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Extended wavelength anisotropy resolved multidimensional emission spectroscopy (ARMES) measurements: better filters, validation standards, and Rayleigh scatter removal methods
Casamayou-Boucau, Yannick; Ryder, Alan G.
Anisotropy resolved multidimensional emission spectroscopy (ARMES) provides valuable insights into multi-fluorophore proteins (Groza et al 2015 Anal. Chim. Acta 886 133-42). Fluorescence anisotropy adds to the multidimensional fluorescence dataset information about the physical size of the fluorophores and/or the rigidity of the surrounding micro-environment. The first ARMES studies used standard thin film polarizers (TFP) that had negligible transmission between 250 and 290 nm, preventing accurate measurement of intrinsic protein fluorescence from tyrosine and tryptophan. Replacing TFP with pairs of broad band wire grid polarizers enabled standard fluorescence spectrometers to accurately measure anisotropies between 250 and 300 nm, which was validated with solutions of perylene in the UV and Erythrosin B and Phloxine B in the visible. In all cases, anisotropies were accurate to better than +/- 1% when compared to literature measurements made with Glan Thompson or TFP polarizers. Better dual wire grid polarizer UV transmittance and the use of excitation-emission matrix measurements for ARMES required complete Rayleigh scatter elimination. This was achieved by chemometric modelling rather than classical interpolation, which enabled the acquisition of pure anisotropy patterns over wider spectral ranges. In combination, these three improvements permit the accurate implementation of ARMES for studying intrinsic protein fluorescence. The funders, Irish Research Council (IRC), had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors also acknowledge financial support from an IRC Postgraduate Scholarship (Grant #GOIPG/2015/3826). Agilent Technologies (Australia) are also thanked for a fluorimeter loan.
Keyword(s): fluorescence; anisotropy; multidimensional; standards; polarizers; Rayleigh scatter; chemometrics; MULTIVARIATE CURVE RESOLUTION; CELL-CULTURE MEDIA; FLUORESCENCE-SPECTRA; EEM SPECTROSCOPY; SERUM-ALBUMIN; RAMAN SCATTER; PARAFAC; MOLECULES; GLYCEROL; DECOMPOSITION
Publication Date:
2018
Type: Journal article
Peer-Reviewed: Yes
Language(s): English
Contributor(s): Irish Research Council
Institution: NUI Galway
Publisher(s): IOP Publishing
File Format(s): application/pdf
First Indexed: 2019-03-23 06:45:16 Last Updated: 2019-03-23 06:45:16