Chapter 2 - Amyloid Fibril Length Quantification by Atomic Force Microscopy

Abstract

The biologic properties of the highly structured protein aggregates known as amyloid fibrils are intimately related to their length. Detailed information regarding amyloid fibrils’ assembly mechanism, functional properties, disease association, and nano-mechanical characteristics can be gained from studies of their length distribution. Such statistical characterization of fibril length will contribute to the future development of improved therapies targeting amyloid-associated disease and novel amyloid-based nano-materials. Nano-scale microscopy visualization of amyloid fibrils allows direct measurement of the length of individual fibril particles deposited on the surface of substrates. However, quantitative characterization of the detailed length distributions of amyloid fibrils in bulk samples is far from straightforward. In this chapter, I describe a method, based on the analysis of atomic force microscopy images, which enables the quantitative characterization of amyloid fibril length distributions in bulk fibril samples. This is illustrated by using amyloid fibril samples generated in vitro from β2-microglobulin. I discuss the practical considerations in determining fibril length distribution, including sample preparation, image acquisition, fibril tracing, length-dependent bias correction, and distribution analysis.