smFRET
What is single-molecule FRET?
Table of Contents
What is smFRET?
Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful biophysical technique that enables researchers to observe molecular interactions and conformational changes at the nanometer scale (2 –10 nm) in real-time. Unlike traditional ensemble FRET, which averages signals over many molecules, smFRET focuses on individual molecules, revealing dynamic behaviours and heterogeneity that bulk methods may obscure.
How does smFRET work?
smFRET involves labelling a biomolecule with two fluorophores: a donor and an acceptor. When the donor is excited by a specific wavelength of light, energy can transfer non-radiatively to the acceptor if they are within close proximity, leading to acceptor fluorescence. The efficiency of this energy transfer is highly sensitive to the distance between the fluorophores, allowing researchers to monitor structural changes within and interactions between molecules.
What are the benefits of smFRET?
- Single-molecule resolution:
Provides insights into the behaviours of individual molecules, avoiding ensemble averaging and revealing rare events - Real-time monitoring:
Captures dynamic processes as they occur - High sensitivity:
Detects subtle conformational changes and interactions - Versatility:
Applicable to a wide range of biological molecules and systems
What applications can smFRET be used for?
smFRET has become a powerful tool in molecular biology, biophysics, and structural biology due to its ability to resolve dynamic molecular processes at the nanometer scale. The ability to resolve molecular heterogeneity, dynamic transitions, and rare intermediates one molecule at a time is no longer a niche advantage – it is rapidly becoming a standard for mechanistic insight. Some of its key applications include:
Protein Folding and Conformational Dynamics
Application:
smFRET tracks how individual protein molecules fold and unfold. It distinguishes intermediate states, folding pathways, and misfolded species that are invisible in ensemble studies.
Use cases:
- Monitoring folding kinetics of small proteins (e.g. villin, chymotrypsin inhibitor)
- Investigating chaperone-assisted folding processes
- Understanding disease-related misfolding (e.g. prions, amyloids)
Enzyme Mechanisms and Catalysis
Application:
By attaching FRET dyes near the active site, smFRET can reveal conformational cycles that enzymes undergo during catalysis.
Use cases:
- Real-time observation of polymerase function (e.g., DNA/RNA synthesis)
- Conformational gating in ribozymes and riboswitches
- Ligand-induced domain motions in kinases and GTPases
Nucleic Acid Structure and Folding
Application:
smFRET can resolve structural transitions in DNA and RNA such as hairpin opening, pseudoknot formation, or hybridisation events.
Use cases:
- Folding pathways of ribozymes and aptamers
- CRISPR-Cas9 target recognition dynamics
- Telomeric DNA structure transitions
Molecular Motors and Mechanical Processes
Application:
smFRET tracks distance changes as motor proteins like helicases, myosins, or kinesins step along filaments or unwind DNA.
Use cases:
- ATP-driven movement of helicases
- Stepping mechanisms of dyneins and kinesins
- Force-dependent conformational changes
Biomolecular Complex Assembly and Binding
Application:
Observing multi-subunit assembly, binding kinetics, and induced-fit dynamics at the single-molecule level.
Use cases:
- Transcription factor-DNA binding
- Assembly of ribosomal subunits
- Protein-protein interactions in signalling cascades
Membrane Protein Dynamics and Signalling
Application:
smFRET enables functional studies of membrane-embedded proteins by tracking conformational states in lipid bilayers or nanodiscs.
Use cases:
- Activation of G-protein-coupled receptors (GPCRs)
- Conformational cycling in ion channels
- Transporter dynamics (e.g. ABC transporters)
Drug Screening and Mechanistic Pharmacology
Application:
smFRET can identify allosteric changes or binding-induced conformational effects of drug candidates.
Use cases:
- Screening inhibitors that stabilise specific conformations
- Identifying slow-binding or transient effectors
- Detecting small molecule-induced folding or unfolding
Live-Cell and In Situ Measurements (Emerging)
Application:
With advances in fluorophore stability and imaging techniques, smFRET is beginning to be applied inside living cells.
Use cases:
- Real-time tracking of biosensors (e.g. calcium sensors).
- In vivo protein-protein interaction studies.
- Intracellular trafficking and localisation of molecular machines.
Studying Heterogeneity in Populations
Application:
smFRET reveals molecule-to-molecule variation that ensemble techniques average out.
Use cases:
- Distinguishing multiple functional states of an enzyme
- Mapping conformational landscapes
- Disentangling kinetic subpopulations
Method Development and Tool Validation
Application:
Researchers also use smFRET to benchmark new fluorescent labels, validate biosensors, or refine analysis algorithms.
