Single-molecule fluorescence (or Förster) resonance energy transfer (or smFRET) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected at a single molecule level. In contrast to "ensemble FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium with no ensemble/bulk signal change. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of intramolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are particularly useful in chemical sensing, bioassays, and biosensing.
Methodology
Single-molecule FRET measurements are typically performed on fluorescence microscopes, either using surface-immobilized or freely-diffusing molecules. Single FRET pairs are illuminated using intense light sources, typically lasers, in order to generate sufficient fluorescence signals to enable single-molecule detection. Wide-field multiphoton microscopy is typically combined with total internal reflection fluorescence microscope (TIRF). This selectively excites FRET pairs on the surface of the measurement chamber and rejects noise from the bulk of the sample. Alternatively, confocal microscopy minimizes background by focusing the fluorescence light onto a pinhole to reject out-of-focus light. The confocal volume has a diameter of around 220 nm, and therefore it must be scanned across if an image of the sample is needed. With confocal excitation, it is possible to measure much deeper into the sample than when using TIRF. The fluorescence signal is detected either using ultra-sensitive CCD or scientific CMOS cameras for wide-field microscopy or SPADs for confocal microscopy. Once the single molecule intensities vs. time are available the FRET efficiency can be computed for each FRET pair as a function of time and thereby it is possible to follow kinetic events on the single molecule scale and to build FRET histograms showing the distribution of states in each molecule. However, data from many FRET pairs must be recorded and combined in order to obtain general information about a sample or a dynamic structure.
Surface-immobilized In surface-immobilized experiments, biomolecules labeled with fluorescent tags are bound to the surface of the coverglass and images of fluorescence are acquired (typically by a CCD or scientific CMOS cameras). Data collection with cameras will produce movies of the specimen which must be processed to derive the single-molecule intensities with time. If a confocal microscope with a SPAD detector is used, molecule searching is done first by scanning the sample. Then the confocal point sits on a molecule and collects intensity-time data directly, giving the sample drifting is negligible, or an active feedback loop is used to keep track of the molecule.
An advantage of surface-immobilized experiments is that many molecules can be observed in parallel for an extended period of time until photobleaching (typically 1-30 s). This allows us to conveniently study transitions taking place on slow time scales. A disadvantage is represented by the additional biochemical modifications needed to link molecules to the surface and the perturbations that the surface can potentially exert on the molecular activity. In addition, the maximum time resolution of single-molecule intensities is limited by the camera acquisition time (typically >1 ms).
Freely-diffusing SmFRET can also be used to study the conformations of molecules freely diffusing in a liquid sample. In freely-diffusing smFRET experiments (or diffusion-based smFRET), the same biomolecules are free to diffuse in solution while being excited by a small excitation volume (usually a diffraction-limited spot). Bursts of photons due to a single-molecule crossing the excitation spot are acquired with SPAD detectors. The confocal spot is usually fixed in a given position (no scanning happens, and no image is acquired). Instead, the fluorescence photons emitted by individual molecules crossing the excitation volume are recorded and accumulated in order to build a distribution of different populations present in the sample. Depending on the complexity of this distribution, acquisition times vary from ~5 min to several hours.
A distinctive advantage of setups employing SPAD detectors is that they are not limited by a "frame rate" or a fixed integration time like when using cameras. In fact, unlike cameras, SPADs produce a pulse every time a photon is detected, while additional electronics are needed to "timestamp" each pulse with 10-50 ns resolution. The high time resolution of confocal single-molecule FRET measurements allows observers to potentially detect dynamics on time scales as low as 10 μs. However, detecting "slow" transitions on timescales longer than the diffusion time (typically ~1 ms) is more difficult than in surface-immobilized experiments and generally requires much longer acquisitions.
Normally, the fluorescent emission of both donor and acceptor fluorophores is detected by two independent detectors and the FRET signal is computed from the ratio of intensities in the two channels. Some setup configurations further split each spectral channel (donor or acceptor) in two orthogonal polarizations (therefore requiring 4 detectors) and are able to measure both FRET and fluorescence anisotropy at the same time. In other configurations, 3 or 4 spectral channels are acquired at the same time in order to measure multiple FRET pairs at the same time. Both CW or pulsed lasers can be used as excitation sources. When using pulsed lasers, suitable acquisition hardware can measure the photon arrival time with respect to the last laser pulse with picosecond resolution, in the so-called time-correlated single photon counting (TCSPC) acquisition. In this configuration, each photon is characterized by a macro-time (i.e. a coarse 10-50 ns timestamp) and a micro-time (i.e. delay with respect to the last laser pulse). The latter can be used to extract lifetime information and obtain the FRET signal.
