X. Michalet edited burst-search.tex  about 8 years ago

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Following background estimation, burst search is the next step of  the analysis.  In FRETBursts, a standard burst search on using  a single photon stream (see section~\ref{sec:burstsearch_intro}) is performed by calling the  \verb|Data.burst_search| method  (\href{http://fretbursts.readthedocs.org/en/latest/data_class.html#fretbursts.burstlib.Data.burst_search}{link}). 

\end{lstlisting}  performs a burst search on all photons  (\verb|ph_sel=Ph_sel('all')|), with a minimum count  rate threshold equal to  6 timeslarger than  the local  background rate (\verb|F=6|) and (\verb|F=6|),  using 10 consecutive photons to compute the local count  rate (\verb|m=10|). A different photon selection, stream,  threshold ($F$) or number of photonsfor rate  computation  $m$ can be selected by passinga  different value. values.  These parameters are generally a good starting point for smFRET analysis but can be adjusted in  specific cases.  %that sound a bit vague...  Note that, in that  the previous burst search, no search does not perform any  burst size selection was performed  (i.e. (however, by definition,  the minimum bursts size is effectively $m$). An additional parameter $L$ can be passed to apply impose  a threshold on the raw minimum  burst size (before before  any correction).  It correction.  However, it  is recommended, however, recommended  to select bursts only afterthe  background correction  is applied corrections  are applied,  as shown discussed  in the next section~\ref{sec:burstsel}. It might sometimes be useful to specify a fixed photon-rate threshold, instead  of a threshold depending on the background rate, as in the previous example. In 

\end{lstlisting}  Finally, to perform a DCBS burst search (or in general an AND gate burst search,  see section~\ref{sec:burstsearch_intro})we use  the function \verb|burst_search_and_gate|  (\href{http://fretbursts.readthedocs.org/en/latest/plugins.html#fretbursts.burstlib_ext.burst_search_and_gate}{link})  is used,  as illustrated  in the following example: \begin{lstlisting}  d_dcbs = bext.burst_search_and_gate(d, F=6, m=10)  \end{lstlisting}  The last command puts the burst search results in a new copy of  the \verb|Data| variable \textit{d} (the (in this example, the  copy ishere  called \verb|d_dcbs|). Since FRETBursts shares thearrays  timestamps and detectors arrays  between different copies of \verb|Data| objects, the memory usage is contained minimized,  even whenusing  several copies. copies are created.  \paragraph{Python details}  Note that, while \verb|.burst_search()| is a method of \verb|Data|, 

The function \verb|burst_search_and_gate| accepts optional arguments,  \verb|ph_sel1| and \verb|ph_sel2|, whose default values correspond to the  classical DCBS photon stream selection (see section~\ref{sec:burstsearch_intro}).  These arguments can be specified to select different photon streams than those used  in a classical DCBS.  The \verb|bext| module (\href{http://fretbursts.readthedocs.org/en/latest/plugins.html}{link})  

\subsubsection{Correction Coefficients}  \label{sec:corrcoeff}  In µs-ALEX µs-ALEX,  there are 3 important correction parameters: γ-factor, donorspectral  leakage into the acceptor channel and acceptor direct excitation by the donor excitation laser~\cite{Lee_2005}. These corrections can be applied by simply assigning values  to the respective \verb|Data| attributes: \begin{lstlisting}  d.gamma = 0.85 

correction parameters.  These correction factors can be used to display corrected FRET distributions.  However, when the goal is fitting to fit  the FRET efficiency of sub-populations, it is more accurate to fit the uncorrected FRET histogram (i.e. background-corrected  proximity ratio) and then correct the fitted FRET efficiency (see SI in~\cite{Lee_2005}, SI). %how is that more accurate?  This procedure avoids   distortion of the FRET distributions due to the corrections, which causes  a departure from the ideal Binomial statistics~\cite{Gopich_2007}.  %fundamentally, if there are contamination beyond FRET, the distribution is NOT binomial anymore.  FRETBursts implements the correction formulas for $E$ and $S$ in the functions  \verb|fretmath.correct_E_gamma_leak_dir| and \verb|fretmath.correct_S|  (\href{http://fretbursts.readthedocs.org/en/latest/fretmath.html}{link}).   A rigorous derivation of these correction formulas (using computer-assisted algebra)   can be found online as an interactive notebook (\href{http://nbviewer.jupyter.org/github/tritemio/notebooks/blob/master/Derivation%20of%20FRET%20and%20S%20correction%20formulas.ipynb}{link}).  %using computer algebra to derive such a simple relation is a bit embarrassing, I would say...