Fluorescence Absorbance Inner-Filter Decomposition: The Role of Emission Shape on Estimates of Free Ca2+ Using Rhod-2

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Abstract:

A method for decomposing complex emission spectra by correcting for known inner-filter effects is described. This approach builds on previous work using a linear combination of model emission spectra and combines the known absorption characteristics of the system to fit the composite emission spectrum. Rhod-2, which has a small Stokes shift and significant self-absorption, was used as the model system. By adding the absorption characteristics of Rhod-2 to the model, the degree of fit was significantly improved, thus minimizing residuals, and accurately predicted the spectral shape changes with increasing concentration, [Rhod-2]. More complex studies were conducted with Rhod-2 in isolated cardiac mitochondria with multiple emission and absorption elements. By including known absorbances to the spectral decomposition, the overall precision increased almost four fold. Moreover, this approach eliminated the significant [Rhod-2] dependence on the apparent K50 and therefore improved the accuracy of free [Ca2+] calculations. These data demonstrate that secondary inner-filter correction can significantly improve spectral decomposition of complex emission spectra, which are used in a variety of biological applications.

Keywords: ABSORBANCE; ALGORITHMS; CALCIUM; FLUORESCENCE; INNER FILTER; RHOD-2; SPECTRAL DECOMPOSITION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370207779947530

Affiliations: 1: Lilly Center for Molecular and Anatomical Imaging, Lilly Research Laboratories, Eli Lilly and Company, 2001 West Main Street, B220/GL54, Greenfield, Indiana 46140; Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, Bldg. 10, Rm. B1D-416, Bethesda, Maryland 20892-1061 2: Lilly Center for Molecular and Anatomical Imaging, Lilly Research Laboratories, Eli Lilly and Company, 2001 West Main Street, B220/GL54, Greenfield, Indiana 46140; Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, Bldg. 10, Rm. B1D-416, Bethesda, Maryland 20892-1061 3: Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, Bldg. 10, Rm. B1D-416, Bethesda, Maryland 20892-1061

Publication date: February 1, 2007

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