Nanoscale imaging and characterisation of Amyloid-β
Creators: name: Tinker-Mill, Claire
Publisher: name: Lancaster University
Year: 2015

description: In this work several novel Scanning Probe Microscopy (SPM) methods have
      been applied to the study of the amyloid peptide implicated in the
      pathogenesis of Alzheimer’s disease (AD). Amyloid-β (Aβ) undergoes a
      hierarchy of aggregation following a structural transition making it an
      ideal subject of studying with SPM.
      
      The application of SPM based techniques to biological samples has become
      increasingly common place. However, these techniques are not always
      immediately suitable for imaging delicate samples of proteins and
      adaptions must be made before imaging can be considered successful. AD is
      the most common form of dementia worldwide, and a growing concern for
      health authorities. As a result it has attracted the attention of a wide
      range of disciplines. There has been much work conducted which combines
      the main pathogenic peptide, Aβ , with Atomic Force Microscopy (AFM) in
      order to elucidate more about its aggregation behaviour, however these
      techniques offer little more than structural comments, with only the most
      advanced forms of cryo-Electron Microscopy (EM) providing more details on
      the nanoscale. Presented here is a method for reliably and robustly
      producing samples of Aβ by capturing them at various stages of
      aggregation, as well as the results of subsequent imaging by various
      methods of AFM. Each of the AFM techniques studied provides additional
      “added value” to the data which can typically be collected by AFM; either
      nanomechanical, elastic, thermal or spectroscopical.
      
      
      By imaging samples of Aβ with Ultrasonic Force Microscopy, a detailed
      substructure to the morphology could be seen, which correlates well with
      the most advanced cryo-EM work. In addition this technique was ideal for
      detecting the most toxic from of Aβ, early aggregates, in a sensitive and
      non-destructive fashion robustly differentiating them from the underlying
      layer of another peptide (poly-L-Lysine) that was designed to reliably
      capture the Aβ aggregates. Early work investigating the potential for
      combining an established method of thermal AFM with a mid-IR laser system
      also shows promise for detecting the response of the protein.
      
      
      It was also the focus of this work to study the aggregation of Aβ using
      Dynamic Light Scattering (DLS), in order to confirm whether the technique
      could identify differences between populations throughout the aggregation
      process. This was applied in conjunction with potential therapeutics which
      target the early aggregates to prevent their accumulation, as well as
      block formation of fibrils.
      
      Ultimately this work aims to shows with care to the initial protocols
      used, physical techniques such as AFM and DLS can be added to the existing
      methods of monitoring aggregation. Synergistic use of these techniques can
      generate a clearer overall picture of the effect of metal ions/developing
      therapeutics on Aβ aggregation and provide more detail than classical
      biological techniques alone.