Abstract
Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm−1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm−1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm−1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.
Highlights
Coherent anti-Stokes Raman scattering (CARS) microscopy can be applied in living cells and provides vibrational contrast at a speed that is orders of magnitude higher than conventional Raman microscopy [1,2]
This is a novel approach for protein structure evaluation in vivo, the various steps required are illustrated in figure 1
Our strategy for the detection and identification of polyQ aggregates in vivo by CARS microscopy relies upon effective characterisation of similar polyQ aggregates in vitro
Summary
Coherent anti-Stokes Raman scattering (CARS) microscopy can be applied in living cells and provides vibrational contrast at a speed that is orders of magnitude higher than conventional Raman microscopy [1,2]. With a knowledge of the potential CARS spectrum, it is possible to establish the optimal vibrational frequency for imaging using our single frequency CARS microscopy method where the aim is to evaluate the structure of the aggregates of polyglutamine in vivo.
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