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What is luminescence? Luminescence definition

The term luminescence refers to the emission of light that is not caused by high temperature. The phenomenon is therefore also known as "cold light".

There are many light-emitting processes all around us. They are either naturally occurring or artificially produced. Examples of natural luminescence include fireflies and phytoplankton (bioluminescence). Light-emitting diodes (LEDs) and computer monitors (electroluminescence) are examples of man-made luminescence.

Types of luminescence

In Life Sciences different types of luminescence are used. The different types of luminescence can be grouped by two different criteria: by method of substance excitation and by duration of signal emission.

A. Types of luminescence by method of generation of high-energy substance:

Most types of luminescence involve a high-energy substance which loses energy in the form of light. The mechanism by which this substance is generated or gets the “extra” energy to be released as light is a useful way to classify luminescence.

 

1. Chemiluminescence

Chemiluminescence describes the emission of light as a result of a chemical reaction. The reaction enthalpy provides the energy required, producing an excited product or intermediate. When the intermediate falls into its ground state, it emits a photon.

1.1 Bioluminescence

If such a reaction takes place in a living organism, e.g. in fireflies, this is called Bioluminescence. This biochemical reaction involves a light-emitting molecule called luciferin and an enzyme called luciferase. Luciferases are a family of photo-proteins that can be found in various insects, marine organisms, and prokaryotes [1]. These enzymes catalyse the oxidation of the luciferin, resulting in photon emission. Depending on the organism, the enzyme and substrate produced as well as the co-factors required are different. As a result, the emission wavelength of the light produced varies producing emission spectra ranging between 400 nm and 620 nm.

One of the most commonly used luciferases in Life Sciences is Firefly Luciferase emitting yellow-green light at approximately 550 – 570 nm. Another great example is Renilla Luciferase from the sea pansy Renilla reniformis, emitting blue light at 480 – 500 nm

2. Photoluminescence

Photoluminescence describes the luminescence of a substance excited (that is, brought to a higher energy level) by light, usually ultraviolet or visible. This is called photoexcitation and is the result of moving electrons to energetically higher levels through the absorption of photons. As a result of the excitation, various relaxation processes usually occur in which typically lower energy photons are re-emitted. Fluorescence and phosphorescence are the main types of photoluminescence, and a molecule with fluorescent properties is called a fluorophore. In Life Sciences, this form of luminescence is most commonly used in fluorescence assays.

BRET and FRET involve a special type of energy transfer which could be considered as a form of photoluminescence. In both methods there are two molecules or groups, a donor and an acceptor, the donor being either a luciferase (in BRET) or a fluorophore (in FRET), and the acceptor being a fluorophore that can be excited at the emission wavelength of the donor. The excitation of the acceptor by the donor is reminiscent of fluorescence, however, in both BRET and FRET, the excess energy is transported to the acceptor through a non-radiative process in the form of the emitted virtual photon — this transfer is facilitated by dipole-dipole couplings between the molecules and is called resonance.  The term virtual is indicative of the fact that the photon is reabsorbed before its properties, such as wavelength, take on physical significance [3].

3. Electroluminescence

Electroluminescence describes the generation of light as a result of an electric current passed through a substance. In Life Sciences this is typically used in some forms of Immunoassay and in Immunochemistry for clinical applications.

The Electrochemiluminescent Immunoassay (ECLIA) [2] is a highly sensitive method in which an electrochemical intermediate is generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. The molecule emits light when relaxed to a lower energy level.

 

4. Radioluminescence

Radioluminescence occurs when specific substances are hit by ionizing radiation like α, β or γ rays. In the recent past (1960s) this phenomenon was being used to make watch dials glow in the dark.

This principle is applied for the separation and detection of radioactive tracers by high-performance liquid chromatography (radio HPLC) in various pharmaceutical and clinical applications. In radio HPLC, an electron hits a scintillator molecule and lifts it to a higher energy level. The scintillator immediately jumps back to its initial energy level by emitting photons. These photons are detected by a photomultiplier tube (PMT).

In the Life Sciences field the term “luminesce” is most often used to refer to chemiluminescence (and, by extension, bioluminescence) as opposed to fluorescence. But, as you have seen above, fluorescence is a subtype of luminescence. Learn more about the differences between luminescence, fluorescence, and phosphorescence. As this is the usual convention in the field, in the rest of the article we are going to use the term luminescence to refer to chemiluminescence and bioluminescence, unless stated otherwise.

B. Types of luminescence by duration of signal emission

1. Flash luminescence

Assays that produce a short but usually strong signal are called flash assays. The signal half-life of these assays is typically in the range of a few minutes or even shorter. The challenge with flash assays is that once you add the test reagent, the signal peaks almost immediately and then quickly declines. This is not a problem when using a single-tube luminometer, as each sample can be measured individually immediately after adding the starter reagent. But what about measuring in a microplate luminometer instead?

If one were to start the reaction in all wells simultaneously in a 96-well plate and then measure sequentially, only the first wells would be measured at the peak of the signal strength. In the later measured wells, however, the intensity of the luminescence signal would already be significantly reduced.

Therefore, the measurement of flash assays in a microplate reader requires the use of so-called injectors. In this way, the starter reagent can be added first in each well and the signal measured immediately. The Dual-Luciferase Reporter™ assay and the SPARCL assays are both flash-type assay examples.

2. Glow luminescence

Glow-type assays, on the other hand, have a signal intensity that lasts for hours. Although their signal intensity is reduced, the much simpler workflow due to the longer signal intensity, which does not require reagent injectors, often makes this form of luminescence assay the method of choice. However, as all wells are emitting light at the same time, it is possible that light coming from adjacent wells interferes in the measurement, in a phenomenon called crosstalk (see below).