Skip to main content Accessibility help

Single-Molecule Localization Super-Resolution Microscopy: Deeper and Faster

  • Sébastien Herbert (a1) (a2), Helena Soares (a3), Christophe Zimmer (a1) and Ricardo Henriques (a1)


For over a decade fluorescence microscopy has demonstrated the capacity to achieve single-molecule localization accuracies of a few nanometers, well below the ∼200 nm lateral and ∼500 nm axial resolution limit of conventional microscopy. Yet, only the recent development of new fluorescence labeling modalities, the increase in sensitivity of imaging hardware, and the creation of novel image analysis tools allow for the emergence of single-molecule-based super-resolution imaging techniques. Novel methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy can typically reach a tenfold increase in resolution compared to standard microscopy methods. Their implementation is relatively easy only requiring minimal changes to a conventional wide-field or total internal reflection fluorescence microscope. The recent translation of these two methods into commercial imaging systems has made them further accessible to researchers in biology. However, these methods are still evolving rapidly toward imaging live samples with high temporal resolution and depth. In this review, we recall the roots of single-molecule localization microscopy, summarize major recent developments, and offer perspective on potential applications.


Corresponding author

* Corresponding author: E-mail:
** Corresponding author: E-mail:


Hide All
Abbe, E. (1882). The relations of aperture and power in the microscope. J Roy Micro Soc 2, 300309.
Abraham, A.V., Ram, S., Chao, J., Ward, E. & Ober, R.J. (2009). Quantitative study of single molecule location estimation techniques. Opt Express 17, 23352.
Ando, T., Uchihashi, T., Kodera, N., Yamamoto, D., Miyagi, A., Taniguchi, M. & Yamashita, H. (2008). High-speed AFM and nano-visualization of biomolecular processes. Eur J Physiol 456, 211225.
Aquino, D., Schönle, A., Geisler, C., Middendorff, C.V., Wurm, C.A., Okamura, Y., Lang, T., Hell, S.W. & Egner, A. (2011). Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat Methods 8, 353359.
Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89, 141145.
Baddeley, D., Crossman, D., Rossberger, S., Cheyne, J.E., Montgomery, J.M., Jayasinghe, I.D., Cremer, C., Cannell, M.B. & Soeller, C. (2011). 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS One 6, e20645.
Bates, M., Huang, B., Dempsey, G.T. & Zhuang, X. (2007). Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 17491753.
Betzig, E. (1995). Proposed method for molecular optical imaging. Opt Lett 20, 237239.
Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J. & Hess, H.F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 16421645.
Biteen, J.S., Thompson, M.A., Tselentis, N.K., Bowman, G.R., Shapiro, L. & Moerner, W.E. (2008). Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat Methods 5, 947949.
Burnette, D.T., Sengupta, P., Dai, Y., Lippincott-Schwartz, J. & Kachar, B. (2011). Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules. Proc Natl Acad Sci USA 108, 2108121086.
Chao, J., Ram, S., Abraham, A.V., Sally Ward, E. & Ober, R.J. (2009a). A resolution measure for three-dimensional microscopy. Optics Comm 282, 17511761.
Chao, J., Ram, S., Ward, E.S. & Ober, R.J. (2009b). A comparative study of high resolution microscopy imaging modalities using a three-dimensional resolution measure. Opt Express 17, 2437724402.
Cheezum, M.K., Walker, W.F. & Guilford, W.H. (2001). Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys J 81, 23782388.
Cox, S., Rosten, E., Monypenny, J., Jovanovic-Talisman, T., Burnette, D.T., Lippincott-Schwartz, J., Jones, G.E. & Heintzmann, R. (2011). Bayesian localization microscopy reveals nanoscale podosome dynamics. Nat Methods 9, 195200.
