Abstract
Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.
Similar content being viewed by others
References
Altman RB, Terry DS, Zhou Z, Zheng Q, Geggier P, Kolster RA, Zhao Y, Javitch JA, Warren JD, Blanchard SC (2012) Cyanine fluorophore derivatives with enhanced photostability. Nat Methods 9(1):68–71
Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94(10):108101
Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317(5845):1749–1753
Chen Y, Lin H, Ludford-Menting MJ, Clayton AH, Gu M, Russell SM (2015) Polarization of excitation light influences molecule counting in single-molecule localization microscopy. Histochem Cell Biol 143(1):11–19
Conley NR, Biteen JS, Moerner WE (2008) Cy3–Cy5 covalent heterodimers for single-molecule photoswitching. J Phys Chem B 112(38):11878–11880
DesMarais V, Ichetovkin I, Condeelis J, Hitchcock-DeGregori SE (2002) Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge. J Cell Sci 115(Pt 23):4649–4660
Doksani Y, Wu J, de Lange T, Zhuang X (2013) Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell 155(2):345–356
Dow LE, Kauffman JS, Caddy J, Zarbalis K, Peterson AS, Jane SM, Russell SM, Humbert PO (2007) The tumour-suppressor Scribble dictates cell polarity during directed epithelial migration: regulation of Rho GTPase recruitment to the leading edge. Oncogene 26(16):2272–2282
Flors C, Ravarani CN, Dryden DT (2009) Super-resolution imaging of DNA labelled with intercalating dyes. ChemPhysChem 10(13):2201–2204
Fu N, Xiong Y, Squier TC (2012) Synthesis of a targeted biarsenical Cy3–Cy5 affinity probe for super-resolution fluorescence imaging. J Am Chem Soc 134(45):18530–18533
Geertsema HJ, Schulte AC, Spenkelink LM, McGrath WJ, Morrone SR, Sohn J, Mangel WF, Robinson A, van Oijen AM (2015) Single-molecule imaging at high fluorophore concentrations by local activation of dye. Biophys J 108(4):949–956
Gunning PW, Hardeman EC, Lappalainen P, Mulvihill DP (2015) Tropomyosin—master regulator of actin filament function in the cytoskeleton. J Cell Sci 128(16):2965–2974
Heidecker M, Yan-Marriott Y, Marriott G (1995) Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin. Biochemistry 34(35):11017–11025
Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47(33):6172–6176
Hess S, Gould T, Gunewardene M, Bewersdorf J, Mason M (2009) Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy. Micro Nano Technol Bioanal 544:483–522
Holden SJ, Uphoff S, Kapanidis AN (2011) DAOSTORM: an algorithm for high-density super-resolution microscopy. Nat Methods 8(4):279–280
Huang ZJ, Haugland RP, You WM, Haugland RP (1992) Phallotoxin and actin binding assay by fluorescence enhancement. Anal Biochem 200(1):199–204
Huang B, Jones SA, Brandenburg B, Zhuang X (2008a) Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5(12):1047–1052
Huang B, Wang W, Bates M, Zhuang X (2008b) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813
Juette MF, Terry DS, Wasserman MR, Zhou Z, Altman RB, Zheng Q, Blanchard SC (2014) The bright future of single-molecule fluorescence imaging. Curr Opin Chem Biol 20C:103–111
Kiuchi T, Higuchi M, Takamura A, Maruoka M, Watanabe N (2015) Multitarget super-resolution microscopy with high-density labeling by exchangeable probes. Nat Methods 12(8):743–746
Kwon J, Hwang J, Park J, Han GR, Han KY, Kim SK (2015) RESOLFT nanoscopy with photoswitchable organic fluorophores. Sci Rep 5:17804
Legant WR, Shao L, Grimm JB, Brown TA, Milkie DE, Avants BB, Lavis LD, Betzig E (2016) High-density three-dimensional localization microscopy across large volumes. Nat Methods 13:359–365
Lukinavičius G, Umezawa K, Olivier N, Honigmann A, Yang G, Plass T, Mueller V, Reymond L, Corrêa I, Luo Z-G, Schultz C, Lemke E, Heppenstall P, Eggeling C, Manley S, Johnsson K (2013) A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chem 5(2):132–139
