Open AccessAutomated smFRET microscope for the quantification of label-free DNA oligos
Author(s) -
Ran Lin,
Yuhong Wang
Publication year - 2019
Publication title -
biomedical optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.10.000682
Subject(s) - förster resonance energy transfer , microscope , oligonucleotide , biological system , computer science , microscopy , dna , single molecule fret , fluorescence microscope , confocal , duplex (building) , nanotechnology , fluorescence , chemistry , materials science , optics , biology , physics , biochemistry
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.