Cremer, C. & Cremer, T. (1978). Considerations on a laser-scanning-microscope with high-resolution and depth of field. Microsc Acta 81, 3144.
Crocker, J.C. & Grier, D.G. (1996). Methods of digital video microscopy for colloidal studies. J Colloid Interf Sci 179, 298310.
Dedecker, P., Mo, G.C.H., Dertinger, T. & Zhang, J. (2012). Widely accessible method for superresolution fluorescence imaging of living systems. Proc Natl Acad Sci USA 109, 1090910914.
de Jonge, N., Peckys, D.B., Kremers, G.J. & Piston, D.W. (2009). Electron microscopy of whole cells in liquid with nanometer resolution. Proc Natl Acad Sci USA 106, 21592164.
Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. (2009). Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci USA 106, 2228722292.
Ehsani, S., Santos, J.C., Rodrigues, C.D., Henriques, R., Audry, L., Zimmer, C., Sansonetti, P., Tran Van Nhieu, G. & Enninga, J. (2012). Hierarchies of host factor dynamics at the entry site of Shigella flexneri during host cell invasion. Infect Immun 80, 25482557.
Erni, R., Rossell, M.D., Kisielowski, C. & Dahmen, U. (2009). Atomic-resolution imaging with a sub-50-pm electron probe. Phys Rev Lett 102, 96101.
Folling, J., Bossi, M., Bock, H., Medda, R., Wurm, C.A., Hein, B., Jakobs, S., Eggeling, C. & Hell, S.W. (2008). Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5, 943945.
Gautier, A., Juillerat, A., Heinis, C., Correa, I.R. Jr., Kindermann, M., Beaufils, F. & Johnsson, K. (2008). An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15, 128136.
Gelles, J., Schnapp, B.J. & Sheetz, M.P. (1988). Tracking kinesin-driven movements with nanometre-scale precision. Nature 331, 450453.
Giessibl, F.J. (1995). Atomic resolution of the silicon (111)-(7×7) surface by atomic force microscopy. Science 267, 6871.
Gordon, M.P., Ha, T. & Selvin, P.R. (2004). Single-molecule high-resolution imaging with photobleaching. Proc Natl Acad Sci USA 101, 6462.
Greenfield, D., McEvoy, A.L., Shroff, H., Crooks, G.E., Wingreen, N.S., Betzig, E. & Liphardt, J. (2009). Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7, e1000137.
Gustafsson, M.G.L. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198, 8287.
Hänninen, P., Hell, S., Salo, J., Soini, E. & Cremer, C. (1995). Two-photon excitation 4Pi confocal microscope: Enhanced axial resolution microscope for biological research. Appl Phys Lett 66, 1698.
Hedde, P.N., Fuchs, J., Oswald, F., Wiedenmann, J. & Nienhaus, G.U. (2009). Online image analysis software for photoactivation localization microscopy. Nat Methods 6, 689690.
Heilemann, M., van de Linde, S., Mukherjee, A. & Sauer, M. (2009). Super-resolution imaging with small organic fluorophores. Angew Chem Int Edit 48, 69036908.
Hell, S.W. & Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Opt Lett 19, 780782.
Henriques, R., Griffiths, C., Hesper Rego, E. & Mhlanga, M.M. (2011). PALM and STORM: Unlocking live-cell super-resolution. Biopolymers 95, 322331.
Henriques, R., Lelek, M., Fornasiero, E.F., Valtorta, F., Zimmer, C. & Mhlanga, M.M. (2010). QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7, 339340.
Henriques, R. & Mhlanga, M.M. (2009). PALM and STORM: What hides beyond the Rayleigh limit? Biotechnol J 4, 846857.
Hess, S.T., Girirajan, T.P. & Mason, M.D. (2006). Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91, 42584272.
Holden, S.J., Uphoff, S. & Kapanidis, A.N. (2011). DAOSTORM: An algorithm for high-density super-resolution microscopy. Nat Methods 8, 279280.
Hu, K., Ji, L., Applegate, K.T., Danuser, G. & Waterman-Storer, C.M. (2007). Differential transmission of actin motion within focal adhesions. Science's STKE 315, 111.
