Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

“…For each spherulite, the spectrum is mainly characterised by two large peaks centered at 1620 cm -1 and one at 1670 cm -1 , respectively. In line with previous reports [49][50][51][52] , the 1620 cm -1 peak is ascribable to the aggregated -sheets characteristic of the amyloid structure, while the other broad peak can arise from the presence of native and aggregate structures including -helices, turns and loops (1660 cm -1 ) and antiparallel aggregated -sheets (1670-1680 cm -1 ) [49][50][51][52] .…”

“…The phasor plots corresponding to these measurements are shown in Figure 5b and 5f together with magnifications of relevant areas (Figure 5c and 5g). Phasors lie inside the universal circle indicating that ThT lifetimes in these conditions are characterised by non-single exponential decay 49,50,56 . The presented analysis immediately reveals a large fluorescence lifetime distribution appearing as a cloud of points extended along a straight line in the phasor plot (see the orange dashed line in Figure 5c and 5g).…”

“…FLIM measurements reported in the following are analysed using the phasor approach 56,75 . This method provides a global view of fluorescence molecules decays at each pixel in the images not imposing any specific model as required by fitting procedures 49,56,75,76 . When using phasor analysis each pixel of the intensity images is mapped to a point in the phasor plot corresponding to the measured fluorescence lifetime.…”

“…When using phasor analysis each pixel of the intensity images is mapped to a point in the phasor plot corresponding to the measured fluorescence lifetime. Single exponential lifetimes lie on the socalled "universal circle"; long lifetimes are located near the origin (0 on the x axis), while short lifetimes are shifted on the circumference toward the bottom right intersection with the x axis (1 on the x axis) 49,56 .…”

“…Finally, extracting information about the mesoscopic physical parameters as viscosity and order of the structure with high spatial resolution may require time-consuming processing of the data. In this context, advanced optical microscopy methods have already shown their potential as they give the possibility to analyze spatially heterogeneous samples, 48,49 reveal intrinsic features of aggregates 50,51 and separate multiple species occurring in protein aggregation 52,53 .…”