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

Abstract

Determining the concentration of nucleic acids in biological samples precisely and reliably still is a challenge. In particular when only very small sample quantities are available for analysis, the established fluorescence-based methods give insufficient results. Photobleaching is seen as the main reason for this. In this paper we present a method to correct for the photobleaching effect. Using confocal microscopy with single molecule sensitivity, we derived calibration curves from DNA solutions with defined fragment length. We analyzed dilution series over a wide range of concentrations (1 pg/μl-1000 pg/μl) and measured their specific diffusion coefficients employing fluorescence correlation spectroscopy. Using this information, we corrected the measured fluorescence intensity of the calibration solutions for photobleaching effects. We evaluated our method by analyzing a series of DNA mixtures of varying composition. For fragments smaller than 1000 bp, our method allows to determine sample concentrations with high precision in very small sample quantities (< 2 μl with concentrations < 20 pg/μl). Once the technical parameters are determined and remain stable in an established process, our improved calibration method will make measuring molecular biological samples of unknown sequence composition more efficient, accurate and sample-saving than previous methods.

The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition

“…The objective lens used in the setup is a long-distance air lens instead of an oil or water immersion lens. We have shown the feasibility of using a long-distance lens for FFS experiments in other study [30]. The main advantage is that the setup is less costly and easier to automate.…”

“…Equal volumes of DNA solution and 0.6x dye solution are mixed and equilibrated for 2h. RiboGreen stains all types of nucleic acids and has been shown in pre-tests to be very bright and to yield accurate results [30].…”

“…The deviation from an ideal straight line is presumably due to size effects of the DNA molecules when traversing the detection volume. However, despite the low excitation powers, increasing photon bleaching effects for larger molecules cannot ruled out either [30]. Subplot C shows the average diffusion times of the dilution series.…”