Optical Super-Resolution Imaging of β-Amyloid Aggregation In Vitro and In Vivo: Method and Techniques

  • Dorothea Pinotsi
  • Gabriele S. Kaminski Schierle
  • Clemens F. Kaminski
Part of the Methods in Molecular Biology book series (MIMB, volume 1303)


Super-resolution microscopy has emerged as a powerful and non-invasive tool for the study of molecular processes both in vitro and in live cells. In particular, super-resolution microscopy has proven valuable for research studies in protein aggregation. In this chapter we present details of recent advances in this method and the specific techniques, enabling the study of amyloid beta aggregation optically, both in vitro and in cells. First, we show that variants of optical super-resolution microscopy provide a capability to visualize oligomeric and fibrillar structures directly, providing detailed information on species morphology in vitro and even in situ, in the cellular environment. We focus on direct Stochastic Optical Reconstruction Microscopy, dSTORM, which provides morphological detail on spatial scales below 20 nm, and provide detailed protocols for its implementation in the context of amyloid beta research. Secondly, we present a range of optical techniques that offer super-resolution indirectly, which we call multi-parametric microscopy. The latter offers molecular scale information on self-assembly reactions via changes in protein or fluorophore spectral signatures. These techniques are empowered by our recent discovery that disease related amyloid proteins adopt intrinsic energy states upon fibrilisation. We show that fluorescence lifetime imaging provides a particularly sensitive readout to report on the aggregation state, which is robustly quantifiable for experiments performed either in vitro or in vivo.

Key words

Amyloid beta Amyloid fibrils In vivo imaging Super-resolution microscopy Multi-parametric imaging 



This work was funded by grants from the Medical Research Council UK (MR/K015850/1 and MR/K02292X/1), Alzheimer Research UK (ARUK-EG2012A-1), the EPSRC (EP/H018301/1) and the Wellcome Trust (089703/Z/09/Z). D.P. wishes to acknowledge support from the Swiss National Science Foundation and the Cambridge Wellcome Trust Senior Internship scheme.


  1. 1.
    Fitzpatrick AWP, Debelouchina GT, Bayro MJ et al (2013) Atomic structure and hierarchical assembly of a cross-β amyloid fibril. Proc Natl Acad Sci U S A 110:5468–5473PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Lomakin A (1997) Kinetic theory of fibrillogenesis of amyloid beta-protein. Proc Natl Acad Sci U S A 94:7942–7947PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Knowles TPJ, Waudby CA, Devlin GL et al (2009) An analytical solution to the kinetics of breakable filament assembly. Science 326:1533–1537PubMedCrossRefGoogle Scholar
  4. 4.
    Cohen SIA, Linse S, Luheshi LM et al (2013) Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism. Proc Natl Acad Sci U S A 110:9758–9763PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Hellstrand E, Boland B, Walsh DM, Linse S (2010) Amyloid β-protein aggregation produces highly reproducible kinetic data and occurs by a two-phase process. ACS Chem Neurosci 1:13–18PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Pinotsi D, Buell AK, Dobson CM et al (2013) A label-free, quantitative assay of amyloid fibril growth based on intrinsic fluorescence. Chembiochem 14:846–850PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Kaminski Schierle GS, Bertoncini CW, Chan FTS et al (2011) A FRET sensor for non-invasive imaging of amyloid formation in vivo. ChemPhysChem 12:673–680PubMedCrossRefGoogle Scholar
  8. 8.
    Chan FTS, Kaminski Schierle GS, Kumita JR et al (2013) Protein amyloids develop an intrinsic fluorescence signature during aggregation. Analyst 138:2156–2162PubMedCrossRefGoogle Scholar
  9. 9.
    Heilemann M, van de Linde S, Schüttpelz M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172–6176PubMedCrossRefGoogle Scholar
  10. 10.
    Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782PubMedCrossRefGoogle Scholar
  11. 11.
    Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645PubMedCrossRefGoogle Scholar
  13. 13.
    Kaminski Schierle GS, van de Linde S, Erdelyi M et al (2011) In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging. J Am Chem Soc 133:12902–12905PubMedCrossRefGoogle Scholar
  14. 14.
    Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5:159–161PubMedCrossRefGoogle Scholar
  15. 15.
    Esbjörner EK, Chan F, Rees EJ, Erdelyi M, Luheshi LM, Bertoncini CW, Kaminski CF, Dobson CM, Kaminski-Schierle GS (2014) Direct observations of the formation of amyloid β self-assembly in live cells provide insights into differences in the kinetics of Aβ(1–40) and Aβ(1–42) aggregation. Chem Biol 21 (6): 732–742Google Scholar
  16. 16.
    Fritschi SK, Langer F, Kaeser SA, Maia LF, Portelius E, Pinotsi D, Kaminski CF, Winkler DT, Maetzler W, Keyvani K, Spitzer P, Wiltfang J, Kaminski Schierle GS, Zetterberg H, Staufenbiel M, Jucker M (2014) Highly potent soluble amyloid-β seeds in human Alzheimer brain but not cerebrospinal fluid. Brain 137(11):2909–2915Google Scholar
  17. 17.
    Pinotsi D, Buell AK, Galvagnion C et al (2014) Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy. Nano Lett 14:339–345PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Michel CH, Kumar S, Pinotsi D et al (2014) Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology. J Biol Chem 289:956–967PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Frank JH, Elder AD, Swartling J et al (2007) A white light confocal microscope for spectrally resolved multidimensional imaging. J Microsc 227:203–215PubMedCrossRefGoogle Scholar
  20. 20.
    van Ham TJ, Esposito A, Kumita JR et al (2010) Towards multiparametric fluorescent imaging of amyloid formation: studies of a YFP model of alpha-synuclein aggregation. J Mol Biol 395:627–642PubMedCrossRefGoogle Scholar
  21. 21.
    Murakami T, Yang SP, Xie L et al (2012) ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism. Hum Mol Genet 21:1–9PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Erdelyi M, Rees E, Metcalf D et al (2013) Correcting chromatic offset in multicolor super-resolution localization microscopy. Opt Express 21:10978–10988PubMedCrossRefGoogle Scholar
  23. 23.
    Rees EJ, Erdelyi M, Pinotsi D et al (2012) Blind assessment of localisation microscope image resolution. Opt Nanoscopy 1:12CrossRefGoogle Scholar
  24. 24.
    Wolter S, Löschberger A, Holm T et al (2012) rapidSTORM: accurate, fast open-source software for localization microscopy. Nat Methods 9:1040–1041PubMedCrossRefGoogle Scholar
  25. 25.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Dorothea Pinotsi
    • 1
  • Gabriele S. Kaminski Schierle
    • 1
  • Clemens F. Kaminski
    • 1
  1. 1.Department of Chemical Engineering and BiotechnologyUniversity of CambridgeCambridgeUK

Personalised recommendations