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...
immobilization or additional functionalization needed for surface
attachment~\cite{Eggeling_1998,Dahan_1999}.
The increasing amount of work using freely-diffusing smFRET has
resulted
in motivated
a growing number of theoretical contributions to the specific topic of data
analysis~\cite{Fries_1998,Eggeling_2001,Zhang_2005,Gopich_2005,Lee_2005,Nir_2006,Antonik2006,Gopich_2007,Gopich_2008,Camley_2009,Santoso_2010,Torella_2011,Tomov_2012}.
Despite this profusion of
methods, publications, most research groups still rely on
their own implementation of a limited number of methods, with very little
collaboration or code sharing.
To clarify this statement, let us point that our own group's past smFRET papers
...
sharing protocols and samples, in the domain of scientific software,
we have relegated ourselves to islands of non-communication.
From a more general standpoint, the non-availability of
codes the code
used to produce scientific results, hinders reproducibility,
makes it impossible to review and validate the software's correctness
and prevents improvements and extensions by other scientists.
...
%(\href{https://www.mozillascience.org/effective-code-review-for-journals}{Mills 2015})
%(\href{http://ivory.idyll.org/blog/2015-we-live-in-a-bubble.html}{Brown 2015} and \href{http://ivory.idyll.org/blog/on-code-review-of-scientific-code.html}{2013}).
Recently, a few
efforts publications have
been made to provide standard implementations included software implementation of methods used
in to analyze
surface immobilized single-molecule experiments~\cite{Bronson_2009,Greenfeld_2012, K_nig_2013}.
In the
field of freely-diffusing
smFRET field, smFRET, although it is common to
find mention of "code available from the
best authors upon request" in publications, there is a dearth of
such open source code, with, to our knowledge,
there is only the notable exception of a
laudable example in this direction~\cite{Murphy2014}. single example~\cite{Murphy2014}.
To address
a similar need in the freely-diffusing field, this issue, we
have developed FRETBursts,
an open source Python software for
burst the analysis of
freely diffusing single-molecule FRET
experiments.
With bursts.
FRETBursts
we provide a tool that is available to any scientist
to use, inspect can be used, inspected and
modify.
FRETBursts is suitable for routine state-of-the-art
analysis of modified by anyone interested in using state-of-the art smFRET
data but also analysis method or implementing modifications or completely new techniques. FRETBursts therefore represents an ideal platform
for quantitative comparison of different methods
in burst analysis,
as well as for
development of new methods.
A smFRET burst analysis.
Technically, a strong
focus is placed on emphasis has been given to the reproducibility of complete analysis
workflows. FRETBursts
execution model is based on uses Jupyter
Notebook~\cite{Shen_2014}, Notebooks~\cite{Shen_2014},
an interactive and executable document containing
textual narrative, input parameters,
code code, and computational
results. results (tables, plots, etc.). A notebook
thus captures the various analysis steps
in a document
that which is easy to share and
re-execute. execute.
To minimize the
inevitable occurrence possibility of
bugs~\cite{Soergel_2015} bugs being introduced inadvertently~\cite{Soergel_2015}
we employ modern software engineering techniques
such as unit testing and continuous integration.
FRETBursts is hosted
and openly developed on GitHub~\cite{Blischak_2016,Prli__2012},
where users can
send write comments, report issues or contribute code.
In a
parallel related effort, we recently introduced Photon-HDF5,
a open file format for timestamp-based single-molecule fluorescence
experiments~\cite{Ingargiola2016}. An other related open source tool is PyBroMo~\cite{Ingargiola_2016},
a freely-diffusing smFRET simulator which produces Photon-HDF5 files that are
...
ecosystem of open tools for reproducible science in the single-molecule field.
\subsection{Paper Overview}
This paper is
written as an introduction to smFRET burst analysis and
FRETBursts usage.
Therefore, after its implementation in FRETBursts.
After a brief overview of FRETBursts features (section~\ref{sec:overview}),
we introduce
core smFRET burst analysis essential concepts and terminology
for smFRET burst analysis
(section~\ref{sec:concepts}).
These concepts are used throughout the paper
so reading section~\ref{sec:concepts} is highly recommended.
In section~\ref{sec:analysis}, we illustrate the
practical steps involved
in smFRET burst analysis:
(i) data loading (section~\ref{sec:dataload}),
defining )ii) definition of the
excitation alternation periods (section~\ref{sec:alternation}),
(iii) background
correction (section~\ref{sec:bg_calc}),
(iv) burst search (section~\ref{sec:burstsearch}),
(v) burst selection (section~\ref{sec:burstsel}) and
(vi) FRET
histogram fitting (section~\ref{sec:fretfit}).
The aim
of this section is
elucidating to illustrate the specificities and trade-off
of involved in various approaches
with
enough sufficient details to
empower enable readers new to the field
(or Jupyter Notebooks) to customize the analysis
to for their own needs.
For the most advanced use-case, section~\ref{sec:bva} Section~\ref{sec:bva} walks the reader thorough implementing
Burst Variance Analysis
(BVA)~\cite{Torella_2011} (BVA)~\cite{Torella_2011}, as an example of
manipulating timestamps
and burst data. implementation of an advanced data processing technique.
Finally,
in section~\ref{sec:conclusions}, we summarize section~\ref{sec:conclusions} summarizes what we believe to be
the strengths of FRETBursts software.
Throughout this paper,
links to relevant sections of documentation and other web resources
are displayed as ``(link)''.
In order to make the text
accessible to the widest number of readers, more legible,
we
have concentrated python-specific details in
special subsections
titled entitled
\textit{Python details}. These subsections provide deeper insights for readers
already familiar with python and can be
safely initially skipped
otherwise. by readers who are not.
Finally, note that all commands here reported can be found in the
accompanying notebooks
(\href{https://github.com/tritemio/fretbursts_paper}{link}).
