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Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes

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We demonstrate three-dimensional (3D) super-resolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable monomeric (m)Cherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we have substantially reduced excitation and bleaching of unlocalized markers, which allows us to use 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms, which are based on cross-correlation, do not rely on idealized noise models or specific optical configurations. This allows instrument design to be flexible. By generating appropriate fusion constructs and expressing them in Cos7 cells, we could image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network and the endoplasmic reticulum at depths of greater than 8 μm.

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Figure 1: Experimental considerations for 3D PALM.
Figure 2: Improvement over existing 3D PALM.
Figure 3: 3D super-resolution imaging of a mitochondrial network.
Figure 4: 3D super-resolution imaging of an endoplasmic reticulum network.
Figure 5: 3D PALM imaging of a vimentin network.
Figure 6: 3D PALM image up to 7.5 μm in depth.

Change history

  • 04 March 2011

    In the version of this article initially published online, the affiliation for Alipasha Vaziri was incorrect. The error has been corrected for the print, PDF and HTML versions of this article.


  1. Larabell, C.A. & Le Gros, M.A. X-ray tomography generates 3-D reconstructions of the yeast, Saccharomyces cerevisiae, at 60-nm resolution. Mol. Biol. Cell 15, 957–962 (2004).

    Article  CAS  Google Scholar 

  2. Hohmann-Marriott, M.F. et al. Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nat. Methods 6, 729–731 (2009).

    Article  CAS  Google Scholar 

  3. Gustafsson, M.G.L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Article  CAS  Google Scholar 

  4. Gustafsson, M.G.L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).

    Article  CAS  Google Scholar 

  5. Donnert, G. et al. Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl. Acad. Sci. USA 103, 11440–11445 (2006).

    Article  CAS  Google Scholar 

  6. Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

    Article  CAS  Google Scholar 

  7. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  8. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  9. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  10. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    Article  CAS  Google Scholar 

  11. Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).

    Article  CAS  Google Scholar 

  12. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001).

    Article  CAS  Google Scholar 

  13. Vaziri, A., Tang, J., Shroff, H. & Shank, C.V. Multilayer three-dimensional super resolution imaging of thick biological samples. Proc. Natl. Acad. Sci. USA 105, 20221–20226 (2008).

    Article  CAS  Google Scholar 

  14. Fölling, J. et al. Photochromic rhodamines provide nanoscopy with optical sectioning. Angew. Chem. Int. Edn. Engl. 46, 6266–6270 (2007).

    Article  Google Scholar 

  15. Mlodzianoski, M.J., Juette, M.F., Beane, G.L. & Bewersdorf, J. Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy. Opt. Express 17, 8264–8277 (2009).

    Article  CAS  Google Scholar 

  16. Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047–1052 (2008).

    Article  CAS  Google Scholar 

  17. Subach, F.V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

    Article  CAS  Google Scholar 

  18. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  Google Scholar 

  19. Zhu, G., van Howe, J., Durst, M., Zipfel, W. & Xu, C. Simultaneous spatial and temporal focusing of femtosecond pulses. Opt. Express 13, 2153–2159 (2005).

    Article  Google Scholar 

  20. Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13, 1468–1476 (2005).

    Article  Google Scholar 

  21. Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    Article  CAS  Google Scholar 

  22. Tal, E., Oron, D. & Silberberg, Y. Improved depth resolution in video-rate line-scanning multiphoton microscopy using temporal focusing. Opt. Lett. 30, 1686–1688 (2005).

    Article  Google Scholar 

  23. Shroff, H. et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc. Natl. Acad. Sci. USA 104, 20308–20313 (2007).

    Article  CAS  Google Scholar 

  24. Juette, M.F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    Article  CAS  Google Scholar 

  25. Pavani, S.R.P. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl. Acad. Sci. USA 106, 2995–2999 (2009).

    Article  CAS  Google Scholar 

  26. Smith, C.S., Joseph, N., Rieger, B. & Lidke, K.A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat. Methods 7, 373–375 (2010).

    Article  CAS  Google Scholar 

  27. Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration, and distance measurements. Nature 466, 647–651 (2010).

