\title{Walkthrough:\Hardware and software for single-molecule fluorescence analysis} \author[1]{Ben Gamari bgamari@physics.umass.edu} \author[2]{Laura Dietz dietz@cs.umass.edu} \author[1]{Lori Goldner lgoldner@physics.umass.edu} \affil[1]{Department of Physics, University of Massachussetts, Amherst} \affil[2]{Department of Computer Science, University of Massachussetts, Amherst}
\maketitle
In this document describe how to carry out a small experimental data analysis using the tools provided in our submission. In the submission package we have provided two datasets from a single-molecule fluorescence experiment taken with our time-stamping hardware. The datasets\footnote{experimental data due to Peker Milas of the Goldner group}, taken for our recent publication [@Milas2013], examine FRET in an RNA 16-mer (Figure \ref{fig:rna}) labelled terminally with Cy3 and Cy5 dyes for comparison against predictions derived from molecular dynamics simulations.
The tools used are provided by four packages, each of which have
associated documentation including installation instructions. The
firmware for the timestamping hardware is provided by the
timetag-fx2
and timetag-fpga
packages. The tools for interacting with the hardware are provided in
the timetag-tools
package. Installation and usage
of these tools is described in the tutorial.
The photon-tools
package provides a variety of
utilities for working with fluorescence timestamp data and computing
and analyzing fluorescence correlation functions.
Finnally, the hphoton
package provides an end-to-end FRET
analysis package.
We will be focusing on the tools provided by these last two packages.
We will begin by examining the first dataset, donor-only.timetag
,
which is a measurement of the 16-mer labelled with only the donor dye
for calibration purposes. We will begin by examining the binned
intensity timeseries of this dataset using the plot-bins
utility
provided by photon-tools
,
$ plot-bins donor.timetag
donor.timetag
Average rates:
acceptor: 125.666360 / second
donor: 222.855797 / second
This will display a plot similar to that shown in Figure \ref{fig:plot-bins}. Each row shows roughly 10 seconds of the experiments where we see a number intensity bursts from labelled RNA passing through the observation volume. As this is a donor-only sample, we see that nearly all of the fluorescence is in the donor (green) channel. In contrast, we can examine the doubly-labelled sample,
$ plot-bins fret.timetag
Here we see more sparse bursts (due to lower sample concentration) but find that a substantial amount of fluorescence is being detected in the acceptor (red) channel. We see that neither of the samples show any indication of long, intense bursts, which are a common sign of contamination.
For reference, passing the -help
option to plot-bins
gives a
help message describing the various flags supported by the tool.
Next we can compute a correlation function to characterize the
diffusive characteristics of the samples, using
fcs-corr
, also provided by photon-tools
. We will ignore lags
beneath 5 µs to avoid seeing photophysical effects,
$ fcs-corr --plot -min-lag=5e-5 donor.timetag
This will produce six files in the current directory: three correlation functions (donor and acceptor autocorrelation, and the donor-acceptor cross-correlation, in tab-separated format) along with a plot of each. Nex, we fit a three-dimensional diffusion model to, for instance, the donor (channel 0) auto-correlation function to extract a characteristic diffusion time from which we can infer the molecule's hydrodynamic radius,
$ fcs-fit plot donor.timetag.acorr-0
This shows a plot of the correlation function along with a fit, its
parameters, and a variety of goodness-of-fit metrics. Most
importantly, we see that the diffusion time, tau_d
has a value of
292 μs. As a sanity check, we can compare this fit against that of
fret.timetag
, where we see that the diffusion times are within the
parameter uncertainty of one another. Furthermore, looking at the
Further uses of the photon-tools
are described in the package
documentation.
Have verified that the sample is clear of contamination and contains the expected species, we can now extract a FRET efficiency. We begin by examining the donor-only sample, fitting the resulting FRET distribution to a single Beta distribution with 5 ms bin width, and a burst acceptance threshold of 10 photons,
$ fret-analysis --fit-comps=1 --bin-width=5e-3 --burst-size=10 --nbins=10 donor.timetag
This produces a few tab-separated files describing the
above-threshold bins, a variety of plots, as well as an HTML file
summarizing the analysis. We see in the summary that the donor-only
sample contains a single population exhibiting low FRET
efficiency. Althouhg there are no true acceptor emissions (as
there is no acceptor dye), the peak is shifted away from
Next we can examine the doubly-labelled FRET sample. Here we will use two fit components (one for the donor-only population and another for the doubly-labelled population) and a higher threshold due to the second dye. Further, we can indicate that the software should correct for crosstalk and gamma, inferring the correction parameters from the donor-only dataset,
$ fret-analysis --fit-comps=2 --burst-size=20 --nbins=20 --donly-file=donor.timetag
--crosstalk=auto --gamma=auto --bin-width=5e-3 fret.timetag
We see, the FRET histogram, the inferred fit for the FRET distribution, and a variant of the fit which shows the distribution that would be expected in the case of a shot-noise limited FRET population exhibiting no slow dynamics.
While sometimes effective, the bin-threshold technique imposes an arbitrary timescale on the data and there struggles to isolate bursts shorter than the significantly shorter than the bin width, as is common in solution FRET experiments like the one described above.
To overcome this, we provide a novel Bayesian inference for burst
identification. In contrast to the bin-threshold method, our approach
exploits the exponential distribution of Poissonian interarrival times
in conjunction with an assumption of the smoothness on the
fluorescence intensity as a function of time. This allows bursts with
duration shorter than one bin width to be isolated and examined. We
include an implementation of this technique in the hphoton
package
which can be easily used to support a simple FRET analysis as seen in
\ref{fig:burstfind}.