauer, P. Tinnefeld, J. Am. Chem. Soc. 127, 3801 (2005)
R. Zondervan, F. Kulzer, M.A. Kolchenko, M. Orrit, J. Phys. Chem. A 108, 1657 (2004)
Z. Huang, D. Ji, A. Xia, Colloid Surf. A 257–258, 203 (2005)
Author information
Authors and Affiliations
Corresponding authors
Additional information
PACS
33.50.Dq; 42.30.-d; 87.64.-t
Rights and permissions
About this article
Cite this article
Bock, H., Geisler, C., Wurm, C. et al. Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Appl. Phys. B 88, 161–165 (2007). https://doi.org/10.1007/s00340-007-2729-0
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00340-007-2729-0