Single-Molecule FRET: Watching a Molecule Switch Shape

Molecule & Dye Pair

Forster radius R₀ (nm) 5.5

Switching rate k (s^-1) 1.2

Closed-state population 50%

Detected photon rate (cps) 120

Live Readout

OPEN conformation

Dye-dye distance r

7.0 nm

True FRET efficiency E

0.00

Measured E (photon window)

0.00

Transitions so far

How it works

Single-molecule FRET (smFRET) tags a biomolecule with a donor dye (green, e.g. Cy3) and an acceptor dye (red, e.g. Cy5) at two points. When the molecule folds or hinges shut, the dye-to-dye distance r shrinks; when it opens, r grows. Forster resonance energy transfer converts that distance into a photon color ratio:

E(r) = 1 / (1 + (r / R0)^6)

R0 = Forster radius: the distance at which
     exactly half the donor's excitation
     energy transfers to the acceptor (E = 0.5)

Because E falls off as the inverse sixth power of distance, it is an extremely sensitive "molecular ruler" over roughly 0.5-1.5 x R0 — the regime most protein and nucleic-acid conformational changes fall into.

This simulator models the molecule as a two-state Markov system (open <-> closed) with rate constants set by the switching-rate and population-bias sliders. Each detected photon is randomly assigned to the acceptor channel with probability E(r) and to the donor channel otherwise — exactly how a real single-photon avalanche diode pair records an smFRET trace. The "Measured E" readout is the acceptor fraction inside a rolling one-second window of simulated photon counts, so at low photon rates it visibly fluctuates around the true E due to shot noise, just as in a real experiment.

R0 — sets how far apart the dyes can be before FRET efficiency collapses; shifts how sharply open vs. closed states separate.
Switching rate k — how often the molecule hops between conformations (mean dwell time is roughly 1/k per state).
Closed-state population — the equilibrium free-energy bias between the two conformations.
Photon rate — detector brightness; low rates reveal photon shot noise, high rates approach the ideal noise-free trace.

Real-world relevance: this is the core readout technique behind decades of protein-folding, riboswitch, and molecular-motor smFRET studies — the same math (Stryer & Haugland, 1967) underlies every commercial and academic TIRF-based single-molecule instrument in use today.

Donor dye Acceptor dye

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