Data analysis Typical smFRET data of a two-dye system are time trajectories of the fluorescent emission intensities of the donor dye and the acceptor dye, called the two channels. Mainly two methods are used to obtain the emission of the two dyes: (1) accumulative measurement uses a fixed exposure time of the cameras for each frame, such as PMT, APD, EMCCD, and CMOS camera; (2) single-photon arriving time sequence measured using PMT or APD detectors. The principle is to use optical filters or dichroic mirrors to separate the emissions of the two dyes and measure them in two channels. For example, a setup using two halves of a charge-coupled device (CCD) camera is explained in the literature.
For a two-dye system, the emission signals are then used to calculate the FRET efficiency between the dyes over time. The FRET efficiency is the number of photons emitted from the acceptor dye over the sum of the emissions of the donor and the acceptor dye. The time trajectories produced in every experiment may contain signals from incomplete or incorrect labeling or even aggregates. To account for this multiple research groups have developed sophisticated tools for the time trace selection based on multiple metrics, or using deep neural network. Usually, only the donor dye is excited, but even more accurate information can be obtained if donor and acceptor dye are alternatingly excited. In a single excitation scheme, the emission of the donor and acceptor dyes is just the number of photons collected for the two dyes divided by the photon collection efficiencies of the two channels respectively which are the functions of the collection efficiency, the filter and optical efficiency, and the camera efficiency of the two wavelength bands. These efficiencies can be calibrated for a given instrumental setup.
where FRET is the FRET efficiency of the two-dye system at a period of time, and are measured photon counts of the acceptor and donor channel respectively at the same period of time, and are the photon collection efficiencies of the two channels, and are quantum yield of the two dyes, and is a correction to the different intrinsic brightness of the two dyes to the microscope setting. Thus, calculates the actual number of photons emitted from the dye. If the photon collection efficiencies of the two channels are similar and the actual FRET distance is not interested, one can set the two = 1, and the two quantum yields to 1 as well, i.e. .
For the accumulative emission smFRET data, the time trajectories contain mainly the following information: (1) state transitions, (2) noise, (3) camera blurring (analog of motion blur), (4) photoblinking and photobleaching of the dyes. The state transition information is the information a typical measurement wants. However, the rest signals interfere with the data analysis and thus have to be addressed.
A list of software packages can be found on the KinSoftChallenge website. A report on the comparison results among these software packages can be found here.
Noise
The noise signal of the dye emission typically contains camera readout noise, shot noise and white noise, and real-sample noise, each following a different noise distribution due to the different sources. The real-sample noise comes from the thermal disturbance of the system that results in the FRET distance broadening, uneven dye orientation distribution, dye emission variations, fast blinking, and faster-than-integration-time kinetics. Slow variations of the dye emission are likely considered false-positive states that should be experimentally avoided by choosing different dyes. The other noises are from the excitation path and the detection path, especially the camera. In the end, the noise of the raw emission data is a combination of noises with Poisson distribution and Gaussian distribution. The noises in each channel sometimes (e.g. for single-photon detectors) can be simplified to the summation of an intensity-dependent Gaussian noise (the higher the count the larger the noise; it is a combination of Gaussian and larger-amplitude Poisson noise that can be represented by the Gaussian function) and a Poisson background noise (signal-count-independent). The latter noise dominates when the channel is in the OFF or low-intensity state (right figure). The noises can be approximated as Gaussian distribution, especially at relatively high signal counts. The noises in the two channels then are combined into the non-linear equation listed above to calculate the FRET values. Thus, the noise on the smFRET trajectories is very complicated. The noise is asymmetric above and below the mean FRET values and its magnitude changes towards the two ends (0 and 1) of the FRET values due to the changing uncertainties of the A and D channels. Most noise can be reduced by binning the data (see Figure) with the cost of losing time resolution. See GitHub (postFRET) for an example of MATLAB codes to simulate smFRET time trajectories without and with noise (GitHub link ).
Camera blurring