PMC:5778504 / 8975-9883 JSONTXT

{"project":"2_test","denotations":[{"id":"29177430-21044609-77171913","span":{"begin":97,"end":99},"obj":"21044609"},{"id":"29177430-23184986-77171914","span":{"begin":168,"end":170},"obj":"23184986"},{"id":"29177430-22404588-77171915","span":{"begin":536,"end":538},"obj":"22404588"}],"text":"Fluorescence intensities were extracted from images using previously described TwoTone software (36). The apparent FRET efficiency, E*, was calculated as described in (37). We manually inspected intensity time trajectories and selected 100 molecules for analysis according to the following criteria: single-step bleaching after at least 105 frames; circular PSF across DD, DA, and AA channels; no donor photoblinking (although fluctuations were permitted); no acceptor photoblinking (although we allowed for different ATTO647N states; (38)); fluorescence intensities within a limited range; no defocusing; and no nearby molecules as measured using a nearest-neighbour criterion. Histograms of E* were constructed using 250 frames of data from each of our 100 selected molecules; these molecules were collected from independent experiments using a single batch of labeled DNA and a single preparation of RNAP."}