Abstract
In a method termed photoactivated localization microscopy (PALM), super-resolution fluorescence imaging can be achieved through the localization of single molecules. This allows the resolution of specific proteins fused to the appropriate fluorescent protein label. Here, we summarize fluorescent proteins suitable for PALM, the technical aspects of multicolor and three-dimensional imaging, and the software packages that are available. Additionally, we highlight several biological applications with an emphasis on neuroscience.
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References
Lewis A, Lieberman K (1991) Near-field optical imaging with a non-evanescently excited high-brightness light source of sub-wavelength dimensions. Nature 354(6350):214–216
Betzig E, Trautman JK (1992) Near-field optics: microscopy, spectroscopy, and surface modification beyond the diffraction limit. Science 257(5067):189–195
Page Faulk W, Malcolm Taylor G (1971) Communication to the editors. An immunocolloid method for the electron microscope. Immunochemistry 8(11):1081–1083
Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780–782
Hell SW (2003) Toward fluorescence nanoscopy. Nat Biotechnol 21(11):1347–1355
Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102(37):13081–13086
Hess et al (2006) Biophysical Journal 91(11):4258–4272
Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795
Heilemann M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47(33):6172–6176
Chmyrov et al (2013) Nature Methods 10:737–740
Shtengel G et al (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106(9):3125–3130
Kanchanawong P et al (2010) Nanoscale architecture of integrin-based cell adhesions. Nature 468(7323):580–584
Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19(11):555–565
Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783
Yildiz A et al (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065
Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120(24):4247–4260
McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6(2):131–133
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(4):260–266
Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5(5):417–423
Bates M, Huang B, Zhuang X (2008) Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes. Curr Opin Chem Biol 12(5):505–514
Shroff H et al (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A 104(51):20308–20313
Subach FV et al (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6(2):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(18):6481–6491
Fernandez-Suarez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9(12):929–943
Henriques R, Mhlanga MM (2009) PALM and STORM: what hides beyond the Rayleigh limit? Biotechnol J 4(6):846–857
Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367
Biteen JS et al (2008) Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat Methods 5(11):947–949
Moerner WE, Fromm DP (2003) Methods of single-molecule fluorescence spectroscopy and microscopy. Rev Sci Instrum 74(8):3597–3619
Greenfield D et al (2009) Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. PLoS Biol 7(6):e1000137
Axelrod D (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2(11):764–774
Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5(2):159–161
Wiedenmann J et al (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A 101(45):15905–15910
Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306(5700):1370–1373
Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877
Chudakov DM et al (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22(11):1435–1439
Andresen M et al (2008) Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26(9):1035–1040
Garini Y, Young IT, McNamara G (2006) Spectral imaging: principles and applications. Cytometry A 69(8):735–747
Keshava N, Mustard JF (2002) Spectral unmixing. IEEE Signal Process Mag 19(1):44–57
Zimmermann T (2005) Spectral imaging and linear unmixing in light microscopy. Adv Biochem Eng 95:245–265
Gunewardene MS et al (2011) Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. Biophys J 101(6):1522–1528
Piatkevich KD et al (2010) Monomeric red fluorescent proteins with a large Stokes shift. Proc Natl Acad Sci U S A 107(12):5369–5374
Gurskaya NG et al (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24(4):461–465
Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813
Juette MF et al (2008) Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5(6):527–529
Pavani SRP et al (2009) Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci U S A 106(9):2995–2999
Tang J, Akerboom J, Vaziri A, Looger LL, Shank CV (2010) Near-isotropic 3D optical nanoscopy with photon-limited chromophores. Proc Natl Acad Sci U S A 107(22):10068–10073
Foelling J et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5(11):943–945
Vaziri A, Tang J, Shroff H, Shank CV (2008) Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci U S A 105(51):20221–20226
York AG, Ghitani A, Vaziri A, Davidson MW, Shroff H (2011) Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat Methods 8(4):327–333
Manley S, Gillette JM, Lippincott-Schwartz J (2010) Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics. Methods Enzymol 475:109–120
Hedde PN, Fuchs J, Oswald F, Wiedenmann J, Nienhaus GU (2009) Online image analysis software for photoactivation localization microscopy. Nat Methods 6(10):689–690
Wolter S et al (2010) Real-time computation of subdiffraction-resolution fluorescence images. J Microsc 237(1):12–22
Krizek P, Raska I, Hagen GM (2011) Minimizing detection errors in single molecule localization microscopy. Opt Express 19(4):3226–3235
Rogers SS, Waigh TA, Zhao X, Lu JR (2007) Precise particle tracking against a complicated background: polynomial fitting with Gaussian weight. Phys Biol 4(3):220–227
Smith CS, Joseph N, Rieger B, Lidke KA (2010) Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7(5):373–375