    Article  CAS  Google Scholar 

  28. Arimoto, R. & Murray, J.M. A common aberration with water-immersion objective lenses. J. Microsc. 216, 49–51 (2004).

    Article  CAS  Google Scholar 

  29. Guizar-Sicairos, M., Thurman, S.T. & Fienup, J.R. Efficient subpixel image registration algorithms. Opt. Lett. 33, 156–158 (2008).

    Article  Google Scholar 

  30. Oliphant, T. Python for scientific computing. Comput. Sci. Eng. 9, 10–20 (2007).

    Article  CAS  Google Scholar 

  31. Gupton, S.L., Collings, D.A. & Allen, N.S. Endoplasmic reticulum targeted GFP reveals ER organization in tobacco NT-1 cells during cell division. Plant Physiol. Biochem. 44, 95–105 (2006).

    Article  CAS  Google Scholar 

  32. Yoon, M., Moir, R.D., Prahlad, V. & Goldman, R.D. Motile properties of vimentin intermediate filament networks in living cells. J. Cell Biol. 143, 147–157 (1998).

    Article  CAS  Google Scholar 

  33. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  CAS  Google Scholar 

  34. Ritter, J.G., Veith, R., Jan-Peter, S. & Kubitscheck, U. High-contrast single-particle tracking by selective focal plane illumination microscopy. Opt. Express 16, 7142–7152 (2008).

    Article  Google Scholar 

  35. Mortensen, K.I., Churchman, L.S., Spudich, J.A. & Flyvbjerg, H. Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat. Methods 7, 377–381 (2010).

    Article  CAS  Google Scholar 

  36. Hunter, J.D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

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We thank N. Morgan and A. Gillespie for training and use of their spin coater; G. Patterson (National Institute of Biomedical Imaging and Bioengineering) for the gift of purified PA-mCherry1 and mEos2 and for the use of his cell culture facilities; A. Jin for measuring the thickness of our quantum dot films; E. Ramko for help with preparing the PA-mCherry1 fusion vectors; K. Kilborn (Intelligent Imaging Innovations) for loaning us the Vector Point Scanning 2P system; and S. Parekh and H. Eden for feedback and suggestions on the manuscript. This work was supported by the Intramural Research Programs of the National Institute of Biomedical Imaging and Bioengineering.

Author information

Authors and Affiliations



A.G.Y., A.G. and H.S. conceived, designed and built the experimental setup. A.G.Y. wrote the analysis code. A.G. and H.S. collected the data. A.G.Y., A.G. and H.S. analyzed the data. M.W.D. and A.V. contributed reagents and materials. A.G.Y., M.W.D. and H.S. wrote the paper. All authors edited and refined the paper.

Corresponding author

Correspondence to Andrew G York.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16, Supplementary Table 1 and Supplementary Notes 1–3 (PDF 2999 kb)

Supplementary Video 1

z-stack of PA-mCherry1-mito fusions, to accompany Figure 3. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.4 pixels in each dimension was applied before plotting data. (MOV 1173 kb)

Supplementary Video 2

z-stack of PA-mCherry1-ER fusions, to accompany Figure 4. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.6 pixels in each dimension was applied before plotting data. (MOV 1519 kb)

Supplementary Video 3

z-stack of PA-mCherry1-vimentin fusions, to accompany Figure 5. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.6 pixels in each dimension was applied before plotting data. (MOV 4243 kb)

Supplementary Video 4

z-stack of PA-mCherry1-lamin fusions, to accompany Figure 6. Histogram bin size is 50 nm, individual frames are separated by 50 nm z steps. Smoothing of σ = 0.75 pixels in each dimension was applied before plotting data. (MOV 7782 kb)

Supplementary Video 5

z-stack of PA-mCherry1-lamin fusions, extending over > 8.5 μm imaging depth. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.75 pixels in each dimension was applied before plotting data. (MOV 3267 kb)

Supplementary Software

Supplementary Software (ZIP 51 kb)

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York, A., Ghitani, A., Vaziri, A. et al. Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat Methods 8, 327–333 (2011).

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