Phasor‐FLIM analysis of Thioflavin T self‐quenching in Concanavalin amyloid fibrils

The formation of amyloid structures has traditionally been related to human neurodegenerative pathologies and, in recent years, the interest in these highly stable nanostructures was extended to biomaterial sciences. A common method to monitor amyloid growth is the analysis of Thioflavin T fluorescence. The use of this highly selective dye, diffused worldwide, allows mechanistic studies of supramolecular assemblies also giving back important insight on the structure of these aggregates. Here we present experimental evidence of self‐quenching effect of Thioflavin T in presence of amyloid fibrils. A significant reduction of fluorescence lifetime of this dye which is not related to the properties of analyzed amyloid structures is found. This result is achieved by coupling Fluorescence Lifetime Imaging Microscopy with phasor approach as suitable model‐free methods and constitute a serious warning that have to be taken in account if is dye is used for quantitative studies. Complex fluorescence decay of Thioflavin T, bound to amyloids, can be readily analyzed by phasor approach without modelling it. Thioflavin T self‐quenches, in presence of amyloid fibrils, as its molar concentration increases. Changes in ThT lifetime represents a serious warning when attempting quantification and comparison between samples in different conditions.