Abstract
Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods—collectively referred to as “single-molecule localization microscopy” (SMLM)—are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.
Key to this strategy is the use of fluorophores that can be switched “on” and “off” in a controllable manner. Here we review the strengths and weaknesses of the wide variety of SMLM-compatible photoswitchable fluorophores and labeling strategies currently available. We also discuss their suitability for live-cell and multicolor imaging, as well as molecular counting.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783
Abbe E (1873) Beitrage zur theorie des mikroskops und der mikroskopischen wahrnehmung. Archive Mikroskop Anat 9:413–420
Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149:243–251
Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175
Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158
McEvoy AL, Greenfield D, Bates M, Liphardt J (2010) Q&A: single-molecule localization microscopy for biological imaging. BMC Biol 8:106
Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645
Rust MJ, Bates M, Zhuang XW (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795
Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47:6172–6176
Folling J, Belov V, Kunetsky R, Medda R, Schonle A et al (2007) Photochromic rhodamines provide nanoscopy with optical sectioning. Angew Chem Int Ed 46:6266–6270
Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272
Folling J, Bossi M, Bock H, Medda R, Wurm CA et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5:943–945
Lemmer P, Gunkel M, Baddeley D, Kaufmann R, Urich A et al (2008) SPDM: light microscopy with single-molecule resolution at the nanoscale. Appl Phys B 93:1–12
Steinhauer C, Forthmann C, Vogelsang J, Tinnefeld P (2008) Superresolution microscopy on the basis of engineered dark states. J Am Chem Soc 130:16840
Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877
van de Linde S, Wolter S, Heilemann M, Sauer M (2010) The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. J Biotechnol 149:260–266
Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J et al (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci USA 104:20308–20313
Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565
Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Phys Rev 90:1103–1163
Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388:355–358
Fuchs J, Bohme S, Oswald F, Hedde PN, Krause M et al (2010) A photoactivatable marker protein for pulse-chase imaging with superresolution. Nat Methods 7:627–630
Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S et al (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22:1435–1439
McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133
Habuchi S, Ando R, Dedecker P, Verheijen W, Mizuno H et al (2005) Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci USA 102:9511–9516
Andresen M, Stiel AC, Folling J, Wenzel D, Schonle A et al (2008) Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26:1035–1040
Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C et al (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101:15905–15910
Annibale P, Scarselli M, Kodiyan A, Radenovic A (2010) Photoactivatable fluorescent protein mEos2 displays repeated photoactivation after a long-lived dark state in the red photoconverted form. J Phys Chem Lett 1:1506–1510
Endesfelder U, Malkusch S, Flottmann B, Mondry J, Liguzinski P et al (2011) Chemically induced photoswitching of fluorescent probes-a general concept for super-resolution microscopy. Molecules 16:3106–3118
Chudakov DM, Lukyanov S, Lukyanov KA (2007) Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc 2:2024–2032
Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008) mKikGR, a monomeric photoswitchable fluorescent protein. PLoS One 3:e3944
Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367
Subach FV, Patterson GH, Manley S, Gillette JM, Lippincott-Schwartz J et al (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6:153–159
Subach FV, Patterson GH, Renz M, Lippincott-Schwartz J, Verkhusha VV (2010) Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J Am Chem Soc 132:6481–6491
Subach OM, Patterson GH, Ting LM, Wang Y, Condeelis JS et al (2011) A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat Methods 8:771–777
Kim SY, Gitai Z, Kinkhabwala A, Shapiro L, Moerner WE (2006) Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc Natl Acad Sci USA 103:10929–10934
Matsuda A, Shao L, Boulanger J, Kervrann C, Carlton PM et al (2010) Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones. PLoS One 5:e12768
Li S, Kimura E, Ng R, Fall BM, Meuse L et al (2006) A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of duchenne muscular dystrophy. Hum Mol Genet 15:1610–1622
Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS et al (2009) Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7:e1000137
Belov VN, Wurm CA, Boyarskiy VP, Jakobs S, Hell SW (2010) Rhodamines NN: a novel class of caged fluorescent dyes. Angew Chem Int Ed Engl 49:3520–3523
Heilemann M, Sauer M, Margeat E, Kasper R, Tinnefeld P (2005) Carbocyanine dyes as efficient reversible single-molecule optical switch. J Am Chem Soc 127:3801–3806
Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317:1749–1753
Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angew Chem Int Ed 48:6903–6908
van de Linde S, Krstic I, Prisner T, Doose S, Heilemann M et al (2011) Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging. Photochem Photobiol Sci 10:499–506
Kottke T, van de Linde S, Sauer M, Kakorin S, Heilemann M (2010) Identification of the product of photoswitching of an oxazine fluorophore using fourier transform infrared difference spectroscopy. J Phys Chem Lett 1:3156–3159
Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M et al (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed 47:5465–5469
Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D et al (2007) Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J 93:3285–3290
Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C et al (2010) Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J 99:2686–2694
Lemmer P, Gunkel M, Weiland Y, Muller P, Baddeley D et al (2009) Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range. J Microsc 235:163–171
Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:108101
Belov VN, Bossi ML, Folling J, Boyarskiy VP, Hell SW (2009) Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy. Chemistry 15:10762–10776
Lidke KA, Rieger B, Jovin TM, Heintzmann R (2005) Superresolution by localization of quantum dots using blinking statistics. Opt Express 13:7052–7062
Goldstein M, Watkins S (2008) Immunohistochemistry. Curr Protoc Mol Biol 14:Unit 14.6.
