A Low Cost and Versatile STED Superresolution Fluorescent Microscope
- 1 Electrical and Computer Engineering Department, Texas A&M University, College Station, USA
- 2 Electrical and Computer Engineering Department, Texas A&M University, College Station, USA
- 3 Electrical and Computer Engineering Department, Texas A&M University, College Station, USA
- 4 Microscopy & Imaging Center, Texas A&M University, College Station, USA
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 equipment s used are given. The excitation and depletion beams are ex tracted 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.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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