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 K<sub>50</sub> and therefore improved the accuracy of free [Ca<sup>2+</sup>] 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.
Vol. 2, Iss. 3 Virtual Journal for Biomedical Optics
Paul R. Territo, Jeremy Heil, Salil Bose, Frank J. Evans, and Robert S. Balaban, "Fluorescence Absorbance Inner-Filter Decomposition: The Role of Emission Shape on Estimates of Free Ca2+ Using Rhod-2," Appl. Spectrosc. 61, 138-147 (2007)