Summary
Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high-sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Representative images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, to significantly advance the understanding of biological systems.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pawley, J. B. (2006). Handbook of Biological Confocal Microscopy, 3rd ed., Springer, New York, NY.
Abbe, E. (1873). Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung, Archive für mikroskopische Anatomie 9, 413–68.
Sandison, D. R., Piston, D. W., Williams, R. M.and Webb, W. W. (1995). Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser-scanning microscopes, Appl Opt 34, 3576–88.
Gu, M. (1999). Advanced Optical Imaging Theory, Springer, Heidelberg.
Sandison, D. R. and Webb, W. W. (1994). Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes, Appl Opt 33, 603–15.
Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y. E. and Selvin, P. R. (2003). Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization, Science 300, 2061–5.
Barak, L. S. and Webb, W. W. (1982). Diffusion of low density lipoprotein-receptor complex on human fibroblasts, J Cell Biol 95, 846–52.
Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York.
Gustafsson, M. G. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy., J Microsc 198, 82–7.
Hell, S. and Steltzer, E. H. K. (1992). Properties of a 4Pi confocal fluorescence microscope, J Opt Soc Am A 9, 2159–67.
Bewersdorf, J., Bennett, B. T. and Knight, K. L. (2006). H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy, Proc Natl Acad Sci U S A 103, 18137–42.
Egner, A., Jakobs, S. and Hell, S. W. (2002). Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast, Proc Natl Acad Sci U S A 99, 3370–5.
Gugel, H., Bewersdorf, J., Jakobs, S., Engelhardt, J., Storz, R. and Hell, S. W. (2004). Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy, Biophys J 87, 4146–52.
Bewersdorf, J., Schmidt, R. and Hell, S. W. (2006). Comparison of I5M and 4Pi-microscopy, J Microsc 222, 105–17.
Gustafsson, M. G., Agard, D. A. and Sedat, J. W.(1995). Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses, Proc SPIE 2412, 147–56.
Gustafsson, M. G., Agard, D. A. and Sedat, J. W. (1999). I5M: 3D widefield light microscopy with better than 100 nm axial resolution, J Microsc 195, 10–6.
Hell, S. W. and Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated-emission – stimulated-emission-depletion fluorescence microscopy, Opt Lett 19, 780–82.
Westphal, V. and Hell, S. W. (2005). Nanoscale resolution in the focal plane of an optical microscope, Phys Rev Lett 94, 143903.
Kittel, R. J., Wichmann, C., Rasse, T. M., Fouquet, W., Schmidt, M., Schmid, A., Wagh, D. A., Pawlu, C., Kellner, R. R., Willig, K. I., Hell, S. W., Buchner, E., Heckmann, M. and Sigrist, S. J. (2006). Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release, Science 312, 1051–4.
Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X. N., Lukyanov, S., Matz, M., Kim, S., Weissman, I. and Siebert, P. (2000). “Fluorescent timer”: protein that changes color with time, Science 290, 1585–88.
Hell, S. W. and Kroug, M. (1995). Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit, Appl Phys B 60, 495–97.
Lidke, K. A., Rieger, B., Jovin, T. M. and Heintzmann, R. (2005). Superresolution by localization of quantum dots using blinking statistics, Opt Express 13, 7052–62.
Gustafsson, M. G. (2005). Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution, Proc Natl Acad Sci U S A 102, 13081–6.
Hofmann, M., Eggeling, C., Jakobs, S. and Hell, S. W. (2005). Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins, Proc Natl Acad Sci U S A 102, 17565–9.
Burns, D. H., Callis, J. B., Christian, G. D. and Davidson, E. R. (1985). Strategies for attaining superresolution using spectroscopic data as constraints, Appl Opt 24, 154.
Hwang, J., Tamm, L. K., Bohm, C., Ramalingam, T. S., Betzig, E. and Edidin, M. (1995). Nanoscale complexity of phospholipid monolayers investigated by near-field scanning optical microscopy, Science 270, 610–14.
Esa, A., Edelmann, P., Kreth, G., Trakhtenbrot, L., Amariglio, N., Rechavi, G., Hausmann, M. and Cremer, C. (2000). Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome, J Microsc 199, 96–105.
Kural, C., Kim, H., Syed, S., Goshima, G., Gelfand, V. I. and Selvin, P. R. (2005). Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?, Science 308, 1469–72.
Qu, X., Wu, D., Mets, L. and Scherer, N. F. (2004). Nanometer-localized multiple single-molecule fluorescence microscopy, Proc Natl Acad Sci U S A 101, 11298–303.
Gordon, M. P., Ha, T. and Selvin, P. R. (2004). Single-molecule high-resolution imaging with photobleaching, Proc Natl Acad Sci U S A 101, 6462–5.
Bates, M., Huang, B., Dempsey, G. T. and Zhuang, X. (2007). Multicolor super-resolution imaging with photo-switchable fluorescent probes, Science 317, 1749–53.
Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution, Science 313, 1642–45.
Rust, M. J., Bates, M. and Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), Nat Methods 3, 793–6.
Egner, A., Geisler, C., von Middendorff, C., Bock, H., Wenzel, D., Medda, R., Andresen, M., Stiel, A. C., Jakobs, S., Eggeling, C., Schonle, A. and Hell, S. W. (2007). Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters, Biophys J 93, 3285–90.
