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)

Abstract

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 

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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

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