A conventional fluorescence microscope was previously constructed for simultaneous imaging of two colors to gain subdiffraction localization. The system is predicated on color separation of overlapping Airy discs, construction of matrices of Cartesian coordinates to determine locations as well as centers of the point spread functions of fluorophores. Quantum dots that are separated by as little as 10 nm were resolved in the x-y coordinates. Inter-fluorophore distances that vary by 10 nm could also be distinguished. Quantum dots are bright point light source emitters that excite with a single laser and can serve as a label for many biomolecules. Here, alterations in the method are described to test the ability to resolve Atto 488 and Atto 647 dyes attached to DNA origami at ~40 nm spacing intervals. Dual laser excitation is used in tandem with multi-wavelength bandpass filters. Notwithstanding challenges from reduced intensity in Atto labeled DNA origami helical bundles compared to quantum dots, preliminary data show a mean inter-fluorophore distance of 56 nm with a range (14-148 nm). The range closely matches published results with DNA origami with other methods of subdiffraction microscopy. Sub-diffraction simultaneous two-color imaging fluorescence microscopy acronymically christened (SSTIFM) is a simple, readily accessible, technique for measurement of inter-fluorophore distances in compartments less than 40 nm. Preliminary results with so called nanorulers are encouraging for use with other biomolecules.
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