Bleaching correction for DNA-measurements in highly diluted solutions using confocal microscopy

Abstract

Precise and reliable determination of the nucleic acid concentration in biological samples still remains a challenge. This is particularly since the established fluorescene-based methods provide insufficient results, when only minute sample quantitites are available for analysis. Among other effects, photobleaching is the main reason for this. Since large molecules diffuse more slowly than small molecules, they are exposed to more excitation cycles and therefore have a higher probability of permanently losing their fluorescence. Solutions with large molecules hence show a reduced fluorescence. In this paper we present a method to correct this effect and thus allow high-precision sample concentration determination in minute sample quantities (< 2 {micro}l drops with concentrations < 20 pg/{micro}l). For this purpose, we used confocal microscopy with single molecule sensitivity. In the first step, we derived calibration curves from DNA solutions with defined fragment length. We analyzed dilution series over a wide concentration range (1 pg/{micro}l - 1000 pg/{micro}l) and measured their specific diffusion coefficients by employing fluorescence correlation spectroscopy. Using this information, we correct the measured fluorescence intensity of the calibration solutions for photobleaching effects. Subsequently, we evaluated our method by analyzing a series of DNA mixtures of varying composition.