Abstract
The optical properties of amyloid fibers are often distinct from those of the source protein in its non-fibrillar form. These differences can be utilized for label-free imaging or characterization of such structures, which is particularly important for understanding amyloid fiber related diseases such as Alzheimer's and Parkinson's disease. We demonstrate that two amyloid forming proteins, insulin and β-lactoglobulin (β-LG), show intrinsic fluorescence with emission spectra that are dependent on the excitation wavelength. Additionally, a new fluorescence peak at about 430 nm emerges for β-LG in its amyloid state. The shift in emission wavelength is related to the red edge excitation shift (REES), whereas the additional fluorescence peak is likely associated with charge delocalization along the fiber backbone. Furthermore, the spherulitic amyloid plaque-like superstructures formed from the respective proteins were imaged label-free with confocal fluorescence, multiphoton excitation fluorescence (MPEF), and second-harmonic generation (SHG) microscopy. The latter two techniques in particular yield images with a high contrast between the amyloid fiber regions and the core of amorphously structured protein. Strong multiphoton absorption (MPA) for the amyloid fibers is a likely contributor to the observed contrast in the MPEF images. The crystalline fibrillar region provides even higher contrast in the SHG images, due to the inherently ordered non-centrosymmetric structure of the fibers together with their non-isotropic arrangement. Finally, we show that MPEF from the insulin spherulites exhibits a spectral dependence on the excitation wavelength. This behavior is consistent with the REES phenomenon, which we hypothesize is the origin of this observation. The presented results suggest that amyloid deposits can be identified and structurally characterized based on their intrinsic optical properties, which is important for probe-less and label-free identification and characterization of amyloid fibers in vitro and in complex biological samples.
Highlights
There are many proteins that misfold and aggregate into amyloid-like fibrillar structures upon mild denaturation. [1, 2] Such fibers vary in size, but are typically 5 to 15 nm in diameter, have typical persistence lengths in the μm regime and are chemically and mechanically stable. [3,4,5] This family of fibers has backbones of intermolecular β-sheets that run along the fiber axis. [6] Amyloid fibers are found in a wide range of severe conditions, many of which are becoming increasingly prevalent
Most studies of amyloid structures by multiphoton excitation fluorescence (MPEF) and secondharmonic generation (SHG) imaging have relied on external probes, [52,53,54,55,56,57] while only a few have been label-free. [58,59,60] In this paper, we show that these techniques produce images with high contrast for the amyloids
The ability to image amyloid spherulites with confocal fluorescence, MPEF, and second-harmonic generation (SHG) microscopy without the use of any probes or labels was demonstrated
Summary
There are many proteins that misfold and aggregate into amyloid-like fibrillar structures upon mild denaturation. [1, 2] Such fibers vary in size, but are typically 5 to 15 nm in diameter, have typical persistence lengths in the μm regime and are chemically and mechanically stable. [3,4,5] This family of fibers has backbones of intermolecular β-sheets that run along the fiber axis. [6] Amyloid fibers are found in a wide range of severe conditions, many of which are becoming increasingly prevalent. [7,8,9,10,11] Other examples of amyloid-forming proteins are insulin and β-lactoglobulin (β-LG), [12,13,14] which to date have not been shown to induce degenerative conditions. As a result, these amyloids are well-suited to be used in basic research that aims to develop strategies for identification and characterization of amyloid structures. The assembly process depends on several factors, such as the protein concentration, temperature, pH and ion concentrations, and has been described elsewhere. [43,44,45,46,47,48,49,50,51] The microscopy efforts in this work focused on spherulites prepared from these proteins and the intrinsic optical properties of these amyloid structures were utilized in label-free imaging applications
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