Fluorescence absorbance inner-filter decomposition: the role of emission shape on estimates of free Ca(2+) using Rhod-2.
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(50) and therefore improved the accuracy of free [Ca(2+)] 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.
Territo
Paul R
PR
Lilly Center for Molecular and Anatomical Imaging, Lilly Research Laboratories, Eli Lilly and Company, 2001 West Main Street, B220/GL54, Greenfield, Indiana 46140, USA. territop@lilly.com
Heil
Jeremy
J
Bose
Salil
S
Evans
Frank J
FJ
Balaban
Robert S
RS
D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry; D066298:In Vitro Techniques; D016018:Least-Squares Analysis; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D009010:Monte Carlo Method; D017711:Nonlinear Dynamics; D013050:Spectrometry, Fluorescence; D013552:Swine
D006575:Heterocyclic Compounds, 3-Ring; C068483:rhod-2; D002118:Calcium; D019793:Fluorescein