Fluorescence quenching data interpretation in biological systems. The use of microscopic models for data analysis and...

In micro-heterogeneous media (e.g. membranes, micelles and colloidal systems), the fluorescence decay in the absence of quencher is usually intrinsically complex, e.g. due to the existence of several sub-populations with different micro-environments. In this case it is impossible to analyze data in detail (accounting for transient effects) and simpler formalisms are needed. The objective of the present work is to present and discuss such simpler formalisms. The goal is to achieve simple data analysis and meaningful, clear data interpretation in complex systems using microscopic models that consider several sub-populations of chromophores. Two points are dealt with in detail. (i) It is shown that the approximation of the transient effects by the quenching sphere-of-action model is not always possible. The quenching sphere-of-action concept can be regarded as a valuable tool, although crude, only in a limited range of experimental conditions, namely time resolution. (ii) The Stern-Volmer equation usually used for data analysis is only valid for a limited range of small and moderate equilibrium association constants, Ka, although this is frequently overlooked in the literature. Self-consistency criteria are presented for the proposed methods. The well-known downward curvature due to a fraction of fluorophores which is not accessible to the quencher is only a limiting case from a set of possible situations which result in deviations to linearity. A systematic classification of the different types of quenching is presented.