Single molecule FRET

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 signal 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. Conversely, 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 the sample in order to generate an image. With confocal excitation, it is possible to measure much deeper into the sample than when using TIRF. 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.

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.

Freely-Diffusing

SmFRET can also be used to study the conformations of molecules freely diffusing in a liquid sample. In freely-diffusing smFRET experiments (also called confocal 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 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. Normally, the fluorescent emission of both donor and acceptor fluorophores is detected by two independent detectors. Some setups configurations further split each spectral channel (donor or acceptor) in two orthogonal polarizations (therefore requiring 4 detectors) and are able to measure also fluorescence anisotropy. 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. A distinctive feature 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 an additional electronics is needed to "timestamps" each pulse with 10-50ns resolution. Both CW or pulsed lasers can be used as excitation source. When using pulsed lasers, a 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 10-50ns timestamp) and a micro-time (i.e. delay with respect the last laser pulse). The latter can be used to extract lifetime information. The high time resolution of confocal single-molecule FRET measurements allows to potentially detect dynamics on time scales as low as 10μs.

Advantages of smFRET

SmFRET allows for a more precise analysis of heterogeneous populations and has a few advantages when compared to ensemble FRET.

One benefit of studying distances in single molecules is that heterogeneous populations can be studied more accurately with values specific for each molecule rather than computing an average based on an ensemble. This allows for the study of specific homogeneous populations within a heterogeneous population. For example, if two existing homologous populations within a heterogeneous population have different FRET values, an ensemble FRET analysis will produce a weighted averaged FRET value to represent the population as a whole. Thus, the obtained FRET value does not produce data on the two distinct populations. In contrast, smFRET would be able to differentiate between the two populations and would allow analysis of the existing homologous populations.

SmFRET also provides dynamic temporal resolution of an individual molecule that cannot be accomplished through ensemble FRET measurements. This allows smFRET to be used to study an RNA’s folding dynamics. Similar to protein folding, RNA folding goes through multiple interactions, folding pathways, and intermediates before reaching its native state. Ensemble FRET has the ability to detect well-populated transition states that accumulate in a population, but it lacks the ability to characterize intermediates that are short-lived and do not accumulate. This limit is addressed by smFRET which offers a direct way to observe the intermediates of single molecules regardless of accumulation. Therefore, smFRET demonstrates the ability to capture transient subpopulations in a heterogeneous environment.

SmFRET is also shown to utilize a three-color system better than ensemble FRET. Using two acceptor fluorophores rather than one, FRET can observe multiple sites for correlated movements and spatial changes in any complex molecule. This is shown in the research on the Holliday Junction. SmFRET with the three-color system offers insights on synchronized movements of junction’s three helical sites and near non-existence of its parallel states. Ensemble FRET can use three-color system as well. However, any obvious advantages are outweighed by three-color system’s requirements which includes a clear separation of fluorophore signals. For a clear distinction of signal, FRET overlaps must be small but that also weakens FRET strength. SmFRET corrects its overlap limitations by using band-pass filters and dichroic mirrors which further the signal between two fluorescence acceptors and solve for any bleed through effects.

Applications

A major application of smFRET is to analyze the minute biochemical nuances that facilitate protein folding. In recent years, multiple techniques have been developed to investigate single molecule interactions that are involved in protein folding and unfolding. Force-probe techniques, using atomic force microscopy and laser tweezers, have provided information on protein stability. smFRET allows researchers to investigate molecular interactions using fluorescence. Forster resonance energy transfer (FRET) was first applied to single molecules by Ha et al. and applied to protein folding in work by Hochstrasser, Weiss, et al. The benefit that smFRET as a whole has afforded to analyzing molecular interactions is the ability to test single molecule interactions directly without having to average ensembles of data. In protein folding analysis, ensemble experiments involve taking measurements of multiple proteins that are in various states of transition between their folded and unfolded state. When averaged, the protein structure that can be inferred from the ensemble of data only provides a rudimentary structural model of protein folding. However, true understanding of protein folding requires deciphering the sequence of structural events along the folding pathways between the folded and unfolded states. It is this particular branch of research that smFRET is highly applicable.

FRET studies calculate corresponding FRET efficiencies as a result of time-resolved observation of protein folding events. These FRET efficiencies can then be used to infer distances between molecules as a function against time. As the protein transitions between the folded and unfolded states, the corresponding distances between molecules can indicate the sequence of molecular interactions that lead to protein folding.

Single-molecule FRET can also be applied to study the conformational changes of the relevant channel motifs in certain channels. For example, labeled tetrameric KirBac potassium channels were labeled with donor and acceptor fluorophores at particular sites in order to understand the structural dynamics within the lipid membrane, thus allowing them to generalize similar dynamics for similar motifs in other eukaryotic Kir channels or even cation channels in general. The use of smFRET in this experiment allows for visualization of the conformational changes that cannot be seen if the macroscopic measurements are simply averaged. This will lead to ensemble analysis rather than analysis of individual molecules and the conformational changes within, allowing us to generalize similar dynamics for similar motifs in other eukaryotic channels.

The structural dynamics of the KirBac channel was thoroughly analyzed in both the open and closed states, dependent on the presence of the ligand PIP2. Part of the results based on smFRET demonstrated the structural rigidity of the extracellular region. The selectivity filter and the outer loop of the selectivity filter region was labeled with fluorophores and conformational coupling was observed. The individual smFRET trajectories strongly demonstrated a FRET efficiency of around 0.8 with no fluctuations, regardless of the state of the channel.

Limitations

Despite making approximate estimates, a limitation of smFRET is the difficulty of obtaining the correct distance involved in energy transfer. Requiring an accurate distance estimate gives rise to a major challenge because the fluorescence of the donor and acceptor fluorophores as well as the energy transfer is dependent on the environment and how the dyes are oriented, which can vary depending on the flexibility of where the fluorophores are bound. This issue, however, is not particularly relevant when the distance estimation of the two fluorophores does not need to be determined with exact and absolute precision.