Huang, B., Bates, M. & Zhuang, X. (2009). Super-resolution fluorescence microscopy. Annu Rev Biochem 78, 9931016.
Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. (2008a). Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5, 10471052.
Huang, B., Wang, W., Bates, M. & Zhuang, X. (2008b). Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810813.
Huang, F., Schwartz, S.L., Byars, J.M. & Lidke, K.A. (2011). Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed Opt Express 2, 13771393.
Jares-Erijman, E.A. & Jovin, T.M. (2003). FRET imaging. Nat Biotechnol 21, 13871395.
Jones, S.A., Shim, S.H., He, J. & Zhuang, X. (2011). Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8, 499505.
Juette, M.F., Gould, T.J., Lessard, M.D., Mlodzianoski, M.J., Nagpure, B.S., Bennett, B.T., Hess, S.T. & Bewersdorf, J. (2008). Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5, 527529.
Kao, H.P. & Verkman, A. (1994). Tracking of single fluorescent particles in three dimensions: Use of cylindrical optics to encode particle position. Biophys J 67, 12911300.
Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. (2000). Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci USA 97, 8206.
Klein, T., Löschberger, A., Proppert, S., Wolter, S., van de Linde, S. & Sauer, M. (2010). Live-cell dSTORM with SNAP-tag fusion proteins. Nat Methods 8, 79.
Kner, P., Chhun, B.B., Griffis, E.R., Winoto, L. & Gustafsson, M.G.L. (2009). Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6, 339342.
Lacoste, T.D., Michalet, X., Pinaud, F., Chemla, D.S., Alivisatos, A.P. & Weiss, S. (2000). Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc Natl Acad Sci USA 97, 9461.
Lelek, M., Di Nunzio, F., Henriques, R., Charneau, P., Arhel, N. & Zimmer, C. (2012). Superresolution imaging of HIV in infected cells with FlAsH-PALM. Proc Natl Acad Sci USA 109, 85648569.
Lidke, K., Rieger, B., Jovin, T. & Heintzmann, R. (2005). Superresolution by localization of quantum dots using blinking statistics. Opt Express 13, 70527062.
Löschberger, A., van de Linde, S., Dabauvalle, M.C., Rieger, B., Heilemann, M., Krohne, G. & Sauer, M. (2012). Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J Cell Sci 125, 570575.
Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. (2005). Innovation: Photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6, 885891.
Ma, H., Long, F., Zeng, S. & Huang, Z.L. (2012). Fast and precise algorithm based on maximum radial symmetry for single molecule localization. Opt Lett 37, 24812483.
Manley, S., Gillette, J.M., Patterson, G.H., Shroff, H., Hess, H.F., Betzig, E. & Lippincott-Schwartz, J. (2008). High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5, 155157.
Matsuda, A., Shao, L., Boulanger, J., Kervrann, C., Carlton, P.M., Kner, P., Agard, D. & Sedat, J.W. (2010). Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP-histones. PLoS One 5, e12768.
Mattheyses, A.L., Simon, S.M. & Rappoport, J.Z. (2010). Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123, 36213628.
Ober, R.J., Ram, S. & Ward, E.S. (2004). Localization accuracy in single-molecule microscopy. Biophys J 86, 11851200.
Parthasarathy, R. (2012). Rapid, accurate particle tracking by calculation of radial symmetry centers. Nat Methods 9, 724726.
Pavani, S.R., Thompson, M.A., Biteen, J.S., Lord, S.J., Liu, N., Twieg, R.J., Piestun, R. & Moerner, W.E. (2009). Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci USA 106, 29952999.
Pertsinidis, A., Zhang, Y. & Chu, S. (2010). Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647651.
Qu, X., Wu, D., Mets, L. & Scherer, N.F. (2004). Nanometer-localized multiple single-molecule fluorescence microscopy. Proc Natl Acad Sci USA 101, 11298.
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. (2012). A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9, 582584.