Nanguneri S, Flottmann B, Herrmannsdörfer F, Thomas K, Heilemann M (2014) Single-molecule super-resolution imaging by tryptophan-quenching-induced photoswitching of phalloidin-fluorophore conjugates. Microsc Res Tech 77:510–516
Nickerson A, Huang T, Lin L-JJ, Nan X (2014) Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein–protein interactions in cells. PLoS One 9(6):e100589
Nieuwenhuizen RP, Lidke KA, Bates M, Puig DL, Grunwald D, Stallinga S, Rieger B (2013) Measuring image resolution in optical nanoscopy. Nat Methods 10:557–562
Oda T, Namba K, Maeda Y (2005) Position and orientation of phalloidin in F-actin determined by X-ray fiber diffraction analysis. Biophys J 88(4):2727–2736
Opazo F, Levy M, Byrom M, Schafer C, Geisler C, Groemer TW, Ellington AD, Rizzoli SO (2012) Aptamers as potential tools for super-resolution microscopy. Nat Methods 9(10):938–939
Parsons JT, Horwitz AR, Schwartz MA (2010) Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat Rev Mol Cell Biol 11(9):633–643
Revelo NH, Kamin D, Truckenbrodt S, Wong AB, Reuter-Jessen K, Reisinger E, Moser T, Rizzoli SO (2014) A new probe for super-resolution imaging of membranes elucidates trafficking pathways. J Cell Biol 205(4):591–606
Ries J, Kaplan C, Platonova E, Eghlidi H, Ewers H (2012) A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9:582–584
Rinnerthaler G, Geiger B, Small JV (1988) Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules. J Cell Biol 106(3):747–760
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795
Sabanayagam CR, Eid JS, Meller A (2005) Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on Forster resonance energy transfer. J Chem Phys 123(22):224708
Sharonov A, Hochstrasser RM (2006) Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci USA 103(50):18911–18916
Shim S-H, Xia C, Zhong G, Babcock H, Vaughan J, Huang B, Wang X, Xu C, Bi G-Q, Zhuang X (2012) Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc Natl Acad Sci USA 109(35):13978–13983
Tojkander S, Gateva G, Schevzov G, Hotulainen P, Naumanen P, Martin C, Gunning PW, Lappalainen P (2011) A molecular pathway for myosin II recruitment to stress fibers. Curr Biol 21(7):539–550
van de Linde S, Aufmkolk S, Franke C, Holm T, Klein T, Loschberger A, Proppert S, Wolter S, Sauer M (2013) Investigating cellular structures at the nanoscale with organic fluorophores. Chem Biol 20(1):8–18
van der Velde J, Ploetz E, Hiermaier M, Oelerich J, de Vries J, Roelfes G, Cordes T (2013) Mechanism of intramolecular photostabilization in self-healing cyanine fluorophores. ChemPhysChem 14(18):4084–4093
von der Ecken J, Muller M, Lehman W, Manstein DJ, Penczek PA, Raunser S (2015) Structure of the F-actin-tropomyosin complex. Nature 519(7541):114–117
Xia T, Li N, Fang X (2013) Single-molecule fluorescence imaging in living cells. Annu Rev Phys Chem 64:459–480
Xu K, Babcock H, Zhuang X (2012) Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods 9:185–188
Zessin PJ, Finan K, Heilemann M (2012) Super-resolution fluorescence imaging of chromosomal DNA. J Struct Biol 177(2):344–348
Acknowledgments
We thank Sara Jones (Harvard University) for technical advice on STORM and Andrew Clayton (Swinburne University of Technology) for helpful discussions. The work was supported by a project grant from the Australian National Health and Medical Research Foundation (APP1061647), the Kids Cancer Project to PWG, and fellowships to SMR from the Australian Research Council (Future Fellowship) and the Australian National Health and Medical Research Foundation (Senior Research Fellowship).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
None of the authors has a conflict of interest related to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Chen, Y., Gu, M., Gunning, P.W. et al. Dense small molecule labeling enables activator-dependent STORM by proximity mapping. Histochem Cell Biol 146, 255–266 (2016). https://doi.org/10.1007/s00418-016-1451-6
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-016-1451-6