Abstract
Stochastic optical fluctuation imaging (SOFI) is a superresolution imaging technique that uses the flickering of fluorescent labels to generate a microscopic image with a resolution better than what the diffraction limit allows. Its adaptation towards fluorescent protein-labeled samples (called photoconversion SOFI or pcSOFI) allows for a straightforward and easily accessible way of generating superresolution images. In this protocol, we will discuss how so-called “smart labels,” and specifically the reversibly switchable fluorescent proteins, have opened doors towards superresolution imaging in general and we provide a protocol on how to perform pcSOFI on HeLa cells expressing human β-actin labeled with the reversibly photoswitchable fluorescent protein Dronpa.
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References
Dedecker P, De Schryver FC, Hofkens J (2013) Fluorescent proteins: shine on, you crazy diamond. J Am Chem Soc 135:2387–2402. doi:10.1021/ja309768d
Heilemann M, Dedecker P, Hofkens J, Sauer M (2009) Photoswitches: key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification. Laser Photon Rev 3:180–202. doi:10.1002/lpor.200810043
Huang B (2010) Super-resolution optical microscopy: multiple choices. Curr Opin Chem Biol 14:10–14. doi:10.1016/j.cbpa.2009.10.013
Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149:243–251. doi:10.1016/j.jbiotec.2010.03.012
Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158. doi:10.1126/science.1137395
Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90:1103–1163. doi:10.1152/physrev.00038.2009
Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV (2005) Innovation: photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6:885–891. doi:10.1038/nrm1741
Hofmann M, Eggeling C, Jakobs S, Hell SW (2005) Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A 102:17565–17569. doi:10.1073/pnas.0506010102
Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optic Lett 19:780–782
Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102:13081–13086. doi:10.1073/pnas.0406877102
Schwentker MA, Bock H, Hofmann M et al (2007) Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching. Microsc Res Tech 70:269–280. doi:10.1002/jemt.20443
Herbert S, Soares H, Zimmer C, Henriques R (2012) Single-molecule localization super-resolution microscopy: deeper and faster. Microsc Microanal 18:1419–1429. doi:10.1017/S1431927612013347
Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645. doi:10.1126/science.1127344
Dertinger T, Colyer R, Iyer G et al (2009) Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci U S A 106:22287–22292. doi:10.1073/pnas.0907866106
Dertinger T, Pallaoro A, Braun G et al (2013) Advances in superresolution optical fluctuation imaging (SOFI). Q Rev Biophys 46(2):210–221. doi:10.1017/S0033583513000036
Dedecker P, Mo GCH, Dertinger T, Zhang J (2012) Widely accessible method for superresolution fluorescence imaging of living systems. Proc Natl Acad Sci U S A 109:10909–10914. doi:10.1073/pnas.1204917109
Dertinger T, Heilemann M, Vogel R et al (2010) Superresolution optical fluctuation imaging with organic dyes. Angew Chem Int Ed Engl 49:9441–9443. doi:10.1002/anie.201004138
Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373. doi:10.1126/science.1102506
Pletnev S, Subach FV, Dauter Z et al (2012) A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP. J Mol Biol 417:144–151. doi:10.1016/j.jmb.2012.01.044
Cubitt AB, Woollenweber LA, Heim R (1999) Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. Meth Cell Biol 58:19–30
Mizuno H, Dedecker P, Ando R et al (2010) Higher resolution in localization microscopy by slower switching of a photochromic protein. Photochem Photobiol Sci 9:239–248. doi:10.1039/b9pp00124g
Dedecker P, Duwé S, Neely RK, Zhang J (2012) Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy. J Biomed Optic 17:126008. doi:10.1117/1.JBO.17.12.126008
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 931–957
Acknowledgments
The authors thank Susana Rocha and Jeroen Vangindertael for setting up and maintaining some of the microscope setups, Doortje Borrenberghs for help with the cell culture, and Hideaki Mizuno for the Dronpa-β-actin/pMC1 construct. B.M. is funded by a Ph.D. grant from the Agency for Innovation by Science and Technology (IWT) Flanders. P.D. is a postdoctoral fellow of the Research Foundation–Flanders (FWO).
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Moeyaert, B., Dedecker, P. (2014). pcSOFI as a Smart Label-Based Superresolution Microscopy Technique. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_17
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DOI: https://doi.org/10.1007/978-1-4939-0470-9_17
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