Use cases:
- Calibrating new FRET dye pairs or unnatural amino acid labelling
- Testing photostability and dye-linker behavior
- Validating AI-based single-molecule trajectory analysis tools
What does a typical smFRET experiment involve?
A typical smFRET experiment involves:
- Fluorophore labelling:
Attaching donor and acceptor fluorophores to specific sites on the molecule of interest - Sample immobilisation or diffusion: Fixing molecules on a surface or allowing them to diffuse freely in solution
- Excitation and detection:
Using a laser to excite the donor fluorophore and detecting emissions from both donor and acceptor - Data analysis: Calculating FRET efficiency to infer distance changes and molecular dynamics
Do I need a dark room to run smFRET experiments myself?
Most single-molecule fluorescence spectrometers require a dedicated dark room and optical table. However, there are some instruments emerging, like the EI-FLEX, that are compact enough to be used on a benchtop in ambient light conditions.
What are leading researchers saying about smFRET?
ADD TESTIMONIAL QUOTES. BELOW IS PLACEHOLDER CONTENT.
Dr Timothy Craggs
Founder & CEO
“By making smFRET experiments accessible to the widest possible range of scientists, we are enabling a future in which single-molecule data will solve previously impossible problems, aiding the development of new drugs and diagnostics, but also providing the fundamental underpinning to complex biological processes.”
What are some typical smFRET configurations?
Fluorophore Pairs
Donors
TAMRA
Cy3
Cy3B
Atto532
Atto550
AlexaFlour 532
Alexa Flour 546
Alexa Flour 555
Acceptors
Cy5
Atto647
Atto647N
Alexa Fluor 647
What kinds of experiments complement smFRET?
1. Cryo-Electron Microscopy (cryo-EM)
Why it complements smFRET:
Provides high-resolution 3D structures of biomolecules.
How it helps:
Cryo-EM can reveal the static conformational states that correspond to the dynamic transitions observed via smFRET.
2. X-ray Crystallography
Why it complements smFRET:
Offers atomic-level resolution of protein and nucleic acid structures.
How it helps:
smFRET can verify whether those static crystallographic conformations exist under more physiological, dynamic conditions.
3. Fluorescence Correlation Spectroscopy (FCS)
Why it complements smFRET:
Provides diffusion and concentration measurements with sub-millisecond resolution.
How it helps:
FCS can identify kinetic intermediates or binding rates that correlate with smFRET-detected conformational changes.
4. Optical Tweezers or Magnetic Tweezers
Why it complements smFRET:
Enables mechanical manipulation of single molecules.
How it helps:
Combines mechanical force with FRET readouts to correlate structural transitions with applied forces (e.g., DNA unzipping, protein unfolding).
5. Super-Resolution Microscopy (STORM, PALM)
Why it complements smFRET:
Breaks the diffraction limit for spatial resolution.
How it helps:
Allows spatial mapping of smFRET-active complexes in cellular or membrane contexts.
6. Single-Particle Tracking (SPT)
Why it complements smFRET:
Tracks individual molecules over time in live cells or vesicles.
How it helps:
Offers insights into mobility and interaction dynamics that can contextualise conformational FRET changes.
7. Fluorescence Lifetime Imaging Microscopy (FLIM)
Why it complements smFRET:
Measures fluorescence lifetimes rather than intensity.
How it helps:
FLIM can improve FRET quantification, especially in complex environments with background fluorescence.
8. Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR)
Why they complement smFRET:
Provide ensemble-level structural and dynamic data.
How they help:
Offer orthogonal validation of secondary structure changes or conformational exchange.
9. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)
Why it complements smFRET:
Measures structural flexibility and solvent accessibility.
How it helps:
Identifies dynamic regions that may show distance fluctuations in smFRET.
10. Crosslinking and Mass Spectrometry
Why it complements smFRET:
Provides proximity information between residues.
How it helps:
Confirms FRET-predicted contacts and validates labelling positions.
How does single-molecule FRET spectroscopy compare to single-molecule FRET microscopy?
Both spectroscopy and microscopy in the smFRET context involve the same core principle: measuring Förster Resonance Energy Transfer (FRET) efficiency between a donor and an acceptor fluorophore on a single molecule to infer nanometer-scale distance changes. The distinction lies in the method of observation and experimental implementation.
smFRET spectroscopy is the quantitative analysis of FRET signals from single molecules, usually in terms of photon time traces.
Key features:
- Often performed using confocal or multiphoton setups.
- Primarily used for freely diffusing molecules in solution.
