Calculator Inputs
Use gaussian mode for smooth modeled spectra or custom mode for measured wavelength-intensity pairs. The input grid uses 3 columns on large screens, 2 on smaller screens, and 1 on mobile.
Example Data Table
Use this sample to understand how donor emission and acceptor absorption are aligned before running the calculator.
| Wavelength (nm) | Donor emission | Acceptor absorption |
|---|---|---|
| 480 | 0.10 | 12000 |
| 500 | 0.35 | 26000 |
| 520 | 0.92 | 48000 |
| 540 | 1.00 | 69000 |
| 560 | 0.74 | 80000 |
| 580 | 0.28 | 64000 |
Formula Used
I(λ) = A × exp[-4 ln(2) × ((λ − λ0)² / FWHM²)]
FD,norm(λ) = FD(λ) / ∫FD(λ)dλ
J = ∫FD,norm(λ) × εA(λ) × λ4 dλ
R0(nm) = 0.02108 × [(κ² × ΦD × J) / n4]1/6
E = 1 / [1 + (r / R0)6]
The calculator also reports normalized intersection overlap and Jaccard overlap. These help compare spectral matching even when absolute extinction values differ.
How to Use This Calculator
- Select gaussian mode for modeled curves or custom mode for measured spectra.
- Enter the wavelength range and sampling step.
- Provide donor emission and acceptor absorption parameters or paste wavelength-value pairs.
- Add donor quantum yield, orientation factor, refractive index, and donor–acceptor distance.
- Press Calculate Spectral Overlap to show results above the form.
- Review the overlap integral, spectral match, Förster radius, FRET efficiency, and peak separation.
- Inspect the Plotly chart to confirm whether the donor tail significantly overlaps the acceptor band.
- Download the full dataset as CSV or PDF for reporting, lab notes, or fluorophore selection studies.
FAQs
1) What does spectral overlap tell me?
It shows how strongly donor emission coincides with acceptor absorption over wavelength. Greater overlap usually improves the chance of energy transfer, provided distance, orientation, and medium conditions are also favorable.
2) Why is λ⁴ included in the overlap integral?
The λ⁴ term weights longer wavelengths more strongly in the Förster overlap expression. This reflects how transfer efficiency depends on the donor emission distribution and the acceptor absorption strength across wavelength.
3) When should I use gaussian mode?
Use gaussian mode when you know approximate peak positions and widths but do not have measured spectra. It is quick for screening dye combinations and testing how shifts or broadening change overlap.
4) When should I use custom data mode?
Use custom mode when you have instrument data or literature tables. It better captures asymmetry, shoulders, and nonideal band shapes that simple gaussian models may miss.
5) Does a higher overlap always mean higher FRET efficiency?
Not always. Strong overlap helps, but efficiency also depends heavily on donor quantum yield, dipole orientation, refractive index, and especially donor–acceptor distance through the sixth-power distance relationship.
6) What units should acceptor absorption use?
For physically meaningful Förster radius estimates, use molar extinction coefficients in customary spectroscopy units. Relative values still help compare shapes, but absolute overlap and radius become scaled rather than strict physical values.
7) What is a good wavelength step size?
Small steps improve numerical integration accuracy. A step around 1–2 nm is usually a solid starting point for smooth spectra, while finer spacing can help if your curves change rapidly.
8) Why are peak separation and centroid both reported?
Peak separation compares only the highest points, while centroids reflect the full band shape. Together they show whether broad tails or skewed spectra create overlap beyond the apparent peak positions.