Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

This protocol enables anyone to conduct single-molecule FRET experiments to measure accurate absolute distances within biomolecules using the smfBox, which is a cheap, robust, open-source instrument. smFRET allows you to look at one molecule at a time, rather than the average many billions of molecules, so that you can characterize the different conformations that they adopt. The beauty of this method is that it could be used to shed light on the structure and dynamics of many biomolecular systems.

For example, protein folding, protein DNA interactions, and DNA allostery. Before beginning an analysis, launch the laser control center and confirm that the continuous wave alternating constant current mode is selected. Power on both of the lasers.

Launch smOTTER acquisition software and confirm that each instrument is configured correctly. To align the emission path, place a drop of immersion oil onto the objective and carefully place a clean cover glass onto the objective lens, lowering at an angle to the oil to prevent trapping air bubbles between the cover glass and the objective. Then add 10 microliters of 100 nanomolar Cy3B dye onto the center of the cover glass.

In the laser duty cycles panel, set the donor laser to zero off, 45 on, and 55 off. Set the acceptor laser to 50 off, 45 on, and five off. Set the alternating laser excitation or ALEX period to 100 microseconds.

Open the Z focus tab. In the acquisition panel, switch the lasers to live and start the camera. Adjust the exposure so that a bright spot appears centrally surrounded by a black background.

Starting from a low Z position, increase the height until the bright spot reaches its minimal size. Then raise the height up to an additional 20 micrometers to focus the laser in the sample above the oil and cover glass. When the sample comes into focus, stop the camera.

In the omicron control center, lower the green laser power while monitoring the y-axis scale in the alignment tab of smOTTER until the readout from both detectors is observed, changing the scale as necessary. Then unscrew the four screws at the front of the smfBox to remove the front panel. To align the pinhole, turn the X knob on the pinhole positioner while watching the signal on the alignment tab to increase the signal in green and red.

Turn the Y knob to align the pinhole in the other direction, then return to the X knob to check for any further increase in signal. To align the pinhole lens, turn the lens X knob in one direction to decrease the signal, then turn the pinhole X knob in the same direction to return the signal to the original level. If the new max signal is higher than before, continue to iteratively move both the pinhole and lens knobs in that direction.

If the signal is lower than before, iteratively move the knobs in the opposite direction, then repeat the alignment using the pinhole and lens Y knobs. For alignment of the avalanche photodiode lens, starting with the green avalanche photodiode, move the X knob until the green signal is at its maximum. Repeat the signal modification for the Y knob.

Return to the X knob, moving back and forth to find the threshold points at which the signal begins to fall. Leave the signal at a position halfway between these two points. Find the halfway position for the Y knob and repeat the alignment for the red avalanche photodiode.

Before analyzing the first sample, clean the stage with methanol-soaked lens cleaning tissue, gently wiping across the objective from one end to the other and apply three to four drops of immersion oil onto the center of the objective. Add 10 microliters of type one ultrapure water to the center of a clean cover glass and monitor the water trace to confirm that no fluorescent signals are observed. Then use rubber-ended tweezers to carefully remove the cover glass and replenish the immersion oil if necessary.

To ensure a single-molecule data is obtained, after diluting the sample, select a concentration at which one to five bursts per second are observed in the live trace panel. When the appropriate concentration has been determined, press eight to nine millimeter diameter silicone isolators onto the center of a new cover glass and carefully add 10 microliters of sample into the center, avoiding contact with the silicone. Firmly place the second cover glass onto the isolators until a seal is formed.

Check the live stoichiometry versus FRET efficiency histogram to observe whether the sample is behaving as expected. In the save settings panel, enter the information for the sample name and details, donor and acceptor labels, buffer, donor and acceptor excitation wavelengths, detection wavelengths and laser power, end-user and user affiliation. In the acquisition panel, enter the experiment length in minutes and select an appropriate save interval to mitigate any potential data loss in the case of an error.

Select save laser powers if required, then click start to begin acquiring the data. In a positive result, between five and one burst will be obtained per second. In a negative result, too many or too few bursts will be observed within the same timeframe.

Following collection and analysis, the alternation plot should match the ALEX period of the experimental setup. The time trace is used to qualitatively assess that the sample concentration is reasonable. The background plot shows the distribution of the inter-photon delay periods with a linear fit to the longer times to estimate the background rate.

The background trace can be used to identify if there were changes in the sample over the duration of the experiment. Stoichiometry versus FRET efficiency histograms are generated, selecting for all species and doubly-labeled species. A 1D FRET efficiency histogram is generated with Gaussian fitting of the data.

So the concentration of the sample here is key. If it's too high, you're not doing a single-molecule experiment anymore, whereas if it's too low, it will take too long to get the data. It's enabled the determination of structures of dynamic biomolecules, which could not be determined by other methods like NMR or crystallography.