Conventional fluorescence microscopy below the diffraction limit with
simultaneous capture of two fluorophores in DNA origami
#MMPMID27307653
Glasgow BJ
Proc SPIE Int Soc Opt Eng
2016[Feb]; 9714
(?): ? PMID27307653
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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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