Puchtler H, Meloan SN (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201–204
Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H et al (2007) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 1:Unit 1.16
Mortensen RM, Kingston RE (2009) Selection of transfected mammalian cells. Curr Protoc Mol Biol 9:Unit 9.5.
Subach FV, Malashkevich VN, Zencheck WD, Xiao H, Filonov GS et al (2009) Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc Natl Acad Sci USA 106:21097–21102
You X, Nguyen AW, Jabaiah A, Sheff MA, Thorn KS et al (2006) Intracellular protein interaction mapping with FRET hybrids. Proc Natl Acad Sci USA 103:18458–18463
Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H et al (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89
Sun X, Zhang A, Baker B, Sun L, Howard A et al (2011) Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. Chembiochem 12:2217–2226
Gautier A, Juillerat A, Heinis C, Correa IR Jr, Kindermann M et al (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136
Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382
Miller LW, Cai Y, Sheetz MP, Cornish VW (2005) In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag. Nat Methods 2:255–257
Klein T, Loschberger A, Proppert S, Wolter S, van de Linde S et al (2011) Live-cell dSTORM with SNAP-tag fusion proteins. Nat Methods 8:7–9
Wombacher R, Heidbreder M, van de Linde S, Sheetz MP, Heilemann M et al (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7:717–719
Lee HLD, Lord SJ, Iwanaga S, Zhan K, Xie HX et al (2010) Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores. J Am Chem Soc 132:15099–15101
Wombacher R, Cornish VW (2011) Chemical tags: applications in live cell fluorescence imaging. J Biophotonics 4:391–402
Henriques R, Griffiths C, Hesper RE, Mhlanga MM (2011) PALM and STORM: unlocking live-cell super-resolution. Biopolymers 95:322–331
Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S et al (2009) Cell culture medium affects GFP photostability: a solution. Nat Methods 6:859–860
Dani A, Huang B, Bergan J, Dulac C, Zhuang X (2010) Superresolution imaging of chemical synapses in the brain. Neuron 68:843–856
van de Linde S, Endesfelder U, Mukherjee A, Schuttpelz M, Wiebusch G et al (2009) Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging. Photochem Photobiol Sci 8:465–469
Löschberger A, van de Linde S, Dabauvalle MC, Rieger B, Heilemann M et al (2011) Super-resolution imaging reveals eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J Cell Sci 125: 570–575
Baddeley D, Crossman D, Rossberger S, Cheyne JE, Montgomery JM et al (2011) 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS One 6:e20645
Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011) Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8:527–528
Acknowledgements
The authors are grateful to Sebastian Malkusch and Patrick Zessin for critical reading of the manuscript and to Prof. G.U. Nienhaus for providing spectral data for the fluorescent protein mEosFPthermo. This work was supported by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315262), the German Science Foundation (DFG, grant nr. HE 6166/2-1), and by contract research “Methoden für die Lebenswissenschaften” of the Baden-Württemberg Stiftung (grant nr. P-LS-SPII/11).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Finan, K., Flottmann, B., Heilemann, M. (2013). Photoswitchable Fluorophores for Single-Molecule Localization Microscopy. In: Sousa, A., Kruhlak, M. (eds) Nanoimaging. Methods in Molecular Biology, vol 950. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-137-0_9
Download citation
DOI: https://doi.org/10.1007/978-1-62703-137-0_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-136-3
Online ISBN: 978-1-62703-137-0
eBook Packages: Springer Protocols