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Erschienen in:
2015 | OriginalPaper | Buchkapitel
Pattern-Based Linear Unmixing for Efficient and Reliable Analysis of Multicomponent TCSPC Data
verfasst von : Ingo Gregor, Matthias Patting
Erschienen in: Advanced Photon Counting
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Abstract
A method for a reliable quantitative analysis of fluorescence lifetime imaging microscopy (FLIM) data is presented. It is based on the linear unmixing of the intensity decay on the basis of selected reference patterns. This approach allows to use decays that are not mono-exponential without increasing the complexity of the analysis. This is a major benefit when working with labeled biomolecules or using autofluorescent cellular chromophores.
The method can be used intuitively and is fast. Furthermore, based on the reference patterns and the amount of recorded photons, one can easily determine confidence levels of the obtained results. We demonstrate that for a decomposition to three patterns of common chromophores, one achieves a standard deviation of better than 10% for as few as 1,000 photons per pixel, where the total amplitude of such a signal will show an error of 3% due to shot noise. Indeed, the accuracy of the results is very close to a maximum-likelihood estimator that defines the absolute limit for this kind of problem.