Introduction to Fluorescence Techniques – Core Vocabulary

Key vocabulary from the Molecular Probes Handbook introduction, covering fundamental fluorescence concepts, instrumentation, and probe properties.

Fluorophore

A molecule (often a polyaromatic hydrocarbon or heterocycle) that can absorb light and re-emit it as fluorescence.

Fluorescent probe

A fluorophore that has been designed or modified to respond to a specific stimulus or localize within a particular region of a biological specimen.

Excitation (Stage 1)

Absorption of a photon (hνEX) that elevates a fluorophore from its ground state (S0) to an excited electronic singlet state (S1′).

Excited-state lifetime (Stage 2)

The brief period (typically 1–10 ns) during which an excited fluorophore undergoes conformational and environmental interactions before returning to a lower state.

Fluorescence emission (Stage 3)

Release of a lower-energy photon (hνEM) as the fluorophore returns from the relaxed excited state (S1) to the ground state (S0).

Stokes shift

The energy (or wavelength) difference between absorbed photons and emitted fluorescence; critical for separating signal from excitation light.

Fluorescence quantum yield (QY)

The ratio of photons emitted to photons absorbed; a measure of fluorescence efficiency.

Jablonski diagram

An energy-level schematic that depicts absorption, excited-state processes, and fluorescence emission of a fluorophore.

Fluorescence excitation spectrum

A plot of excitation wavelength versus emitted photon number; mirrors the absorption spectrum for a single fluorophore in dilute solution.

Fluorescence emission spectrum

A plot of emission wavelength versus fluorescence intensity; largely independent of the excitation wavelength.

Extinction coefficient (EC)

A measure of a fluorophore’s light-absorbing capacity at a specific wavelength (cm⁻¹ M⁻¹); used in the Beer–Lambert law.

Brightness

Fluorescence output per molecule, proportional to the product EC × QY.

Spectrofluorometer

An instrument that measures fluorescence of bulk samples over continuous ranges of excitation and emission wavelengths.

Fluorescence microscope

Optical system that resolves spatial distribution of fluorescence in microscopic specimens (<0.1 mm) in two or three dimensions.

Fluorescence scanner

Device (e.g., microarray reader) that maps fluorescence across macroscopic, planar samples like gels or blots.

Flow cytometer

Instrument that measures fluorescence (and light scatter) of individual cells in a flowing stream to identify and quantify subpopulations.

Autofluorescence

Background fluorescence arising from endogenous components of biological samples.

Reagent background

Fluorescence from unbound or nonspecifically bound probes that can obscure true signal.

Rayleigh scattering

Elastic scattering of excitation light; must be separated from emission photons for accurate detection.

Photobleaching

Irreversible loss of fluorescence due to photochemical destruction of excited fluorophores, often via reactive oxygen species.

Antifade reagent

Chemical formulation (e.g., ProLong or SlowFade) added to samples to reduce photobleaching, especially in fixed specimens.

Photostability

Resistance of a fluorophore to photobleaching under illumination; higher photostability permits longer imaging.

Signal amplification

Strategies that increase detectable fluorescence, such as avidin–biotin layering, enzyme-linked fluorogenic substrates, or multi-fluorophore probes.

Self-quenching

Fluorescence loss caused by close proximity of identical fluorophores, common at high labeling densities.

Quenching

Any bimolecular process that reduces quantum yield without altering the emission spectrum; includes collisional and static mechanisms.

Fluorescence Resonance Energy Transfer (FRET)

Distance-dependent transfer of excited-state energy from a donor fluorophore to an acceptor, used to study molecular interactions.

Ratiometric measurement

Analytical approach that monitors the ratio of two fluorescence signals (e.g., free vs. bound indicator forms) to compensate for probe concentration and photobleaching.

Multicolor labeling

Simultaneous use of multiple spectrally distinct fluorophores to visualize different targets within one specimen.

Beer–Lambert law

Relationship A = EC × c × l linking absorbance to concentration and path length; applies to fluorescence intensity in dilute solutions.

Inner-filter effect

Non-linear fluorescence decrease at high absorbance due to re-absorption of excitation or emission photons.

BODIPY dyes

Borondipyrromethene fluorophores prized for narrow emission bandwidths and high photostability, useful in multicolor experiments.

Qdot nanocrystals

Semiconductor quantum dots with very high extinction coefficients and tunable, narrow emissions—ideal for multiplexing.

Phycobiliproteins

Bright, multi-chromophore proteins (e.g., R-phycoerythrin) with extremely high extinction coefficients used for signal amplification.

Alexa Fluor 488

Green-fluorescent dye with enhanced photostability over fluorescein, compatible with standard FITC filter sets.

Environment-sensitive dye

Fluorophore whose spectral properties change with factors like polarity, pH, or binding; enables sensing of local environments.

Fluorescence SpectraViewer

Online tool from Molecular Probes/Thermo Fisher that allows users to plot and compare excitation and emission spectra of >250 dyes.