Direct observation of amyloid growth monitored by total internal reflection fluorescence microscopy.
Most morphological investigations of amyloid fibrils have been performed with various microscopic methods. Among them, direct observation of fibril growth is possible using atomic force microscopy and fluorescence microscopy. Direct observation provides information about the rate and direction of growth at the single fibril level, which cannot be obtained from averaged ensemble measurements. In this chapter, we describe a new technique for the direct observation of amyloid fibril growth using total internal reflection fluorescence microscopy (TIRFM) combined with amyloid-specific thioflavin T (ThT) fluorescence. TIRFM has been developed to monitor single molecules by effectively reducing the background fluorescence in an evanescent field. One of the advantages of TIRFM is that one can selectively monitor fibrils lying along a glass slide, so that one can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent fibril growth of amyloid beta (1-40). The fibril growth was a highly cooperative process, with the fibril ends extending at a constant rate. Because ThT binding is common to all amyloid fibrils, the present method will have general applicability to the real-time analysis of amyloid fibrils.