Fluorescence

Fluorescence is light emitted when an electronically excited molecule or crystal returns to its ground state through a spin-allowed transition. The absorbed photon lifts the molecule to an excited singlet state; within picoseconds it relaxes to the lowest vibrational level of the first excited state (Kasha's rule), and after a lifetime of typically 1–10 ns it emits a photon of lower energy. For fluorescein in basic solution the absorption peak is near 490 nm, the emission peak near 515 nm, the quantum yield about 0.95 and the lifetime about 4.1 ns. It is one form of photoluminescence; the other, phosphorescence, comes from a spin-forbidden triplet state and lasts microseconds to seconds.

Stokes shift, quantum yield and lifetime

Because vibrational energy is lost before emission, the emission spectrum lies at longer wavelength than the absorption, the Stokes shift. For fluorescein, $10^7/490 - 10^7/515 \approx 990$ cm⁻¹. The shift is what allows excitation light to be separated from fluorescence with filters, and emission spectra are often an approximate mirror image of the lowest absorption band.

The excited state decays by a radiative rate $k_r$ and a non-radiative rate $k_{nr}$. The measured lifetime and the quantum yield (photons emitted per photon absorbed) are

$$\tau = \frac{1}{k_r + k_{nr}}, \qquad \Phi = \frac{k_r}{k_r + k_{nr}}.$$

With $\Phi = 0.95$ and $\tau = 4.1$ ns, $k_r = \Phi/\tau = 2.3 \times 10^8$ s⁻¹ and $k_{nr} = 1.2 \times 10^7$ s⁻¹; the natural radiative lifetime $1/k_r$ is 4.3 ns. The emission itself is spontaneous emission, so the decay after a short pulse is exponential for a single emitting species.

Quenching and energy transfer

Collisions with a quencher such as oxygen, iodide or acrylamide open an extra non-radiative channel, described by the Stern–Volmer relation

$$\frac{F_0}{F} = 1 + k_q \tau_0 [Q].$$

For a diffusion-limited $k_q \approx 10^{10}$ M⁻¹s⁻¹ and $\tau_0 = 4.1$ ns, the Stern–Volmer constant is 41 M⁻¹, and 10 mM of quencher reduces the fluorescence by a factor of 1.41. Förster resonance energy transfer (FRET) is non-radiative transfer to a nearby acceptor with efficiency

$$E = \frac{1}{1 + (r/R_0)^6},$$

where the Förster distance $R_0$ is typically 2–6 nm. For $R_0 = 5$ nm, the efficiency is 0.96 at 3 nm, 0.50 at 5 nm and 0.12 at 7 nm; this steep distance dependence makes FRET a molecular ruler.

Measurement

A spectrofluorometer excites the sample through one monochromator and analyses emission at 90° through a second, so that transmitted light does not enter the detector. Quantum yields are measured relative to a standard, such as quinine sulfate in 0.5 M sulfuric acid ($\Phi = 0.546$), or absolutely with an integrating sphere. Lifetimes are measured by time-correlated single-photon counting with a pulsed laser and a photomultiplier or single-photon detector, or by the phase shift under modulated excitation.

Where it matters

Fluorescence microscopy separates excitation and emission with a filter cube built around a dichroic mirror; confocal microscopy adds a pinhole to reject out-of-focus fluorescence. Fluorescent proteins and dyes label cells, DNA sequencing reads fluorescently tagged bases, and flow cytometry counts labelled cells. In optics labs, fluorescence is a background to be filtered: glass, cements and plastics fluoresce under blue or UV excitation, and Raman measurements often need strong notch filters and near-infrared excitation to avoid it. Fluorescence also appears as the emission step in phosphors and in the spontaneous output of laser gain media.

Pitfalls

At absorbance above about 0.05, the inner-filter effect distorts spectra: excitation light is absorbed before the observed volume, and emission is reabsorbed. Photobleaching destroys fluorophores under continuous illumination, so intensity falls during measurement. Raman scattering from the solvent appears as a peak that moves with the excitation wavelength, whereas true fluorescence does not move. Dissolved oxygen quenches long-lifetime emitters, and fluorescence yields depend on pH, temperature and solvent.

Common questions

What is the difference between fluorescence and phosphorescence?

Fluorescence comes from a singlet excited state and decays in nanoseconds. Phosphorescence comes from a triplet state reached by intersystem crossing; the transition to the ground state is spin-forbidden, so it lasts microseconds to seconds.

Why is fluorescence at a longer wavelength than the excitation?

The molecule loses part of the absorbed energy as vibrational energy before emitting, so the emitted photon carries less energy. Anti-Stokes emission occurs only in special cases such as upconversion or thermally assisted excitation.

Is fluorescence the same as scattering?

No. Scattering redirects light without it being absorbed and follows the excitation instantly; fluorescence involves absorption, a nanosecond excited-state lifetime and emission at a different wavelength.

References: J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006); B. Valeur, M. N. Berberan-Santos, Molecular Fluorescence, 2nd ed. (Wiley-VCH, 2012).