Atomic force fluorescence microscopy in the characterization of amyloid fibril assembly and oligomeric intermediates.

Atomic force microscopy (AFM) has become a conventional tool for elucidation of the molecular mechanisms of protein aggregation and, specifically, for analysis of assembly pathways, architecture, aggregation state, and heterogeneity of oligomeric intermediates or mature fibrils. AFM imaging provides useful information about particle dimensions, shape, and substructure with nanometer resolution. Conventional AFM methods have been very helpful in the analysis of polymorphic assemblies formed in vitro from homogeneous proteins or peptides. However, AFM imaging on its own provides limited insight into conformation or composition of assemblies produced in the complex environment of a cell, or prepared from a mixture of proteins as a result of cross-seeding. In these cases, its combination with fluorescence microscopy (AFFM) increases its resolution.