Henriques R et al (2010) QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7(5):339–340
Matsuda A et al (2010) Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP-histones. PLoS One 5(9):1–12
Niu L, Yu J (2008) Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking. Biophys J 95(4):2009–2016
Högbom JA (1974) Aperture synthesis with a non-regular distribution of interferometer baselines. Astron Astrophys Suppl 15:417–426
Holden SJ, Uphoff S, Kapanidis AN (2011) DAOSTORM: an algorithm for high-density super-resolution microscopy. Nat Methods 8(4):279–280
Mlodzianoski MJ et al (2011) Sample drift correction in 3D fluorescence photoactivation localization microscopy. Opt Express 19(16):15009–15019
Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2012
Blanpied TA, Kerr JM, Ehlers MD (2008) Structural plasticity with preserved topology in the postsynaptic protein network. Proc Natl Acad Sci U S A 105(34):12587–12592
Newpher TM, Ehlers MD (2009) Spine microdomains for postsynaptic signaling and plasticity. Trends Cell Biol 19(5):218–227
Penzes P, Cahill ME, Jones KA, VanLeeuwen JE, Woolfrey KM (2011) Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14(3):285–293
Sheng M, Hoogenraad CC (2007) The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu Rev Biochem 76:823–847
Dani A, Huang B, Bergan J, Dulac C, Zhuang X (2010) Superresolution imaging of chemical synapses in the brain. Neuron 68(5):843–856
Wilt BA et al (2009) Advances in light microscopy for neuroscience. Annu Rev Neurosci 32:435–506
Gaietta G et al (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296(5567):503–507
Shu X et al (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9(4):e1001041
Watanabe S et al (2011) Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods 8(1):80–84
Hoze N et al (2012) Heterogeneity of AMPA receptor trafficking and molecular interactions revealed by superresolution analysis of live cell imaging. Proc Natl Acad Sci U S A 109(42):17052–17057
Derkach VA, Oh MC, Guire ES, Soderling TR (2007) Regulatory mechanisms of AMPA receptors in synaptic plasticity. Nat Rev Neurosci 8(2):101–113
Kandel ER, Schwarts JH, Jessell TM (2000) Principles of neural science. McGraw-Hill Medical, New York
Matsuzaki M, Honkura N, Ellis-Davies GCR, Kasai H (2004) Structural basis of long-term potentiation in single dendritic spines. Nature 429(6993):761–766
Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44(1):5–21
Okamoto KI, Nagai T, Miyawaki A, Hayashi Y (2004) Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat Neurosci 7(10):1104–1112
Vanharreveld A, Fifkova E (1975) Swelling of dendritic spines in fascia dentata after stimulation of perforant fibers as a mechanism of post-tetanic potentiation. Exp Neurol 49(3):736–749
Holtmaat A, Svoboda K (2009) Experience-dependent structural synaptic plasticity in the mammalian brain. Nat Rev Neurosci 10(9):647–658
Kopec CD, Li B, Wei W, Boehm J, Malinow R (2006) Glutamate receptor exocytosis and spine enlargement during chemically induced long-term potentiation. J Neurosci 26(7):2000–2009
Naegerl UV, Willig KI, Hein B, Hell SW, Bonhoeffer T (2008) Live-cell imaging of dendritic spines by STED microscopy. Proc Natl Acad Sci U S A 105(48):18982–18987
Urban NT, Willig KI, Hell SW, Naegerl UV (2011) STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys J 101(5):1277–1284
Izeddin I et al (2011) Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe. PLoS One 6(1):e15611
Wilson CJ, Groves PM, Kitai ST, Linder JC (1983) 3-Dimensional structure of dendritic spines in the rat neostriatum. J Neurosci 3(2):383–398
Harris KM, Jensen FE, Tsao B (1992) 3-Dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day-15 and adult ages—implications for the maturation of synaptic physiology and long-term potentiation. J Neurosci 12(7):2685–2705
Naegerl UV, Bonhoeffer T (2010) Imaging living synapses at the nanoscale by STED microscopy. J Neurosci 30(28):9341–9346
Halpain S, Hipolito A, Saffer L (1998) Regulation of F-actin stability in dendritic spines by glutamate receptors and calcineurin. J Neurosci 18(23):9835–9844
Honkura N, Matsuzaki M, Noguchi J, Ellis-Davies GCR, Kasai H (2008) The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines. Neuron 57(5):719–729
Hotulainen P et al (2009) Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. J Cell Biol 185(2):323–339
Manley S et al (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5(2):155–157
Frost NA, Shroff H, Kong H, Betzig E, Blanpied TA (2010) Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines. Neuron 67(1):86–99
Tatavarty V, Das S, Yu J (2012) Polarization of actin cytoskeleton is reduced in dendritic protrusions during early spine development in hippocampal neuron. Mol Biol Cell 23(16):3167–3177
Tatavarty V, Kim E-J, Rodionov V, Yu J (2009) Investigating sub-spine actin dynamics in rat hippocampal neurons with super-resolution optical imaging. PLoS One 4(11):e7724
Hirokawa N (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279(5350):519–526
Morfini GA et al (2009) Axonal transport defects in neurodegenerative diseases. J Neurosci 29(41):12776–12786
Nakata T, Niwa S, Okada Y, Perez F, Hirokawa N (2011) Preferential binding of a kinesin-1 motor to GTP-tubulin-rich microtubules underlies polarized vesicle transport. J Cell Biol 194(2):245–255
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Achurra, P., Holden, S., Pengo, T., Manley, S. (2014). Photoactivated Localization Microscopy for Cellular Imaging. In: Fornasiero, E., Rizzoli, S. (eds) Super-Resolution Microscopy Techniques in the Neurosciences. Neuromethods, vol 86. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-983-3_5
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DOI: https://doi.org/10.1007/978-1-62703-983-3_5
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