Classification of fluorescent anisotropy decay based on the distance approach in the frequency domain.

Frequency-domain (FD) fluorometry is a widely utilized tool to probe unique features of complex biological structures, which may serve medical diagnostic purposes. The conventional data analysis approaches used today to extract the fluorescence intensity or fluorescence anisotropy (FA) decay data suffer from several drawbacks and are inherently limited by the characteristics and complexity of the decay models. This paper presents the squared distance (D) technique, which categorized samples based on the direct frequency response data (FRD) of the FA decay. As such, it improves the classification ability of the FD measurements of the FA decay as it avoids any distortion that results from the challenged translation into time domain data. This paper discusses the potential use of the D approach to classify biological systems. Mathematical formulation of D technique adjusted to the FRD of the FA decay is described. In addition, it validates the D approach using 2 simulated data sets of 6 groups with similar widely and closely spaced FA decay data as well as in experimental data of 4 samples of a fluorophore-solvent (fluorescein-glycerol) system. In the simulations, the classification accuracy was above 95% for all 6 groups. In the experimental data, the classification accuracy was 100%. The D approach can help classify samples whose FA decay data are difficult to extract making FA in the FD a realistic diagnostic tool. The D approach offers an advanced method for sorting biological samples with differences beyond the practical temporal resolution limit in a reliable and efficient manner based on the FRD of their time-resolved fluorescence measurements thereby achieving better diagnostic quality in a shorter time.