A Low Cost and Versatile STED Superresolution Fluorescent Microscope

Daniel McBride, Chin Su, Jun Kameoka, Stanislav Vitha

Electrical and Computer Engineering Department, Texas A&M University, College Station, USA.
Microscopy & Imaging Center, Texas A&M University, College Station, USA.

DOI: 10.4236/mi.2013.23007                

Abstract

A versatile and inexpensive super-resolution fluorescent microscope that functions as easily as a conventional confocal microscope is described. Components of the microscope were designed on a platform which was placed atop a surplus microscope frame. All optical components and equipments used are given. The excitation and depletion beams are extracted from a compact low-cost supercontinuum light source. The focal spot of the depletion beam at the focal plane is studied and imaged by a 100 nm aperture near-field fiber tip. The collinear excitation and depletion beam focused by a 0.9 numerical aperture microscope objective produce a 90 nm lateral super-resolution as verified by imaging 100 nm diameter fluorescent beads.

Keywords

Stimulated Emission-Depletion Microscopy; STED

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S. Hell and J. Wichmann, “Breaking the Diffraction Resolution Limit by Stimulated Emission: Stimulated- Emission-Depletion Fluorescence Microscopy,” Optics Letters, Vol. 19, No. 11, 1994, pp. 780-782. doi:10.1364/OL.19.000780
[2]	B. Hein, K. Willig and S. Hell, “Stimulated Emission Depletion (STED) Nanoscopy of a Fluorescent Protein-Labeled Organelle Inside a Living Cell,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 38, 2008, pp. 14271-14276. doi:10.1073/pnas.0807705105
[3]	R. Schmidt, C. Wurm, S. Jakobs, J. Engelhardt, A. Egner and S. Hell, “Spherical Nanosized Focal Spot Unravels the Interior of Cells,” Nature Methods, Vol. 5, 2008, pp. 539-544. doi:10.1038/nmeth.1214
[4]	M. Reuss, J. Engelhardt and S. Hell, “Birefringent Device Converts a Standard Scanning Microscope into a STED Microscope that Also Maps Molecular Orientation,” Optics Express, Vol. 18, No. 2, 2010, pp. 1049-1058. doi:10.1364/OE.18.001049
[5]	N. Heckenberg, R. McDuff, C. Smith and A. White, “Generation of Optical Phase Singularities by Computer-Generated Holograms,” Optics Letters, Vol. 17, No. 3, 1992, pp. 221-223. doi:10.1364/OL.17.000221
[6]	R. Heintzmann, T. Jovin and C. Cremer, “Saturated Patterned Excitation Microscopy—A Concept for Optical Resolution Improvement,” Journal of the Optical Society of America A, Vol. 19, No. 8, 2002, pp. 1599-1609. doi:10.1364/JOSAA.19.001599
[7]	M. Gustafsson, “Nonlinear Structured-Illumination Microscopy: Wide-Field Fluorescence Imaging with Theoretically Unlimited Resolution,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 37, 2005, pp. 13081-13085. doi:10.1073/pnas.0406877102
[8]	S. Hess, T. Girirajan and M. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophysical Journal, Vol. 91, No. 11, 2006, pp. 4258-4272. doi:10.1529/biophysj.106.091116
[9]	E. Betzig, G. Patterson, R. Sougrat, O. Lindwasser, S. Olenych, J. Bonifacino, M. Davidson, J. Lippincott-Schwartz and H. Hess, “Imaging Intracellular Fluorescent Proteins at Nanometer Resolution,” Science, Vol. 313, No. 5379, 2006, pp. 1642-1645. doi:10.1126/science.1127344
[10]	M. Rust, M. Bates and X. Zhuang, “Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM),” Nature Methods, Vol. 3, 2006, pp. 793-796. doi:10.1038/nmeth929
[11]	M. Bates, B. Huang, G. Dempsey and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science, Vol. 317, No. 5845, 2007, pp. 1749-1753. doi:10.1126/science.1146598
[12]	B. Huang, W. Wang, M. Bates and X. Zhuang, “Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy,” Science, Vol. 319, No. 5864, 2008, pp. 810-813. doi:10.1126/science.1153529
[13]	D. Wildanger, E. Rittweger, L. Kastrup and S. Hell, “STED Microscopy with a Supercontinuum Laser Source,” Optics Express, Vol. 16, No. 13, 2008, pp. 9614-9621. doi:10.1364/OE.16.009614
[14]	Y. Iketaki, T. Watanabe, N. Bokor and M. Fujii, “Investigation of the Center Intensity of First-and Second-Order Laguerre-Gaussian Beams with Linear and Circular Polarization,” Optics Letters, Vol. 32, No. 16, 2007, pp. 2357-2359. doi:10.1364/OL.32.002357
[15]	B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems I. An Integral Representation of the Image Field,” Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences, Vol. 253, No. 1274, pp. 358-379. doi:10.1098/rspa.1959.0200
[16]	V. Westphal and S. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Physical Review Letters, Vol. 94, 2005, Article ID: 143903. doi:10.1103/PhysRevLett.94.143903