This tutorial shows step-by-step, how to calculate a FRET histogram from single FRET pairs detected under single molecule conditions. The script requires a spectrally resolved fluorescence time trace. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations.
Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.
The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as “bursts”. Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.
To record such traces, usually single molecule sensitive detectors as SPAD or Hybrid PMTs are necessary to successfully detect single molecule fluorescence.
Note: The “Samples” workspace is delivered with the SymPhoTime 64 and on the DVD-ROM and contains example data to show the function of the SymPhoTime 64 data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
Cy3+Cy5_diff_PIE-FRET.ptuby a single mouse click.
Note: The window contains three different regions:
Note: If you are unsure which binning to select, calculate the FCS curve and check the diffusion time to see the average residence time of the sample in the laser focus.
Note: Two channels need to be active in order to calculate spectral ratios.
The data in this file were obtained under PIE (Pulsed Interleaved Excitation) conditions: first, a 530 nm pulse excited the Cy3 dye and consequently, the fluorescence of Cy3 and Cy5 – the latter mainly excited via FRET - was detected in the two different channels. In this trace, donor and acceptor channel were recorded with a slight time delay, therefore two peaks can be seen in the green marked area. This region is used to calculate the FRET efficiency histogram. The unmarked white area shows the fluorescence response after the second pulse (at 635 nm), which was used to excite the Cy5 dye directly. This information is not used in this script (in fact, for this script also cw excitation could be used), but in the PIE-FRET script, which is explained in another tutorial (Calculate Ratiometric Single Pair FRET Distributions Using the PIE-FRET Script).
Note: The check box “burst integration” should be checked for measurements of freely diffusing single molecules as in this example file. For observing conformational fluctuations of a single immobilized molecule, an analysis of the time bins set in the “Trace Settings” is appropriate. In that case, just deactivate the “Burst Integration” check box.
The “Cross Talk” can be determined by measuring a solution containing only the donor dye to check which percentage leaks into the acceptor channel.
The “Direct Ext.” parameter is determined by exciting a solution containing only the acceptor with just the laser line normally used to excite the donor.
“Gamma” corrects for the different detection efficiencies in the donor and the acceptor channel, influences e.g. by detector quantum efficiency as well as filter sets.
The equations used for the FRET calculations and an explanation of the parameters can be found when clicking on the “Help” button.
Note: The parameter “Min. Photons per FRET Event” is by default set to 25. In order to calculate distinct FRET efficiencies, a much lower parameter does not make sense, as the possible values for the FRET efficiencies are otherwise too much determined by the photon numbers of the event. For example, in a burst with just 10 photons, only 11 FRET values are numerically possible, which is quite rough.
Response: The FRET efficiencies are calculated and the “FRET Efficiency” and “Distance” histograms are displayed in the lower part of the image.
Note: These values are only correct, if the correct parameters for spectral bleed through, direct excitation and channel sensitivity have been determined and entered into the script.
Response: A window pops up asking for a file name and a place to store the user configuration.
Note: Usually, when a system is delivered, a folder “C:\User_Configurations” is already present on the hard disc. If not, create it and save the profile there.
Response: Software restarts, but applies the user profile
Note: Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop receptor tertiary interaction, Biochemistry (2009), 48(11), 2550-8. (http://pubs.acs.org/doi/abs/10.1021/bi8019788).