Fluorescence absorbance inner-filter decomposition: The role of emission shape on estimates of free Ca2+ using Rhod-2

Paul R. Territo, Jeremy Heil, Salil Bose, Frank J. Evans, Robert S. Balaban

Research output: Contribution to journalArticle

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)138-147
Number of pages10
JournalApplied Spectroscopy
Volume61
Issue number2
DOIs
StatePublished - Feb 2007
Externally publishedYes

Keywords

  • Absorbance
  • Algorithms
  • Calcium
  • Fluorescence
  • Inner filter
  • Rhod-2
  • Spectral decomposition

ASJC Scopus subject areas

  • Instrumentation
  • Spectroscopy

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