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A STED Microscope Designed for Routine Biomedical Applications (Invited Paper)

By Frederik Gorlitz, Patrick Hoyer, Henning Falk, Lars Kastrup, Johann Engelhardt, and Stefan W. Hell
Progress In Electromagnetics Research, Vol. 147, 57-68, 2014


We present a multi-color STED fluorescence microscope providing far-field optical resolution down to 20 nm for biomedical research. The optical design comprises fiber lasers, beam scanners, and a set of active and passive polarizing elements that cooperatively yield an optically robust system for routinely imaging samples at subdiffraction length scales.


Frederik Gorlitz, Patrick Hoyer, Henning Falk, Lars Kastrup, Johann Engelhardt, and Stefan W. Hell, "A STED Microscope Designed for Routine Biomedical Applications (Invited Paper)," Progress In Electromagnetics Research, Vol. 147, 57-68, 2014.


    1. Hell, S. W. and J. Wichmann, "Breaking the diffraction resolution limit by stimulated-emission --- Stimulated-emission-depletion fluorescence microscopy," Optics Letters, Vol. 19, No. 11, 780-782, 1994.

    2. Klar, T. A., et al., "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 15, 8206-8210, 2000.

    3. Westphal, V. and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Physical Review Letters, Vol. 94, 143903, 2005.

    4. Hell, S. W., "Toward fluorescence nanoscopy," Nature Biotechnology, Vol. 21, No. 11, 1347-1355, 2003.

    5. Hell, S. W. and M. Kroug, "Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit," Applied Physics B: Lasers and Optics, Vol. 60, 495-497, 1995.

    6. Hell, S. W., S. Jakobs, and L. Kastrup, "Imaging and writing at the nanoscale with focused visible light through saturable optical transitions," Applied Physics A: Materials Science & Processing, Vol. 77, 859-860, 2003.

    7. Rust, M. J., M. Bates, and X. W. Zhuang, "Sub-diffraction-limit imaging by stochastic optical econstruction microscopy (STORM)," Nature Methods, Vol. 3, 793-795, 2006.

    8. Betzig, E., et al., "Imaging intracellular fluorescent proteins at nanometer resolution," Science, Vol. 313, No. 5793, 1642-1645, 2006.

    9. Hess, S. T., T. P. K. Girirajan, and M. D. Mason, "Ultra-high resolution imaging by fluorescence photoactivation localization microscopy," Biophysical Journal, Vol. 91, No. 11, 4258-4272, 2006.

    10. Dertinger, T., et al., "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. Phys. Chem., Vol. 8, No. 3, 433-443, 2007.

    11. Hell, S. W., "Far-field optical nanoscopy," Science, Vol. 316, No. 5828, 1153-1158, 2007.

    12. Willig, K. I., et al., "STED microscopy with continuous wave beams," Nature Methods, Vol. 4, No. 11, 915-918, 2007.

    13. Voloshinov, V. B., L. N. Magdich, and G. A. Knyazev, "Tunable acousto-optic filters with the multiple interaction of light and sound," Quantum Electronics, Vol. 35, No. 11, 1057-1063, 2005.

    14. Gottfert, F., et al., "Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20nm resolution," Biophysical Journal, Vol. 105, No. 1, L01-L03, 2013.

    15. Moffitt, J. R., C. Osseforth, and J. Michaelis, "Time-gating improves the spatial resolution of STED microscopy," Optics Express, Vol. 19, No. 5, 4242-4254, 2011.

    16. Vicidomini, G., et al., "STED nanoscopy with time-gated detection: theoretical and experimental aspects," PLoS One,, Vol. 8, No. 1, e54421-1-e54421-12, 2013.

    17. Wildanger, D., et al., "A STED microscope aligned by design," Optics Express, Vol. 17, No. 18, 16100-16110, 2009.

    18. Reuss, M., J. Engelhardt, and S. W. Hell, "Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation," Optics Express, Vol. 18, No. 2, 1049-1058, 2010.

    19. Bingen, P., et al., "Parallelized STED fluorescence nanoscop," Optics Express, Vol. 19, No. 24, 23716-23726, 2011.

    20. Schreiber, F., Device and Method for Distributing Illumination Light and Detection Light in a Microscope, 2013.

    21. Donnert, G., et al., "Two-color far-field fluorescence nanoscopy," Biophysical Journal, Vol. 92, No. 8, L67-L69, 2007.

    22. Buckers, J., et al., "Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses," Optics Express, Vol. 19, No. 4, 3130-3143, 2011.

    23. Salthouse, C. D., R. Weissleder, and U. Mahmood, "Development of a time domain fluorimeter for fluorescent lifetime multiplexing analysis," IEEE Transactions on Biomedical Circuits and Systems, Vol. 2, No. 3, 204-211, 2008.

    24. Demandolx, D. and J. Davoust, "Multicolour analysis and local image correlation in confocal microscopy," Journal of Microscopy-Oxford, Vol. 185, 21-36, 1997.

    25. Dickinson, M. E., et al., "Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy," Biotechniques, Vol. 31, No. 6, 1272, 2001.

    26. Neher, R. A., et al., "Blind source separation techniques for the decomposition of multiply labeled fluorescence images," Biophys. J., Vol. 96, No. 9, 3791-3800, 2009.