Single-molecule FRET

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.

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.

However, data from many FRET pairs must be recorded and combined in order to obtain general information about a sample [3] or a dynamic structure.

[5] Data collection with cameras will produce movies of the specimen which must be processed to derive the single-molecule intensities with time.

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).

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.

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.

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 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 typical open source-code software packages can be found online such as HaMMy, vbFRET, ebFRET, SMART, SMACKS, MASH-FRET, etc.

However, the rate constants can be calculated from the probability functions, the number of each transition over the total time of the state it transfers from.

A system under equilibrium with each state parallel transfers to all others in first-order reactions is a special case for easier understanding.

[23] SmFRET also provides dynamic temporal resolution of an individual molecule that cannot be accomplished through ensemble FRET measurements.

[24] Kinetic information in a system under equilibrium is lost at the ensemble level because none of the concentrations of the reactants and products change over time.

However, at the single-molecule level, the transfer between the reactants and products can happen at a measurable high rate and be balanced over time stochastically.

Thus, tracing the time trajectory of a particular molecule enables the direct measurement of the rate constant of each transition step, including the intermediates that are hidden at the ensemble level due to their low concentrations.

Using two acceptor fluorophores rather than one, FRET can observe multiple sites for correlated movements and spatial changes in any complex molecule.

SmFRET with the three-color system offers insights into synchronized movements of the junction's three helical sites and the near non-existence of its parallel states.

However, any obvious advantages are outweighed by the three-color system's requirements, including a clear separation of fluorophore signals.

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.

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.

The structural dynamics of the KirBac channel has been thoroughly analyzed in both the open and closed states, dependent on the presence of the ligand PIP2.

[28] Recently, single-molecule FRET has been applied to quantitatively detect target DNA and to distinguish single nucleotide polymorphism.

Despite making approximate estimates, a limitation of smFRET is the difficulty of obtaining the correct distance involved in energy transfer.

[6] Extracting kinetic information from a complicated biological system with a transition rate of around a few milliseconds or below remains challenging.

A scheme of a typical smFRET experiment (A), FRET distance curve. r is the average distance between the two dyes during the time in collecting one data point. (B), and a time trajectory (C).
(A) An example time trajectory of the acceptor (red) and donor (blue) channel photon counts collected at 1 ms time resolution. (B) Data were binned into 10 ms time resolution to reduce the noise. The inset shows the smFRET time trajectory calculated with the equation. The larger photocounts part has large noise dominated by count-dependent Gaussian noise and the small photocounts area is dominated by the Poisson noise.
Camera blur simulated with an example 3-state smFRET using the postFRET simulator [ 14 ] (two simulations). The signal-to-noise ratio is set at about 20 for both simulations. The time resolution is simulated at 1 ms and then binned to 10 ms, the integration time of the simulated experiments. The simulation rate constants are listed on the left, a small part of the trajectory is shown in the middle (alternative colors represent different molecules), and the FRET histogram of all molecules (100) is shown on the right.
Example reaction networks with different number of smFRET states. Rate constants can equal zero when the transitions are forbidden among some states which can be measured in smFRET.
An example state identified trajectory and the dwell-time histogram of each transition. Lifetime is the average of all dwell times, which is the inverse of the sum of all rate constants of the state to other states.
Comparing schematic results of bulk (orange), ensemble FRET (black), and smFRET measurement of a system with three states (1-3). Bulk measurement averages and ensemble measurement adds all states, and smFRET resolves each state by counting the population of molecules on each state over time. The kinetic information is hidden in the bulk and ensemble measurement but can be observed in the smFRET measurement by analyzing the transitions between each state over time.