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. 2012 Oct 17;103(8):1691–1697. doi: 10.1016/j.bpj.2012.09.014

Figure 2.

Figure 2

Characterization of the line-scan confocal microscope. (a) Comparison of the optical-sectioning capabilities of epifluorescence microscopy (31.7 μm), HILO (6.7 μm), and the line-scan confocal microscopy (1.0 μm). (b) A line-scan confocal image of Cy3-labeled single-stranded DNA on a coverslip in the presence of 10 nM free Cy3. To make the images, 10 frames with 100-ms exposure time were averaged and background fluorescence was subtracted. Scale bar, 3μm. (c) Same as in (b), except that the image was acquired in a HILO microscope. (d) Observation of Holliday junction dynamics via line-scan confocal microscopy. (left) A model of two-state conformational dynamics of the Holliday junction. For FRET measurements, donor (green circle) and acceptor (red circle) dye labels were attached to the ends of the Holliday junction. (right) Representative fluorescence intensity (green for donor and red for acceptor) and corresponding FRET-efficiency (black) time traces. Exposure time, 100 ms. (e) FRET histogram generated from 44 molecules. The oil-immersion objective was used for all experiments.