Methods for Kinetic Modeling of Temporally Resolved Hyperspectral Confocal Fluorescence Images

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Abstract:

Elucidating kinetic information (rate constants) from temporally resolved hyperspectral confocal fluorescence images offers some very important opportunities for the interpretation of spatially resolved hyperspectral confocal fluorescence images but also presents significant challenges, these being (1) the massive amount of data contained in a series of time-resolved spectral images (one time course of spectral data for each pixel) and (2) unknown concentrations of the reactants and products at time = 0, a necessary precondition normally required by traditional kinetic fitting approaches. This paper describes two methods for solving these problems: direct nonlinear (DNL) estimation of all parameters and separable least squares (SLS). The DNL method can be applied to reactions of any rate law, while the SLS method is restricted to first-order reactions. In SLS, the inherently linear and nonlinear parameters of first-order reactions are solved in separate linear and nonlinear steps, respectively. The new methods are demonstrated using simulated data sets and an experimental data set involving photobleaching of several fluorophores. This work demonstrates that both DNL and SLS hard-modeling methods applied to the kinetic modeling of temporally resolved hyperspectral images can outperform traditional soft-modeling and hard/soft-modeling methods which use multivariate curve resolution–alternating least squares (MCRALS) methods. In addition, the SLS method is much faster and is able to analyze much larger data sets than the DNL method.

Keywords: DIRECT NONLINEAR ESTIMATION; DNL; FLUORESCENCE IMAGING; HARD MODELING; HYPERSPECTRAL CONFOCAL MICROSCOPY; KINETIC MODELING; MCR; MULTIVARIATE CURVE RESOLUTION; PHOTOBLEACHING; SEPARABLE LEAST SQUARES; SLS; SOFT MODELING

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370209787391950

Affiliations: 1: Department of Chemistry, East Carolina University, Greenville, North Carolina 27858 2: Sandia National Laboratories, Albuquerque, New Mexico 87185-0895 3: Department of Mathematics and Pathology, University of New Mexico, Albuquerque, New Mexico 87131

Publication date: February 1, 2009

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