Folling, J., Belov, V., Kunetsky, R., Medda, R., Schonle, A., Egner, A., Eggeling, C., Bossi, M. and Hell, S. W. (2007). Photochromic rhodamines provide nanoscopy with optical sectioning, Angew Chem Int Ed Engl 46, 6266–70.
Thompson, R. E., Larson, D. R. and Webb, W. W. (2002). Precise nanometer localization analysis for individual fluorescent probes, Biophys J 82, 2775–83.
Lacoste, T. D., Michalet, X., Pinaud, F., Chemla, D. S., Alivisatos, A. P. and Weiss, S. (2000). Ultrahigh-resolution multicolor colocalization of single fluorescent probes, Proc Natl Acad Sci U S A 97, 9461–6.
Michalet, X. and Weiss, S. (2006). Using photon statistics to boost microscopy resolution, Proc Natl Acad Sci U S A 103, 4797–98.
Lukyanov, K. A., Chudakov, D. M., Lukyanov, S. and Verkhusha, V. V. (2005). Photoactivatable fluorescent proteins, Nat Rev Mol Cell Biol 6, 885–91.
Tsien, R. Y. (1998). The green fluorescent protein, Annu Rev Biochem 67, 509–44.
Giepmans, B. N., Adams, S. R., Ellisman, M. H. and Tsien, R. Y. (2006). The fluorescent toolbox for assessing protein location and function, Science 312, 217–24.
Hess, S. T., Girirajan, T. P. and Mason, M. D. (2006). Ultra-high resolution imaging by fluorescence photoactivation localization microscopy, Biophys J 91, 4258–72.
Hess, S. T., Sheets, E. D., Wagenknecht-Wiesner, A. and Heikal, A. A. (2003). Quantitative Analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells, Biophys J 85, 2566–80.
Heikal, A. A., Hess, S. T. and Webb, W. W. (2001). Multiphoton molecular spectroscopy and excited–state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity, Chem Phys 274, 37–55.
Haupts, U., Maiti, S., Schwille, P. and Webb, W. W. (1998). Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy, Proc Natl Acad Sci U S A 95, 13573–78.
Hess, S. T., Heikal, A. A. and Webb, W. W. (2004). Fluorescence photoconversion kinetics in novel green fluorescent protein pH sensors, J Phys Chem B 108, 10138–48.
Heikal, A. A., Hess, S. T., Baird, G. S., Tsien, R. Y. and Webb, W. W. (2000). Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine), Proc Natl Acad Sci U S A 97, 11996–2001.
Schwille, P., Kummer, S., Heikal, A. A., Moerner, W. E. and Webb, W. W. (2000). Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins, Proc Natl Acad Sci U S A 97, 151–56.
Brasselet, S., Peterman, E. J. G., Miyawaki, A. and Moerner, W. E. (2000). Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon, J Phys Chem B 104, 3676–82.
Patterson, G. H. and Lippincott-Schwartz, J. (2002). A photoactivatable GFP for selective photolabeling of proteins and cells, Science 297, 1873–77.
Chudakov, D. M., Verkhusha, V. V., Staroverov, D. B., Souslova, E. A., Lukyanov, S. and Lukyanov, K. A. (2004). Photoswitchable cyan fluorescent protein for protein tracking, Nat Biotechnol 22, 1435–9.
Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K. D. and Nienhaus, G. U. (2004). EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion, Proc Natl Acad Sci U S A 101, 15905–10.
Gurskaya, N. G., Verkhusha, V. V., Shcheglov, A. S., Staroverov, D. B., Chepurnykh, T. V., Fradkov, A. F., Lukyanov, S. and Lukyanov, K. A. (2006). Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light, Nat Biotechnol 24, 461–65.
Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. and Miyawaki, A. (2002). An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein, Proc Natl Acad Sci U S A 99, 12651–56.
Ando, R., Mizuno, H. and Miyawaki, A. (2004). Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting, Science 306, 1370–73.
Axelrod, D. (2001) Total internal reflection fluorescence microscopy in cell biology, Traffic 2, 764–74.
Axelrod, D., Thompson, N. L. and Burghardt, T. P. (1983). Total internal inflection fluorescent microscopy, J Microsc 129, 19–28.
Hess, S. T., Gould, T. J., Gudheti, M. V., Maas, S. A., Mills, K. D. and Zimmerberg, J. (2007). Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories, Proc Natl Acad Sci U S A 104, 17370–75.
Lakowicz, J. R. (1983). Principles of Fluorescence Spectroscopy, Plenum, New York.
Bass, M. and Optical Society of America. (1995).Handbook of Optics, 2nd ed., McGraw-Hill, New York.
Sternberg, S. R. (1983). Biomedical image processing, IEEE Comput 22–34.
Acknowledgments
The authors thank J. Zimmerberg and P. Blank for useful discussions and loaned equipment, G. Patterson for PA-GFP protein and constructs, J. Wiedenmann and U. Nienhaus for EosFP protein and constructs, J. Gosse for helpful discussions, V. Verkhusha for Dendra2 constructs, T. Tripp for machining services, and J. Lozier for molecular biology services. This work was supported in part by National Institutes of Health (NIH) grant K25-AI65459, National Science Foundation (NSF) grant CHE-0722759, and University of Maine startup funds.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hess, S.T., Gould, T.J., Gunewardene, M., Bewersdorf, J., Mason, M.D. (2009). Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_32
Download citation
DOI: https://doi.org/10.1007/978-1-59745-483-4_32
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-40-4
Online ISBN: 978-1-59745-483-4
eBook Packages: Springer Protocols