Proceedings Article

STED super-resolution microscopy in Drosophila tissue and in mammalian cells

[+] Author Affiliations
Lana Lau, Yin Loon Lee, Maja Matis, Jeff Axelrod, Tim Stearns, W. E. Moerner

Stanford Univ. (USA)

Proc. SPIE 7910, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III, 79101N (February 11, 2011); doi:10.1117/12.881221
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From Conference Volume 7910

  • Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III
  • Samuel Achilefu; Ramesh Raghavachari
  • San Francisco, California, USA | January 22, 2011

abstract

Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

references

Hell, S. W. "Far-field Optical Nanoscopy," Science. 316, 1153-5 (2007).
"Method of the Year 2008," Nat. Methods 6 (1), 1 (2008).
Moerner, W. E., "New directions in single-molecule imaging and analysis," Proc. Natl. Acad. Sci. U.S.A. 104, 12596-12602 (2007).
Rust, M. J., Bates, M. and Zhuang, X. "Sub-diffraction-limited imaging by stochastic optical reconstruction microscopy (STORM)," Nat. Methods 3, 793-795 (2006).
Betzig, E., Patterson, G. H., Sougrat, R. Lindwasser, O. W., Olenych, S., Bonifacino, J.S. Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. R. "Imaging intracellular fluorescent proteins at nanometer resolution," Sciences 313(5793), 1642-1645 (2006).
Hess S. T., Girirajan, T. P. K. and Mason M. D. "Ultra-high resolution imaging by fluorescence photoactivation localization microscopy," Biophys. J. 91, 4258-4272 (2006).
Thompson, R. E., Larson, D. R., Webb W. W. "Precise Nanometer Localization Analysis for Individual Fluorescent Probes," Biophys. J. 82, 2775-2783 (2002).
Pavani, S. R. P., Thompson, M. A., Biteen, J. S., Lord, S. J., Liu, N., Twieg, R. J., Piestun, R. and Moerner, W. E. "Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function," Proc. Nat. Acad. Sci. U. S. A. 106(9), 2995-2999 (2009).
Gutsafsson, M. G. L. "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy," J. Microsc. 198(20), 82-87 (2000).
Hell S. and Wichmann J. "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy" Opt. Lett. 19(11), 780-781 (1994).
Punge, A., Rizzoli, S., Jahn, R., Wildanger, J., Meyer, L., Schonle, A., Kastrup, L., and Hell, S. "3D reconstruction of high-resolution STED microscope images," Microsc. Res. Tech. 71, 644-650 (2008).
Hein, B., Willig, K., and Hell, S. "Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell," Proc. Nat. Acad. Sci. U. S. A. 105(38), 14271-14276 (2008).
Westphal, V., Rizzoli, S., Lauterbach, M., Kamin, D., Jahn, R., and Hell, S. "Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement," Science 320, 246-249 (2008).
Hell, S. W., Topics in Fluorescence Spectroscopy; Volume 5: Nonlinear and Two-Photon-Induced Fluorescence, Plenum Press, New York, Ch. 9 (1997).
Clark, J. L. and Rumbles, G. "Laser cooling in the condensed phase by frequency up-conversion," Phys. Rev. Lett. 76(12), 2037-2040 (1996).
Peterson, O. G., Webb, J. P., and McColgin, W. C., "Organic Dye Laser Threshold," J. Appl. Phys. 42(5), 1917-1928 (1971).
Westphal, V. and Hell, S. W. "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94(14), 143903-1-4 (2005).
Hotta, J. Fron, E., Dedecker, P., Janssen, K. P. F., Li, C., Muellen, K., Harke, B., Buekers, J., Hell, S. W., and Hofkens, J. "Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores," J. Am. Chem. Soc., 132(14), 5021-5023 (2010).
Donnert, G., Keller, J., Medda, R., Andrei, M. A., Rizzoli, S. O., Luehrmann, R., Jahn, R., Eggeling, C., and Hell, S. W. "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Nat. Acad. Sci. U. S. A., 103(31), 11440-11445 (2006).
Kasper, R., Harke, B., Forthmann, C., Tinnefeld, P., Hell, S. W., and Sauer, M. "Single-Molecule STED Microscopy with Photostable Organic Fluorophores," 6(13), 1379-1384 (2010).
Wildanger, D., Medda, R., Kastrup, L., and Hell, S. W. "A compact STED microscope providing 3D nanoscale resolution," J. Microsc. 232, 35-43 (2009).
Avlasevich, Y., Müller, S., Erk, P., and Müllen, K., "Novel Core-Expanded Rylenebis(Dicarboximide) Dyes Bearing Pentacene Units: Facile Synthesis and Photophysical Properties," Chem. Eur. J., 13, 6555 - 6561 (2007).
"Resolution, Imaging with the Leica TCS STED- A Practical Guide," Leica Microsystems, Feb. 2009 No. 32 (2009).
Axelrod, J. D., "Progress and challenges in understanding planar cell polarity signaling," Sem. Cell Dev. Bio. 20 964-971 (2009).
Pokutta, S. and Weis, W. I., "Structure and Mechanism of Cadherins and Catenins in Cell-Cell Contacts," Annu. Rev. Cell Dev. Biol. 23 237-261 (2007).
Dawe, H. R., Farr, H., Gull, K. "Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells," J. Cell Sci., 120(1), 7-15 (2007).
Graser, S., York-Dieter, S., Lavoie, S. B., Gassner, O. S., Lamla, S., Le Clech, M., and Nigg, E. A. "Cep164, a novel centriole appendage protein required for primary cilium formation," J. Cell Biol., 179(2), 321-330 (2007).
Ibrahim, R., Messaoudi, C., Chichon, F. J., Celati, C., and Marco S. "Electron Tomography Study of Isolated Human Centrioles," Microsc. Res. Tech. 72, 42-48 (2009).
Full text of this article:
Citation

