Thioflavin T fluoresces as excimer in highly concentrated aqueous solutions and as monomer being incorporated in amyloid fibrils

Fluorescence of thioflavin T (ThT) is a proven tool for amyloid fibrils study. The correct model of ThT binding to fibrils is crucial to clarify amyloid fibrils structure and mechanism of their formation. Although there are convincing evidences that ThT has molecular rotor nature, implying it’s binding to fibrils in monomer form, speculations concerning ThT binding to fibrils in aggregated forms appear in literature so far. The elaborated approach for fluorescence intensity correction on the inner filter effects applied to ThT aqueous solutions with a wide range of concentration allowed characterizing ThT excimers fluorescence and showing its difference from that of ThT bound to fibrils. Obtained results experimentally prove the monomer model of ThT binding to amyloid fibrils and demonstrate wide capacity of the used approach in the spectroscopy of other fluorescent dyes for examination of concentration self-quenching and deformation of fluorescence spectra, dye molecules interaction, dimers and excimers formation.


Fluorescence intensity correction for the primary inner filter effect
The nonlinearity of the concentration dependence of the fluorescence intensity is caused by the so-called primary inner filter effect. The reasons for this effect include both the attenuation of the excitation light flux on its path through an absorbing solution (Beer-Lambert law) and the difference between the area that is illuminated by the excitation light and the working area from which the fluorescence light is gathered.
It is generally accepted that for low concentration solutions (low absorbance), the fluorescence intensity is proportional to the concentration of the fluorescence substance, and primary inner filter effects are negligible; however, this assumption is not valid. 1 In reality, the total fluorescence intensity is only proportional to the absorbance (A) at one point, when A = 0.
Even at A = 0.1, the deviation from linearity is 12%, and at A = 0.3, the deviation is 38%.
In an ideal case when the area illuminated by the excitation light coincides with the working area from which the fluorescence light, the recorded total fluorescence intensity where F(λ ex ,λ em ) is the fluorescence intensity that is excited at the wavelength λ ex and recorded at the wavelength  em , is proportional to the fraction of the excitation light that is absorbed by the ). If only one substance is responsible for the absorption and fluorescence of a solution, then: Here, is the intensity of the excitation light at  ex , k' is a proportionality factor, ex   is the spectral width of the slits of the monochromator in the excitation pathway, and  is a normalization factor determined using a standard (a fluorescent substance with known fluorescence quantum yield) at the same experimental conditions (i.e., slits widths, photomultiplier voltage, and other factors) that were used in the experiment with the sample. The coefficient k is chosen such that the total fluorescence intensity of the standard and the sample give physical meaning to the product of absorbance and the fluorescence quantum yield: The fluorescence excitation spectra recorded at 490 and 570 nm and corrected on the primary inner filter effect using the calculated coefficient W are shown in Fig. 2, Panels b and c.
Obviously, both fluorescence excitation spectra recorded at 490 nm and at 570 nm are close to the corresponding absorption spectrum. Therefore, there are reasons to suggest that the calculated correction factor W could be used over a wider range of absorbance, than it was shown for NATA solutions of different concentration. 1

Particularities of Cary Eclipse spectrofluorimeter
In most spectrofluorimeters, the detected fluorescence intensity is not proportional to the fraction of light that is absorbed by the solution. Thus, the correction factor cannot be calculated according to Eq. 4; instead, it must be determined experimentally. 1 Moreover, because the fluorescence intensity measured by these spectrofluorimeters decreases as the absorbance of the investigated solutions increases, the fluorescence of solutions with high absorbance (A > 5.0 for most spectrofluorimeters with a cell with an optical path length of 1 cm) cannot be recorded at all.
We showed experimentally 1 that the Cary Eclipse spectrofluorimeter enables recordings of fluorescent solutions with very high absorbance; in contrast to all known spectrofluorimeters, this spectrofluorimeter has horizontal slits (Fig. S1). 2 Using this spectrofluorimeter and rectangular cells with 10 mm optical path lengths in the present work, we recorded the fluorescence spectrum of a solution with absorbance A 412 =880 at an excitation wavelength of 412 nm. The unique feature of this spectrofluorimeter is that the area illuminated by the excitation light coincides with the working area from which the fluorescence light is gathered (Fig. S1).  . Figure S2. Fluorescence spectra of ThT in aqueous solutions with different absorbances (different concentrations) after correction for the primary and both primary and secondary inner filter effects. Left Panels. Fluorescence spectra corrected for primary inner filter effect. Right Panels. Fluorescence spectra corrected for both primary and secondary inner filter effects. Panels A-E show the fluorescence spectra of ThT with absorbance ranging from 0.1 to 1, from 2 to 10, from 20 to 50, from 60 to 90 and from 100 to 880, respectively, λ ex =412 nm. The dotted curves are the red edges of the absorption spectra. The spectral slits width was 10 nm.