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M-BLANK: a program for the fitting of X-ray fluorescence spectra.

ABSTRACT: The X-ray fluorescence data from X-ray microprobe and nanoprobe measurements must be fitted to obtain reliable elemental maps. The most common approach in many fitting programs is to initially remove a per-pixel baseline. Using X-ray fluorescence data of yeast and glial cells, it is shown that per-pixel baselines can result in significant, systematic errors in quantitation and that significantly improved data can be obtained by calculating an average blank spectrum and subtracting this from each pixel.

SUBMITTER: Crawford AM

PROVIDER: S-EPMC6412182 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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M-BLANK: a program for the fitting of X-ray fluorescence spectra.

Crawford Andrew M AM Deb Aniruddha A Penner-Hahn James E JE

Journal of synchrotron radiation 20190219 Pt 2

The X-ray fluorescence data from X-ray microprobe and nanoprobe measurements must be fitted to obtain reliable elemental maps. The most common approach in many fitting programs is to initially remove a per-pixel baseline. Using X-ray fluorescence data of yeast and glial cells, it is shown that per-pixel baselines can result in significant, systematic errors in quantitation and that significantly improved data can be obtained by calculating an average blank spectrum and subtracting this from each ...[more]

PMID: 30855260

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Project description:Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

Dataset Information

M-BLANK: a program for the fitting of X-ray fluorescence spectra.

Publications

M-BLANK: a program for the fitting of X-ray fluorescence spectra.

Similar Datasets

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