Fluorescence spectra viewer
Overlay excitation and emission spectra and see which pairs will separate.
Up to six. Ordered by emission wavelength within each group.
| Fluorophore | Ex | Em | Stokes | Brightness | 488 nm | 561 nm |
|---|---|---|---|---|---|---|
| EGFP | 488 | 507 | 19 | 33.5 | 100% | 0% |
| mCherry | 587 | 610 | 23 | 15.8 | 8% | 64% |
Ex, Em and Stokes shift in nanometres. Brightness is ε × Φ ÷ 1000, a property of the molecule alone. The laser columns give the fraction of peak absorptivity at that line — multiply by the extinction coefficient to get ε where you are actually exciting.
curves are peak-normalised; laser column = EX(λ) as a fraction of the excitation maximum- Fluorophore spectra, extinction coefficients and quantum yields — FPbase, Nature Methods, 2019
- Fluorescent protein properties and the trade-offs between them — Nature Methods, 2016
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Every curve is scaled to its own maximum, so a tall peak does not mean a bright fluorophore — compare the brightness column for that. Spectra are measured on purified protein or free dye in buffer; inside a cell, pH, chloride and the fusion partner all shift them a little, and a few of these are shifted enough to matter. Where FPbase carries an absorption spectrum but no separate excitation spectrum, the absorption curve is drawn in its place.
When to use this
Use this when you are choosing fluorophores and need to see whether they will separate, or when you want to know how well a laser line you actually have excites something. It answers questions about the molecules. If the question is about your filters — what a given cube will collect, or how much of one channel is really another — the filter set checker is the tool, and if you are choosing between fluorophores on brightness rather than colour, use the brightness comparison.
Worked example
You are planning a two-colour experiment on a confocal with 488 and 561 nm lines.
- Fluorophores
- EGFP, mCherry
- Laser lines
- 488, 561
Result
The pair is clean in one direction and not the other: 561 cannot excite EGFP at all, but 488 does excite mCherry to 8% of its peak, so some red signal will appear in a 488-only image and it will not be a filter fault.
What people get wrong
- Reading peak height as brightness. Every curve is normalised to its own maximum, so a fluorophore with a tenth of the extinction coefficient draws exactly as tall a peak. The brightness column is the number that compares them, and it can differ by a factor of ten between two curves that look identical.
- Judging separation from the two emission maxima alone. What causes bleed-through is the tail, not the peak — EGFP still emits about 1% of its photons beyond 625 nm, which is enough to see in a red channel from a bright sample.
- Assuming the excitation spectrum is negligible away from its peak. EGFP is still at about 17% of maximum on a 405 nm line, because its chromophore has a protonated form absorbing near 400 nm, so a violet channel is never as clean as the maxima suggest.
- Treating a published spectrum as what your sample will do. These are measured on purified protein or free dye in buffer; pH, chloride and the fusion partner all shift things, and for a pH-sensitive protein such as EYFP inside an acidic compartment the shift is large enough to change the experiment.
Questions
+Where do the spectra come from?
FPbase, whose data terms place it under no copyright restriction for commercial or non-commercial use, asking only that the original authors of each measurement are credited. The set here is curated to 45 fluorophores people actually image with rather than mirrored wholesale.
+Why is the excitation curve dashed and the emission filled?
So the two are distinguishable without colour, since fluorophores are drawn in colours derived from their own emission wavelength and several of them are close together. The fill also makes overlapping emission — the thing that causes trouble — visible where two outlines would cross confusingly.
+What is a Stokes shift and does a big one help?
It is the gap between the excitation and emission maxima. A large one makes a fluorophore easier to separate from its own excitation light and lets it share a laser line with something else, which is why large-shift proteins such as mPlum are chosen despite being dim.
+Why do some entries have no brightness figure?
Because no extinction coefficient or quantum yield is published for them in the source database. Several older dyes are in that position. The tool shows a dash rather than a guess, and those entries also cannot be used as a FRET acceptor.
Related tools
- Filter set and channel checker — Check a multi-colour panel against your filters before you stain anything.
- FRET pair calculator — Förster radius from real spectra, and the artefacts that will spoil the measurement.
- Fluorophore brightness comparison — Rank fluorophores by what your setup will actually detect, not by ε × Φ alone.
Science last reviewed .