PMC:5115869 / 64213-66135 JSONTXT

{"project":"2_test","denotations":[{"id":"27858593-20006957-27104485","span":{"begin":549,"end":553},"obj":"20006957"}],"text":"The smFRET response IAD for each ZMW was corrected for cross talk due to donor emission in the acceptor channel by subtracting 10% of IDD, as determined from donor emission in the acceptor camera in the absence of donor. Next, IAD was baseline corrected by subtracting the mean intensity after acceptor bleaching, and in some cases where it was visually apparent also subtracting a very slow exponential decay (t ~100 s). Binding dynamics were determined by idealization of IAD prior to acceptor bleaching using the software vbFRET (Bronson et al., 2009) and allowing for a maximum of two idealized levels. vbFRET accepts as input both a donor (ID) and acceptor (IA) time series, and idealizes the smFRET response given by IA/(IA+ID). However, IDD was not anticorrelated with IAD due to the fact that the donor was on the freely diffusing ligand, and therefore not present in the excitation volume throughout the experiment. Thus, to avoid adding noise during the idealization, IAD was idealized directly by providing vbFRET with IA=IAD and ID= max(IAD)-IAD, which results in a smFRET response that is an arbitrarily scaled copy of IAD. The idealized records were overlaid on the raw IAD data series after applying the reverse scaling and baseline shifting, and examined visually. ZMWs with either no idealized events or whose idealization did not pass visual inspection were removed from further analysis. The signal to noise ratio (S/N) for IAD was computed as the ratio of the rescaled idealized amplitude to the RMS noise for all unbound time points. The distribution of S/N for all ZMWs was well described by a gamma function, and ZMWs with S/N \u003c 2 were rejected from further analysis. The idealization and HMM analyses described here were able to reproduce known models given simulated data at this S/N cutoff. The idealized smFRET time series were interpreted as a binary unbound versus bound time-dependent signal."}

{"project":"MyTest","denotations":[{"id":"27858593-20006957-27104485","span":{"begin":549,"end":553},"obj":"20006957"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"The smFRET response IAD for each ZMW was corrected for cross talk due to donor emission in the acceptor channel by subtracting 10% of IDD, as determined from donor emission in the acceptor camera in the absence of donor. Next, IAD was baseline corrected by subtracting the mean intensity after acceptor bleaching, and in some cases where it was visually apparent also subtracting a very slow exponential decay (t ~100 s). Binding dynamics were determined by idealization of IAD prior to acceptor bleaching using the software vbFRET (Bronson et al., 2009) and allowing for a maximum of two idealized levels. vbFRET accepts as input both a donor (ID) and acceptor (IA) time series, and idealizes the smFRET response given by IA/(IA+ID). However, IDD was not anticorrelated with IAD due to the fact that the donor was on the freely diffusing ligand, and therefore not present in the excitation volume throughout the experiment. Thus, to avoid adding noise during the idealization, IAD was idealized directly by providing vbFRET with IA=IAD and ID= max(IAD)-IAD, which results in a smFRET response that is an arbitrarily scaled copy of IAD. The idealized records were overlaid on the raw IAD data series after applying the reverse scaling and baseline shifting, and examined visually. ZMWs with either no idealized events or whose idealization did not pass visual inspection were removed from further analysis. The signal to noise ratio (S/N) for IAD was computed as the ratio of the rescaled idealized amplitude to the RMS noise for all unbound time points. The distribution of S/N for all ZMWs was well described by a gamma function, and ZMWs with S/N \u003c 2 were rejected from further analysis. The idealization and HMM analyses described here were able to reproduce known models given simulated data at this S/N cutoff. The idealized smFRET time series were interpreted as a binary unbound versus bound time-dependent signal."}