Rino, J., Braga, J., Henriques, R. & Carmo-Fonseca, M. (2009). Frontiers in fluorescence microscopy. Int J Dev Biol 53, 15691579.
Rogers, S.S., Waigh, T.A., Zhao, X. & Lu, J.R. (2007). Precise particle tracking against a complicated background: Polynomial fitting with Gaussian weight. Phys Biol 4, 220.
Rust, M.J., Bates, M. & Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793795.
Schermelleh, L., Heintzmann, R. & Leonhardt, H. (2010). A guide to super-resolution fluorescence microscopy. J Cell Biol 190, 165175.
Schmidt, R., Wurm, C.A., Jakobs, S., Engelhardt, J., Egner, A. & Hell, S.W. (2008). Spherical nanosized focal spot unravels the interior of cells. Nat Methods 5, 539544.
Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. (2008). Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5, 417423.
Shtengel, G., Galbraith, J.A., Galbraith, C.G., Lippincott-Schwartz, J., Gillette, J.M., Manley, S., Sougrat, R., Waterman, C.M., Kanchanawong, P. & Davidson, M.W. (2009). Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci USA 106, 3125.
Simonson, P.D., Rothenberg, E. & Selvin, P.R. (2011). Single-molecule-based super-resolution images in the presence of multiple fluorophores. Nano Lett 11(11), 50905096.
Smith, C.S., Joseph, N., Rieger, B. & Lidke, K.A. (2010). Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7, 373375.
Steinhauer, C., Forthmann, C., Vogelsang, J. & Tinnefeld, P. (2008). Superresolution microscopy on the basis of engineered dark states. J Am Chem Soc 130, 1684016841.
Thompson, R.E., Larson, D.R. & Webb, W.W. (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82, 27752783.
Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. (2008). Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5, 159161.
Truong, T.V., Supatto, W., Koos, D.S., Choi, J.M. & Fraser, S.E. (2011). Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat Methods 8, 757760.
Van Oijen, A., Köhler, J., Schmidt, J., Müller, M. & Brakenhoff, G. (1998). 3-Dimensional super-resolution by spectrally selective imaging. Chem Phys Lett 292, 183187.
Vaziri, A., Tang, J., Shroff, H. & Shank, C.V. (2008). Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci USA 105, 2022120226.
Vogelsang, J., Kasper, R., Steinhauer, C., Person, B., Heilemann, M., Sauer, M. & Tinnefeld, P. (2008). A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl 47, 54655469.
Wolter, S., Schüttpelz, M., Tscherepanow, M., Van de Linde, S., Heilemann, M. & Sauer, M. (2010). Real-time computation of sub-diffraction-resolution fluorescence images. J Microsc 237, 1222.
Wombacher, R., Heidbreder, M., van de Linde, S., Sheetz, M.P., Heilemann, M., Cornish, V.W. & Sauer, M. (2010). Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7, 717719.
Xu, K., Babcock, H.P. & Zhuang, X. (2012). Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods 9, 185188.
Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E. & Selvin, P.R. (2003). Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science 300, 20612065.
York, A.G., Ghitani, A., Vaziri, A., Davidson, M.W. & Shroff, H. (2011). Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat Methods 8, 327333.
Zanacchi, F.C., Lavagnino, Z., Donnorso, M.P., Del Bue, A., Furia, L., Faretta, M. & Diaspro, A. (2011). Live-cell 3D super-resolution imaging in thick biological samples. Nat Methods 8, 10471049.
Zhu, L., Zhang, W., Elnatan, D. & Huang, B. (2012). Faster STORM using compressed sensing. Nat Methods 9(7), 721723.


Single-Molecule Localization Super-Resolution Microscopy: Deeper and Faster

  • Sébastien Herbert (a1) (a2), Helena Soares (a3), Christophe Zimmer (a1) and Ricardo Henriques (a1)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed