Ultrafast decay (A) Chemical scheme and fibrils of phenylalanine (macroscopic and microscopic photos). (B) Fluorescence emission of phenylalanine assemblies at excitations between 370 and 420 nm. (C) Autofluorescence lifetime imaging of phenylalanine fibrils. Excitation was performed in a two-photon regime at 700 nm. (D) Fluorescence decay curve of the phenylalanine fibrils measured with (sub)nanosecond resolution. (E) Fluorescence decay curve of phenylalanine fibrils measured using fluorescence up-conversion technique. Excitation and emission were set to 380 and 450 nm, respectively. The inset shows the parameters of fluorescence decay obtained using the biexponential decay model (D and E). (F) Schematic decrease in the energy gap between the excited np* and ground state of the model fibrils. (G) Scheme of the shift of the aggregation-induced fluorescence spectrum with time, demonstrating the presence of the ultrafast decay and spectral migration.

Ultrafast decay (A) Chemical scheme and fibrils of phenylalanine (macroscopic and microscopic photos). (B) Fluorescence emission of phenylalanine assemblies at excitations between 370 and 420 nm. (C) Autofluorescence lifetime imaging of phenylalanine fibrils. Excitation was performed in a two-photon regime at 700 nm. (D) Fluorescence decay curve of the phenylalanine fibrils measured with (sub)nanosecond resolution. (E) Fluorescence decay curve of phenylalanine fibrils measured using fluorescence up-conversion technique. Excitation and emission were set to 380 and 450 nm, respectively. The inset shows the parameters of fluorescence decay obtained using the biexponential decay model (D and E). (F) Schematic decrease in the energy gap between the excited np* and ground state of the model fibrils. (G) Scheme of the shift of the aggregation-induced fluorescence spectrum with time, demonstrating the presence of the ultrafast decay and spectral migration.

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Biomolecule luminescence in the visible range of the spectrum has been experimentally observed upon aggregation, contrary to their monomeric state. However, the physical basis for this phenomenon is still elusive. Here, we systematically examine all coded amino acids to provide non-biased empirical insights. Several amino acids, including non-aroma...

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... self-assembly of phenylalanine in aqueous solution was initiated by cooling a supersaturated solution of phenylalanine (40 mg/ml) at 90 C to 20 C, similar to previous work (Shaham-Niv et al., 2018). This leads to the formation of elongated fibrillary aggregates, as shown by bright-field microscopy ( Figure 4A). The phenylalanine fibrils exhibited relatively high fluorescence emission in the visible spectral range, which is absent in the monomeric state of phenylalanine, and were characterized by the wavelength-dependent Stokes shift, i.e., the spectra measured at longer excitation wavelength exhibited red-shifted emission (Fig- ure 4B). ...
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... leads to the formation of elongated fibrillary aggregates, as shown by bright-field microscopy ( Figure 4A). The phenylalanine fibrils exhibited relatively high fluorescence emission in the visible spectral range, which is absent in the monomeric state of phenylalanine, and were characterized by the wavelength-dependent Stokes shift, i.e., the spectra measured at longer excitation wavelength exhibited red-shifted emission (Fig- ure 4B). Such a behavior of the fluorescence emission band is known as the red-edge excitation shift effect and is characteristic for the self-assembly-induced fluorescence of amino acids, peptides, and proteins (Arnon et al., Berger et al., 2015;Kumar et al., 2020). ...
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... a behavior of the fluorescence emission band is known as the red-edge excitation shift effect and is characteristic for the self-assembly-induced fluorescence of amino acids, peptides, and proteins (Arnon et al., Berger et al., 2015;Kumar et al., 2020). The presence of elongated fibrillar structures was detectable by fluorescence lifetime imaging, without the use of any external dyes, only by using the autofluorescence signal of the sample ( Figure 4C). The autofluorescence decay curve obtained for the phenylalanine fibrils revealed the presence of 0.35-and 2.53-ns decay times after fitting to a biexponential model ( Figure 4D). ...
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... presence of elongated fibrillar structures was detectable by fluorescence lifetime imaging, without the use of any external dyes, only by using the autofluorescence signal of the sample ( Figure 4C). The autofluorescence decay curve obtained for the phenylalanine fibrils revealed the presence of 0.35-and 2.53-ns decay times after fitting to a biexponential model ( Figure 4D). However, the temporal resolution, or the width of the instrument response function (IRF) of the fluorescence lifetime imaging setup, which employs the standard time-correlated single photon counting technique, is $50 ps and does not allow for detecting any ultrafast decay components, which will be completely masked by the 50-ps IRF ( Rovnyagina et al, 2019Rovnyagina et al, , 2020. ...
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... fluorescence up-conversion, it was observed that the phenylalanine fibrils exhibited ultrafast decay lifetime component as fast as 0.67 ps ( Figure 4E). The second decay component was characterized by a 12.6 ps decay. ...
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... the range of 15-40 fs after excitation UV fluorescence from S 1 state is observed and is peaked at 3.3 eV ($370 nm). After that, on the timescale of $100-200 fs, internal vibration redistribution occurs that leads to additional redshift of the emission spectrum, down to 2.0 eV (620 nm) ( Figure 4F). This evolution occurs on the timescale of $100 fs and is accompanied by the gradual shift of the fluorescence emission from the UV to the blue-green region ( Figure 4G). ...
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... that, on the timescale of $100-200 fs, internal vibration redistribution occurs that leads to additional redshift of the emission spectrum, down to 2.0 eV (620 nm) ( Figure 4F). This evolution occurs on the timescale of $100 fs and is accompanied by the gradual shift of the fluorescence emission from the UV to the blue-green region ( Figure 4G). By setting the registration emission wavelength to the blue-green region (in our case, to 500 nm [$2.5 eV]), the ultrafast relaxation along the potential curve shown in Figure 4F can be detected. ...
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... evolution occurs on the timescale of $100 fs and is accompanied by the gradual shift of the fluorescence emission from the UV to the blue-green region ( Figure 4G). By setting the registration emission wavelength to the blue-green region (in our case, to 500 nm [$2.5 eV]), the ultrafast relaxation along the potential curve shown in Figure 4F can be detected. This process is illustrated in the inset of Figure 4G and corresponds to the fluorescence decay curve presented in Figure 4E. ...
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... setting the registration emission wavelength to the blue-green region (in our case, to 500 nm [$2.5 eV]), the ultrafast relaxation along the potential curve shown in Figure 4F can be detected. This process is illustrated in the inset of Figure 4G and corresponds to the fluorescence decay curve presented in Figure 4E. The ultrafast relaxation occurs on the timescale of $100 fs, corresponding to the non-radiative decay rate of $10 12 -10 13 s À1 , which is, however, lower than in the case of monomeric proteins, where the lack of stabilization of the np* states results in the absence of near-UV and visible absorption and blue-green fluorescence ( Grisanti et al., 2020). ...
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... setting the registration emission wavelength to the blue-green region (in our case, to 500 nm [$2.5 eV]), the ultrafast relaxation along the potential curve shown in Figure 4F can be detected. This process is illustrated in the inset of Figure 4G and corresponds to the fluorescence decay curve presented in Figure 4E. The ultrafast relaxation occurs on the timescale of $100 fs, corresponding to the non-radiative decay rate of $10 12 -10 13 s À1 , which is, however, lower than in the case of monomeric proteins, where the lack of stabilization of the np* states results in the absence of near-UV and visible absorption and blue-green fluorescence ( Grisanti et al., 2020). ...
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... the presence of the ultrafast decay may result in low fluorescence quantum yield of the aggregation-induced emission of proteins ($0.01) ( Niyangoda et al., 2017). Further relaxation from the lowest energy np* min state may then occur on a longer timescale owing to the stabilization of the geometry of the aggregate, and this relaxation corresponds to the 1 to 2-ns fluorescence lifetime, which was observed for the self-assembly-induced fluorescence in this paper ( Figure 4D) and other previous works ( Tikhonova et al., 2018). Hence, by detecting the ultrafast decay of the fibrillar structures formed as the result of phenylalanine self-assembly, the model of Grisanti et al. can be extended from peptides to amino acids, therefore elucidating the origin of their enigmatic fluorescence emission upon packing ( Grisanti et al., 2020). ...

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