Fluorimetry: 3D EEM and Emission Spectrum
Explore a teaching excitation-emission matrix (EEM) interactively and read the emission spectrum at a chosen excitation wavelength.
Fluorimetry: 3D EEM and emission spectrum
Choose a fluorophore, inspect the excitation-emission matrix and pick λexc from the plot or type it in. The tool shows the matching emission slice and the relative excitation efficiency at that wavelength.
Filter by structural groups / chromophores
The structural filter compares families in the database. It does not quantitatively predict a spectrum from molecular structure.
In 2D map view, tap anywhere to choose λexc. In 3D view, tap or click the excitation axis at the base of the surface.
Emission spectrum at the selected λexc
Curve height is referred to the fluorophore’s absolute maximum: exciting away from the maxima keeps the slice low, which is what the instrument shows too. Below the diagonal — emission wavelengths shorter than λexc — the curve is deliberately suppressed.
How to read the map
An EEM is a series of emission spectra recorded while the excitation wavelength is stepped across a range. In the 3D plot the excitation axis identifies the scan, the emission axis gives the observed wavelength, and height and colour represent relative intensity.
In this simulator the molecular surface is the product of three factors, \(I = E \cdot F \cdot A\). The excitation profile E depends only on λexc and the emission profile F only on λem: both are sums of Gaussian bands centred on the maxima reported in the database, each normalised to its own maximum. The third factor A attenuates the region where λem ≤ λexc, in which a real EEM holds no fluorescence. It is a visual model, not a reconstruction of a measured spectrum.
Why the bands do not merge into a single hump
Gaussian width is capped by the spacing between the maxima listed for that fluorophore. Without that constraint the closely spaced vibronic maxima of fused aromatics — anthracene, pyrene, perylene — would be smoothed into one broad feature, and the plot would erase exactly the information the data carries. Fluorophores with a single emission maximum still get a plain Gaussian band.
Why do diagonal bands appear in a real EEM?
Elastic light scattering creates a Rayleigh band near λem = λexc; a second-order feature may also appear near λem ≈ 2λexc. The tool can display these so students learn to recognise them, but keeps them separate from simulated molecular fluorescence. Below the diagonal nothing is simulated at all: emission at a wavelength shorter than the excitation would need energy the absorbed photon does not carry, and that region of a real EEM contains only scattered light.
Structural groups and fluorescence
Conjugation, rigidity and extended aromatic systems can favour fluorescence, while substituents, protonation, solvent and environment can shift or quench emission. Structural filtering here is for comparison only: it does not convert a list of functional groups into a quantitative spectrum.
Limits of the model
The database reports maxima, not the real shape of the bands: relative intensities, asymmetries and tails are reconstructed, not measured. Relative excitation efficiency describes where λexc sits with respect to the absorption maxima — it is neither a quantum yield nor an absorbance. Solvent, pH and temperature shown in the card are the conditions of the source: change them at the bench and the maxima move too.