- Focuses on photon counting and burst analysis as molecules pass through the excitation volume.
- High temporal resolution (microsecond to millisecond timescales).
- Excellent for kinetic studies and rapid dynamic transitions.
Applications:
- Protein folding/unfolding.
- Transient interactions.
- Diffusion-based dynamics.
smFRET microscopy refers to the spatial imaging of single molecules labelled for FRET, usually fixed or immobilised on surfaces, using wide-field or total internal reflection fluorescence (TIRF) microscopy.
Key features:
- Molecules are immobilised on coverslips or within supported lipid bilayers.
- Allows longer observation windows (seconds to minutes).
- Uses camera-based detection (e.g., EMCCD or sCMOS).
- Spatial resolution enables visual localisation and tracking of individual molecules.
- Can observe multiple molecules simultaneously (parallelisation).
Applications:
- Real-time conformational changes.
- Multi-state dynamics in motor proteins.
- Imaging molecular interactions in membrane environments.
Comparison between confocal smFRET and TIRF
Feature
Molecule state
Spatial resolution
Temporal resolution
Parallel molecule tracking
Setup
Output
Use cases
smFRET Spectroscopy
Typically diffusing
Low
Very high (μs-ms)
Limited (usually one at a time)
Confocal/multiphoton
Fluorescence bursts, FRET histograms
Fast kinetics, solution-phase dynamics
smFRET Microscopy
Immobilised
High (can localise molecule positions)
Moderate (ms-s)
High (hundreds in field of view)
Wide-field/TIRF
Image sequences, trajectory-based FRET data
Long-term conformational tracking, surfaces
What are the challenges and considerations of smFRET?
While smFRET offers numerous advantages, researchers must consider:
- Photobleaching: Fluorophores can degrade over time, limiting observation periods
- Labelling efficiency: Ensuring specific and efficient labelling of molecules is critical
- Data interpretation: Analysing smFRET data requires sophisticated analysis tools to accurately interpret molecular behaviours (something that the EI-FLEX can support you with)
Who can use smFRET?
smFRET is a versatile technique used by researchers across structural biology, biochemistry, biophysics, and cell biology to study dynamic molecular processes at the single-molecule level. It’s being widely adopted in both academic and industrial settings by scientists aiming to uncover the mechanisms of molecular interactions, conformational changes, and complex assemblies. Emerging core users include:
1. Biophysicists
- Use smFRET to study the structural dynamics of biomolecules (like proteins, RNA, and DNA) at the single-molecule level
- Investigate conformational changes, folding/unfolding pathways, or interaction dynamics
2. Structural Biologists
- Apply smFRET to supplement cryo-EM or X-ray crystallography data with dynamic, real-time conformational information
- Useful in mapping distance distributions and motions between domains in biomolecules
3. Biochemists and Molecular Biologists
- Use it to explore protein-protein, protein-nucleic acid, or receptor-ligand interactions
- Suitable for investigating complex biochemical processes like enzymatic activity or transcription
4. Cell Biologists
- Employ smFRET for live-cell imaging to monitor dynamic molecular events, such as signalling cascades or receptor activation
5. Nanotechnologists and Bioengineers
- Use smFRET for studying synthetic biomolecular devices, DNA origami, or molecular motors
- Important in evaluating conformational switching or mechanical motion
6. Pharmaceutical and Drug Discovery Scientists
- Useful in characterising binding mechanisms of small molecules to targets
- Enables screening of compounds affecting macromolecular dynamics
7. Academic Labs and Core Facilities
- Many university-based labs, especially in chemistry, biology, and physics departments, routinely use smFRET
- Core facilities often provide access to the specialised equipment and training needed for smFRET experiments
- See how second-year undergraduate student Hermia Kung at the University of York used smFRET to explore the conformational landscape of the DNA hairpins as a function of ionic strength, revealing how electrostatic interactions govern structural transitions
What does the future of smFRET hold?
The field of smFRET continues to evolve with advancements in fluorophore chemistry, detection technologies, and data analysis methods. Emerging applications include in vivo studies, high-throughput screening, and integration with other single-molecule techniques to provide a more comprehensive understanding of molecular mechanisms.
Exciting Instrument’s CEO and co-founder, Tim Craggs, reflects on the future of smFRET with the following comment:
Dr Timothy Craggs
Founder & CEO
“By making smFRET experiments accessible to the widest possible range of scientists, we are enabling a future in which single-molecule data will solve previously impossible problems, aiding the development of new drugs and diagnostics, but also providing the fundamental underpinning to complex biological processes.”