Using Ab-Space to Remove Background Components from Images in Systems of Multiple Fluorophores

Abstract

The linearization of the mixing of fluorescence intensity afforded by the AB-space formalism, simplifies a number of Frequency domain lifetime imaging tasks. Biological systems often consist of multiple components due to the a presence of Donor, Donor-Acceptor complex, and fluorescent background. Tools allowing a third component to be removed from lifetime imaging data would represent a significant advance. We will describe a simplified treatment for resolving binary mixtures and a novel approach to ternary and higher mixtures using frequency domain procedures. For binary and ternary mixtures there is no requirement for single exponential decay, meaning that each component can represent a multi-fluorophore mix. For many applications in biology, resolution of the fractional fluorescence contributions from the donor and donor-acceptor components is desirable as this allows activation and related parameters to be observed. From frequency domain data, this can be done in a straightforward fashion without computing lifetimes by using the linear mixing characteristics of the AB-coordinate system. We present the theory and demonstrate the approach using solutions and apply it to a simple biological system. Ternary mixtures work well using the technique, however, the advantages of additional frequencies is limited. We have applied the formalism to A431 cells labelled with quantum dots (QDs) which have three components: QDs, cellular autofluorescence and plate background. The method allowed us to strip the autofluoresence and plate background image leaving only the QDs.