Fluorescence Spectra Overlap Calculator

Compare excitation/emission spectra, spectral overlap, and filter crosstalk.

Fluorophore 1 (FRET donor)

Fluorophore 2 (FRET acceptor)

Laser line (nm)Emission filter centre (nm)Emission filter width (nm)Orientation factor κ²Refractive index nDonor–acceptor distance r (nm)

Results

EGFP

Ex/Em peak (FPbase)488/507 nm

ε, Φ, brightness εΦ55,900 M⁻¹cm⁻¹, 0.6, 33.5 mM⁻¹cm⁻¹

Stokes shift19 nm

Excitation at the laser99.8 % of peak

Emission in the filter70.6 %

mCherry

Ex/Em peak (FPbase)587/610 nm

ε, Φ, brightness εΦ72,000 M⁻¹cm⁻¹, 0.22, 15.8 mM⁻¹cm⁻¹

Stokes shift23 nm

Excitation at the laser7.6 % of peak

Emission in the filter0.0 %

mCherry signal / EGFP signal in the filter0.00 %

EGFP signal / mCherry signal in the filter230520328.88 %

Förster overlap J1.944e+15 M⁻¹cm⁻¹nm⁴

Förster radius R₀5.11 nm

FRET efficiency at r53.2 %

Spectra

Model

signal ∝ ε f_ex(λ_laser) Φ ∫_filter F(λ)dλ / ∫F(λ)dλ

J = ∫F_D ε_A λ⁴ dλ / ∫F_D dλ, R₀ = 0.02108 (κ² Φ_D n⁻⁴ J)^(1/6) nm, E = 1/(1 + (r/R₀)⁶)

Peaks, extinction coefficients, quantum yields and spectra come from FPbase (spectra CC BY-SA 4.0), sampled every 1 nm and interpolated linearly, so the weak anti-Stokes tails are kept; the excitation spectrum stands in for the absorption. A spectrum falls to 0 within 1 nm past its record, and each record ends at 6 % of the peak or less, or below the lowest laser line (350 nm). miRFP670's excitation record starts at 520 nm and omits its Soret band near 390 nm, so its blue excitation reads 0. The filter is an ideal top hat and the two dyes are compared at equal concentration and equal laser photon flux. The orientation factor κ² = 2/3 assumes freely rotating dipoles, which fusion proteins often are not.

Model and references

Textbook approximation. A standard approximation from the literature (for example paraxial, plane-wave or Gaussian-beam), valid within the range the page states. About the tiers

Eight FPbase proteins with their FPbase spectra sampled every 1 nm, anti-Stokes tails included; the excitation spectrum stands in for absorption. Ideal top-hat filter, equal concentration and photon flux.

Lambert T. J. (2019). FPbase: a community-editable fluorescent protein database. Nat. Methods 16, 277–278. Peaks, extinction coefficients and quantum yields (read 2026-10-18); spectra from the FPbase API, CC BY-SA 4.0 (read 2026-11-05). doi:10.1038/s41592-019-0352-8
Lakowicz J. R. (2006). Principles of Fluorescence Spectroscopy, 3rd ed. Springer, §13.2. Overlap integral J and R₀ = 0.02108 (κ² Φ_D n⁻⁴ J)^(1/6) nm. doi:10.1007/978-0-387-46312-4

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