Fluorescence Quantum Yield of Thioflavin T in Rigid Isotropic Solution and Incorporated into the Amyloid Fibrils

Abstract

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils.

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

“…5). This finding indicated that an increase in the ThT emission The observed emission enhancement phenomenon, induced by the endospore uptake, is consistent with the sensitivity of the photophysical properties of ThT to the viscosity of the environment: i.e., the emission quantum yield of ThT increases with the increase in the medium viscosity (72,74,81,82,88,90). Due to the single carbon-carbon bond between the two ring systems and the single carbon-nitrogen bond between the phenyl ring and the dimethyl amine (Fig.…”

“…7). As previously reported, an increase in the medium viscosity causes an increase in the ThT fluorescence quantum yield (82), which is expected given the properties of chromophores, such as ThT, classified as molecular rotors (4,81). A viscosity-induced increase in the emission quantum yield of ThT amounts to about three orders of magnitude (82).…”

“…Similar to other chromophores that are molecular rotors (3,4,28,39) within the lifetime of the lowest singlet-excited state of the dye, a viscous microenvironment slows and/or completely restrains the attainment of ThT conformers (i.e., rotamers) that have the density of vibrational states allowing efficient internal conversion to the ground state. Such viscosity-induced suppression of efficient nonradiative deactivation results in an increase in the emission quantum yield of ThT, as demonstrated by photophysical studies of this staining agent for solvent media with differing viscosities (82). Binding of ThT to protein aggregates (or other macromolecular assemblies) has a restrictive effect on its modes of molecular motion that is similar to the effect of increased medium viscosity: i.e., the binding sites provide a microenvironment with relatively high effective viscosity and cause an increase in the fluorescence quantum yield of ThT (82).…”

“…Such viscosity-induced suppression of efficient nonradiative deactivation results in an increase in the emission quantum yield of ThT, as demonstrated by photophysical studies of this staining agent for solvent media with differing viscosities (82). Binding of ThT to protein aggregates (or other macromolecular assemblies) has a restrictive effect on its modes of molecular motion that is similar to the effect of increased medium viscosity: i.e., the binding sites provide a microenvironment with relatively high effective viscosity and cause an increase in the fluorescence quantum yield of ThT (82).…”

Nanophotonics of protein amyloids

“…Recently, we have performed super-resolution imaging of fluorescently-stained amyloid fibrils derived from the low pH form of β 2 -microglobulin that is known to be associated with dialysis-related amyloidosis [43]. In our NSOM experiments, we used Nile Red as the fluorescent marker rather than more commonly used ThT due to the following reasons: (i) ThT has also been shown to bind to amorphous protein aggregates owing to the presence of inherent charge [44], (ii) ThT assay at neutral pH cannot be performed to detect amyloid fibrils that are formed at low pH and disintegrate at neutral pH [45], (iii) the low quantum yield of ThT prevents it from being utilized in single-molecule/particle imaging experiments [46] and (iv) ThT exhibits an emission maximum at ~482 nm upon binding to amyloid fibrils and hence, commonly used laser lines such as 488 and 532 nm cannot be used as excitation wavelengths. All these problems can be circumvented if Nile Red is used [47].…”