Abstract
Single-molecule FRET (smFRET) spectroscopy is a powerful tool for studying inhomogeneous dynamics in biological systems. However, because of the intrinsic variations that accompany the sample sizes, massive data sets are essential to extract statistically reliable information. In this aspect, a simple motorized stage and autofocusing modification can save time without the expense of a high-end automated microscope. In this report, we describe a simple and economical modification of a commercial inverted microscope with a manual stage to automate the data acquisition and measurement process. We collected 8000 images with a 100 ms exposure time in 1000 fields of view in approximately 13 min, where it would take more than 8 h by manual collection. We demonstrated the method with a DNA oligo quantification experiment. In this experiment, the measurement platform is a FRET signal from a dye-labeled DNA duplex containing unmatched base pairs. The target DNA replaces one of the strands because of the formation of a perfect duplex. This thermodynamically driven exchange reaction causes FRET to disappear, which correlated with the DNA concentration. The data are batch processed with the freeware ImageJ. These modifications are feasible and economical for general smFRET experiments.
Highlights
Single-molecule Forster resonance energy transfer spectroscopy measures the fluorescence intensities of two energy-tangled dye molecules at close spatial locations
The reconstruction of signals from individual biomolecules provides inhomogeneous population information and rare event dynamics that cannot be revealed with ensemble methods [1]
DNA strands of surface-bound duplexes, which were dissociated by the target oligo due to the energy levels. This dissociation decreased the FRET pair numbers, which were proportional to the concentration of the microRNA analog
Summary
Single-molecule Forster resonance energy transfer (smFRET) spectroscopy measures the fluorescence intensities of two energy-tangled dye molecules at close spatial locations. To demonstrate our automation system, we quantified a DNA oligo using the smFRET signal as the measurement platform. DNA strands of surface-bound duplexes, which were dissociated by the target oligo (microRNA let-7g’s DNA analog) due to the energy levels This dissociation decreased the FRET pair numbers, which were proportional to the concentration of the microRNA analog. A time as short as 30 s is enough to change the field of view for a skillful researcher, this step amounts to more than 8 h if 1000 videos are needed. To solve this problem, we implement an automotive stage-scanning setup. Our method provides an alternative assay to directly quantify nucleic acids
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