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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 times
larger 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 photons
for rate
computation $m$ can be selected by passing
a 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 after
the 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 is
here called \verb|d_dcbs|).
Since FRETBursts shares the
arrays timestamps and detectors
arrays between
different copies of \verb|Data| objects, the memory usage is
contained minimized, even when
using
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, donor
spectral 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...