Lana Lau ; Yin Loon Lee ; Maja Matis ; Jeff Axelrod ; Tim Stearns and W. E. Moerner
"STED super-resolution microscopy in Drosophila tissue and in mammalian cells", Proc. SPIE 7910, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III, 79101N (February 11, 2011); doi:10.1117/12.881221; http://dx.doi.org/10.1117/12.881221


Figures

Tables

Hell, S. W. "Far-field Optical Nanoscopy," Science. 316, 1153-5 (2007).
"Method of the Year 2008," Nat. Methods 6 (1), 1 (2008).
Moerner, W. E., "New directions in single-molecule imaging and analysis," Proc. Natl. Acad. Sci. U.S.A. 104, 12596-12602 (2007).
Rust, M. J., Bates, M. and Zhuang, X. "Sub-diffraction-limited imaging by stochastic optical reconstruction microscopy (STORM)," Nat. Methods 3, 793-795 (2006).
Betzig, E., Patterson, G. H., Sougrat, R. Lindwasser, O. W., Olenych, S., Bonifacino, J.S. Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. R. "Imaging intracellular fluorescent proteins at nanometer resolution," Sciences 313(5793), 1642-1645 (2006).
Hess S. T., Girirajan, T. P. K. and Mason M. D. "Ultra-high resolution imaging by fluorescence photoactivation localization microscopy," Biophys. J. 91, 4258-4272 (2006).
Thompson, R. E., Larson, D. R., Webb W. W. "Precise Nanometer Localization Analysis for Individual Fluorescent Probes," Biophys. J. 82, 2775-2783 (2002).
Pavani, S. R. P., Thompson, M. A., Biteen, J. S., Lord, S. J., Liu, N., Twieg, R. J., Piestun, R. and Moerner, W. E. "Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function," Proc. Nat. Acad. Sci. U. S. A. 106(9), 2995-2999 (2009).
Gutsafsson, M. G. L. "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy," J. Microsc. 198(20), 82-87 (2000).
Hell S. and Wichmann J. "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy" Opt. Lett. 19(11), 780-781 (1994).
Punge, A., Rizzoli, S., Jahn, R., Wildanger, J., Meyer, L., Schonle, A., Kastrup, L., and Hell, S. "3D reconstruction of high-resolution STED microscope images," Microsc. Res. Tech. 71, 644-650 (2008).
Hein, B., Willig, K., and Hell, S. "Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell," Proc. Nat. Acad. Sci. U. S. A. 105(38), 14271-14276 (2008).
Westphal, V., Rizzoli, S., Lauterbach, M., Kamin, D., Jahn, R., and Hell, S. "Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement," Science 320, 246-249 (2008).
Hell, S. W., Topics in Fluorescence Spectroscopy; Volume 5: Nonlinear and Two-Photon-Induced Fluorescence, Plenum Press, New York, Ch. 9 (1997).
Clark, J. L. and Rumbles, G. "Laser cooling in the condensed phase by frequency up-conversion," Phys. Rev. Lett. 76(12), 2037-2040 (1996).
Peterson, O. G., Webb, J. P., and McColgin, W. C., "Organic Dye Laser Threshold," J. Appl. Phys. 42(5), 1917-1928 (1971).
Westphal, V. and Hell, S. W. "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94(14), 143903-1-4 (2005).
Hotta, J. Fron, E., Dedecker, P., Janssen, K. P. F., Li, C., Muellen, K., Harke, B., Buekers, J., Hell, S. W., and Hofkens, J. "Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores," J. Am. Chem. Soc., 132(14), 5021-5023 (2010).
Donnert, G., Keller, J., Medda, R., Andrei, M. A., Rizzoli, S. O., Luehrmann, R., Jahn, R., Eggeling, C., and Hell, S. W. "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Nat. Acad. Sci. U. S. A., 103(31), 11440-11445 (2006).
Kasper, R., Harke, B., Forthmann, C., Tinnefeld, P., Hell, S. W., and Sauer, M. "Single-Molecule STED Microscopy with Photostable Organic Fluorophores," 6(13), 1379-1384 (2010).
Wildanger, D., Medda, R., Kastrup, L., and Hell, S. W. "A compact STED microscope providing 3D nanoscale resolution," J. Microsc. 232, 35-43 (2009).
Avlasevich, Y., Müller, S., Erk, P., and Müllen, K., "Novel Core-Expanded Rylenebis(Dicarboximide) Dyes Bearing Pentacene Units: Facile Synthesis and Photophysical Properties," Chem. Eur. J., 13, 6555 - 6561 (2007).
"Resolution, Imaging with the Leica TCS STED- A Practical Guide," Leica Microsystems, Feb. 2009 No. 32 (2009).
Axelrod, J. D., "Progress and challenges in understanding planar cell polarity signaling," Sem. Cell Dev. Bio. 20 964-971 (2009).
Pokutta, S. and Weis, W. I., "Structure and Mechanism of Cadherins and Catenins in Cell-Cell Contacts," Annu. Rev. Cell Dev. Biol. 23 237-261 (2007).
Dawe, H. R., Farr, H., Gull, K. "Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells," J. Cell Sci., 120(1), 7-15 (2007).
Graser, S., York-Dieter, S., Lavoie, S. B., Gassner, O. S., Lamla, S., Le Clech, M., and Nigg, E. A. "Cep164, a novel centriole appendage protein required for primary cilium formation," J. Cell Biol., 179(2), 321-330 (2007).
Ibrahim, R., Messaoudi, C., Chichon, F. J., Celati, C., and Marco S. "Electron Tomography Study of Isolated Human Centrioles," Microsc. Res. Tech. 72, 42-48 (2009).
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