An Introduction to Fluorescence

May 31, 2011

Fluorescence is an effect which was first described by George Gabriel Stokes in 1852. He observed that fluorite begins to glow after being illuminated with ultraviolet light. Fluorescence is a form of photoluminescence which describes the emission of photons by a material after being illuminated with light. The emitted light is of longer wavelength than the exciting light. This effect is called the Stokes shift.

Fluorescence as a tool in microscopy

Fluorescence is widely used in microscopy and an important tool for observing the distribution of specific molecules. Most molecules in cells do not fluoresce. They therefore have to be marked with fluorescing molecules called fluorochromes. The molecules of interest can be labeled directly, (e.g. DNA with DAPI) or they can be immunostained with fluorochromes that are coupled to specific antibodies. Fixation of the cells is usually necessary for immunostaining.

Fluorescence microscopy also allows time lapse imaging of living cells or tissue. For this purpose proteins of interest can be tagged with genetically encoded fluorescing molecules like GFP (green fluorescing protein). Molecules of interest (e.g. Ca2+) can also be tagged using reversibly binding synthetic dyes (e.g. fura-2) or genetically modified naturally occurring proteins (e.g. GFP-derivates).

Fig. 1: When light of a certain wavelength (excitation wavelength) hits a molecule (e.g. in a fluorophore) the photons are absorbed by electrons of the molecule. The electrons are then lifted from their ground state (S0) to a higher energy level, the excited state (S1’). This process is called excitation (1). The excited-state lifetime is short (typically 10–9/–10–8 seconds) and some of the energy of the electron is lost during that time (2). When the electrons leave the excited state (S1) and return to the groundstate (3), they lose the remaining energy taken up during excitation. In the case of fluorophores, the energy is emitted as light (fluorescence emission) of a longer wavelength with (and therefore with less energy) than the excitation light. This phenomenon is called Stokes’ shift.

Changes in the energy states of electrons lead to luminescence

Luminescence describes the occurrence of luminous effects that are caused by the change of an electron from an excited state to a state with lower energy. Electrons can exist in different energy states. The ground state is a very stable state for an electron and has the lowest energy level. If electrons absorb energy, they can be elevated to a higher energy level, an excited state. As the excited state is of higher energy than the ground state, energy has to be released when an electron returns to its ground state. This energy can be released in the form of emitted photons.

There are several forms of luminescence differing in the way the system is excited, e.g. in electroluminescence the system is excited via an electric current, chemiluminescence occurs due to a chemical reaction and photoluminescence results from the excitation via photons.

Photoluminescence can further be divided into two sub-groups, fluorescence and phosphorescence. The main difference between fluorescence and phosphorescence is the duration of their luminescence. Fluorescence immediately ends when illumination is stopped. In contrast, phosphorescence can last for hours after the excitation has ended.

The mechanism of fluorescence

Fluorochromes will only fluoresce if they are illuminated with light of the corresponding wavelength. The wavelength depends on the absorption spectrum of the fluorophore and it has to be ensured that an appropriate quantity of energy is delivered to elevate the electrons to the excited state. After the electrons are excited they can dwell in this high energy state for a very short time only. When the electrons relax to their ground state or another state with a lower energy level, energy is released as a photon. As some of the energy is lost during this process, light with an increased wavelength and lower energy is emitted by the fluorochrome compared to the absorbed light.

Fig. 2: COS 7 cells, Green: uncharacterized protein, GFP, Red: α-Tubulin, Cy3, Blue: Nuclei, DAPI. Courtesy of Prof. Wei Bian, Cell Research Center, Institute of Biochemistry and Cell Biology, SIBS, CAS, Shanghai, China
Fig. 3: Mouse fibroblasts, Green: F-Actin, FITC, Red: Tubulin, Cy5, Blue: Nuclei, DAPI. Courtesy of Dr. Günter Giese, Max Planck Institute for Medical Research, Heidelberg, Germany
Fig. 4: Murine primary hippocampal neurons, Blue: Marker for transfected cells, Green: Actin, TRITC, Red: GluA Ampa receptor unit, Texas Red, Grey: Synaptic vesicle protein, Cy 5
Fig. 5: D. melanogaster Larvae, Green: RNA-binding protein, Alexa 488

The mechanism of phosphorescence

As phosphorescing molecules can luminesce for a much longer time than fluorochromes, there must be a difference in the way they store the excitation energy. The basis for this discrepancy is found in the two forms of excitation levels, the singlet excited state and the triplet excited state, which are based on different spin alignments.

Spins are an attribute of electrons. In simplified terms, the spin describes the angular momentum of the electron caused by its rotation. The orientation of an electron’s spin can be positive (+1/2) or negative (–1/2). Spin pairs of higher energy levels can either be parallel or antiparallel in their orientation to each other. In antiparallel spin pairs the individual angular momentums compensate each other and the total spin gets a value of zero. This spin alignment is called singlet state. Two parallel spins do not compensate and get a value different from zero. In this case the spins are said to be in a triplet state.

Fluorescence occurs when electrons go back from a singlet excited state to the ground state.  But in some molecules the spins of the excited electrons can be switched to a triplet state in a process called inter system crossing. These electrons lose energy until they are in the triplet ground state. This state is of higher energy than the ground state but also of lower energy than the singlet excited state. The electrons can therefore not switch back to the singlet state, nor can they easily go back to the ground state, as only total spins with a value of zero are allowed due to quantum mechanics. The molecules are therefore trapped in their energy state. 

But a few changes from the triplet ground state to the ground state are possible at a time. These changes give rise to the emission of photons and the phosphorescence. As only a few events are possible at a time, the triplet ground state presents a kind of energy reservoir, making phosphorescence possible over a longer time period.

Fig. 6: Drosophila melanogaster, larval stadium, Green: Feb211 positive neurons and their axons, Alexa 488, Red: fibrous part of the cns (i.e all axons), Cy3, Blue: Nuclei, DAPI. Courtesy of Dr. Christoph Melcher, Research Institute Karlsruhe, Institute for Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany
Fig. 7: Mouse kidney section, Green: glomeruli and convoluted tubules, Alexa 488 WGA, Red: F-Actin (prevalent in glomeruli and brush border), Blue: Nuclei, DAPI
Fig. 8: Neonatal cardiac myocyte, Yellow: DNA, DAPI, Green: Myomesin, Cy3, Red: Cadherin, Texas Red, Blue: Actin, Alexa 633
Fig. 9: Metaphase-spread FISH - stained chromosomes acquired with a fluorescence Stereomicroscope. Courtesy of Dr. Yumiko Suto, Laboratory of Human Evolution, Graduate School of Frontier Sciences, The University of Tokyo

Luminescence in microscopy

For microscopy, fluorescence is the most useful kind of luminescence. Fluorochromes can easily be excited with their specific wavelength via specific light sources (e.g. lamps and filter systems or lasers) and the emitted light can be distinguished from the excitation light by the wavelength (Stokes’ shift).

Using fluorescence imaging, the experimenter can characterize the amount and the localization of a molecule inside a cell. Another advantage of fluorescence microscopy is that several fluorochromes can be used simultaneously. The fluorochromes only have to vary in their excitation and emission wavelength. Hence, different target molecules can be observed simultaneously, allowing a huge variety of experiments e.g. colocalization studies, to be performed.