principles of fluFluorescence describes a phenomenon where a molecular system absorbs, then emits light. In absorption high energy (short wavelength) light excites the system, promoting electrons within the molecule to transition from the ground state, to the excited state (see below). Once in this state, and after a lag period of several nano-seconds (the fluorescence lifetime), the electrons will relax back to the ground state, releasing their stored energy in an emitted photon. Due to the higher energy relaxation mechanism this emitted light is of a lower energy (longer wavelength) than the absorbed light. The difference between the excitation and the emission energy (wavelength) is termed the Stokes shift.

Certain proteins (fluorophores) can undergo fluorescence. They emit light over a range of wavelengths typically a few 10’s nanometres wide (bandwidth). Emission spectra are shown in the diagram below for some of the most commonly used fluorophores. In general fluorophores are categorised by two main types, fluorescent proteins and fluorescent dyes. Proteins can be genetically encoded for expression and specific protein labelling within the cell under study. On the other hand small molecule fluorescent dyes have been developed (typically from cyanine or fluorescein compounds) for use in immunolabelling of endogenous molecules. Together these fluorophores, along with quantum dots and other optical nanoparticles, have revolutionised biological microscopy. By tagging a molecule of interest with a fluorophore that molecule can be visualised with remarkable contrast, tracked and analysed within a live sample.

spectrum

The revolution in fluorescence microscopy began with the identification and cloning of the green fluorescent protein (GFP) from Aequorea Victoria (the box jellyfish). This breakthrough enabled the tagging of molecules through protein expression, allowing for imaging in live cell samples. The manipulation of these proteins has produced fluorophores which stretch across the visible light spectrum from the cerulean fluorescent protein (CFP) through to mCherry (red). Their application in biological research has continually broadened with the development of fluorophores that change colour over time, those that are pH-sensitive, through new measurement techniques that utilise lifetime or polarisation of the emitted photons and more recently the photoactivatable fluorophores central to super-resolution methodologies.