What are fluorescence, phosphorescence and luminescence? We all associate them with the glowing effect but what sets them apart from each other? While these mechanisms by which things glow are all physically related and can all be grouped within luminescence, they are fundamentally different in the underlying physical mechanism as well as in the effects.

Luminescence

Luminescence – The Simple Explanation

There are three main forms of luminescence at play: fluorescence, phosphorescence and chemiluminescence. Fluorescence and phosphorescence are two forms of photoluminescence. In photoluminescence, a substance’s glow is triggered by light, in contrast to chemiluminescence, where the glow is caused by a chemical reaction. Both fluorescence and phosphorescence are based on the ability of a substance to absorb light and emit light of a longer wavelength and therefore lower energy. The main difference is the time in which it takes to do so. In fluorescence, the emission is basically immediate and therefore generally only visible, if the light source is continuously on (such as UV lights); while phosphorescent material can store the absorbed light energy for some time and release light later, resulting in an afterglow that persists after the light has been switched off. Depending on the material, this afterglow can last anywhere from a few seconds to hours.

So if it disappears immediately, it’s fluorescence. If it lingers, it’s phosphorescence. And if it needs activation, it’s chemiluminescence. Simple!

Picture a scene at the night club: teeth, eyes and fabric glowing under the black light are fluorescent, the emergency exit sign is phosphorescent and the glow sticks are chemiluminescent. The light produced by the glow stick is a result of two chemicals that were mixed when a small capsule in the stick was broken and mixed by shaking the stick.

Luminescence – The Complex Explanation

“But how exactly does it work?” –you may ask? Be prepared to dive into your remnant knowledge of physical chemistry. To understand the principles behind why molecules absorb and emit light, we need to first look at electrons and understand their ground and excited states and electron spin, concepts that are still not fully understood by modern quantum mechanics.

Photoexcitation
A pre-requisite for photoluminescence, regardless of whether it is fluorescence or phosphorescence, is the ability of a molecule to absorb light radiation leading to electronic excitation. A molecule-bound electron absorbs a photon and therefore its energy and becomes excited. It transforms from ground state (S₀) into an excited state (S_n) instantaneously (~10^-15 sec). Excitation might result in the electron occupying a variety of different vibrational levels (v=n) of the excited state, depending on the exact wavelength of the absorbed photon. The resulting electronically excited states are intrinsically unstable and electrons will relax back to their ground state by several combinations of mechanical steps, dissipating energy in different ways in the process.

Vibrational relaxation is extremely rapid (10-¹² to 10^-10 sec) and leads to dissipation of the energy within one excited state through vibrational energy, which is quickly dissipated as heat to neighboring molecules. As the energy is not dissipated by the emission of light, vibrational relaxation is a non-radiative transition.

Internal Conversion is another non-radiative transition, which is iso-energetic and also rapid (10^-14 to 10^-11 sec). It describes the transition of one electronically excited state (S_n+1) into a vibrational state of a lower excited state (S_n) of the same energetic level. No energy is dissipated during the transition. An electron can fully dissipate the initially absorbed energy through vibrational relaxation and internal conversion alone. In this case the relaxation process will be entirely non-radiative and the molecule will neither fluoresce nor phosphoresce and all absorbed energy will be dissipated through heat. The probability at which radiative events occur versus fully non-radiative relaxation defines the quantum yield of a fluorophore and therefore how bright it will shine.

Fluorescence

One radiative mechanism by which excited electrons may relax is a light-emitting transition from the lowest excited state (S₁) to ground state (S₀) in a fast (10^-9 to 10^-6 sec) process called fluorescence. The energy difference is dissipated by emitting a photon. Due to the electron having shed some of the original excitation energy by vibrational relaxation, the emitted photon will be of lower energy and thus of longer wavelength.

Similar to excitation, emission may generally relax to a variety of vibrational levels (v=n) of the ground state (S₀), resulting in a bandwidth of possible wavelengths of the emitted photon. Electrons relax from higher vibrational levels (v=n) further to vibrationless state (v=0) by non-radiative vibrational relaxation, if necessary. The resulting emitted wavelength is independent of the excitation wavelength, as usually excited molecules decay to the lowest vibrational level of the lowest excited state by non-radiative processes before fluorescence emission takes place. Depending on the molecule, non-radiative decay might be responsible for dissipating a smaller or bigger portion of the excitation energy, resulting in molecule-specific shifts between the excitation wavelengths and the wavelengths being emitted. This phenomenon is termed Stokes shift. The possible wavelength of a photon that might be absorbed in the excitation, as well as the possible emitted wavelengths –which vary due to fluorescent decay onto different vibrational levels– define together a molecule’s excitation and emission spectra. As frequently the same electronic transitions are involved in excitation and emission of a fluorescent molecule, the excitation and emission spectra often resemble reflections of each other, which is referred to as the mirror image rule of fluorescence.

Phosphorescence

To understand the difference between fluorescence and phosphorescence, we need to take a little detour into electron spin. Spin is a fundamental, unvarying property of the electron and a form of angular momentum that defines behavior in an electromagnetic field. Electron spin can only have the value of ½ and the spin orientation is either up or down. An electron’s spin is therefore designated as +½ or -½, or alternatively as ↑ or↓. Two electrons in a single orbital will always have antiparallel spin at singlet ground state (S₀). Upon promotion of one electron into excited state, the electron maintains its spin orientation and a singlet excited state (S₁) is formed, where the both spin orientations remain paired as antiparallel. All relaxation events in fluorescence are spin neutral and the spin orientation of the electron is maintained at all times.

However, this is different for phosphorescence. Fast (10^-11 to 10^-6 sec) Intersystem crossing from singlet exited state (S₁) to an energetically favorable triplet excited state (T₁) leads to inversion of the electron spin. Triplet excited states are characterized by parallel spin of both electrons and are metastable. Relaxation occurs via phosphorescence, which results in another flip of the electron spin and the emittance of a photon. The return to relaxed singlet ground state (S₀) might occur after considerable delay (10^-3 to >100 sec). Additionally, more energy is dissipated by non-radiative processes during phosphorescent relaxation than in fluorescence, therefore the energy difference between the absorbed and emitted photon is bigger and the wavelength shift more pronounced. Thus, phosphorescence is characterized by a bigger Stokes shift than fluorescence.

Chemiluminescence

While the mechanisms involved in relaxation of the electronically excited state are in principle the same as in fluorescence, chemiluminescence differs remarkably in the initial generation of the excited state. While most exoergic chemical reactions release the excess energy of the reaction through heat and are exothermic, certain chemical reactions can lead to a product in electronic exited state. If the excited state relaxes through luminescence by emitting a photon, the process is called chemiluminescence. If the reaction is catalyzed by biologic enzymes, we speak of bioluminescence, although the mechanisms are identical.

If the energy generating the excited state stems from mechanical energy (e.g. the pulling off of an adhesive tape), the resulting light emittance is termed triboluminescence.

Fluorescence and (bio-)luminescence are vital means of detection and quantification in a broad variety of products within the portfolios of Enzo Life Sciences. Our CELLESTIAL® product range offers a wide range of fluorescent dyes to visualize a broad variety of cellular compartments and processes. Detection of target DNA by fluorescent in situ hybridization can be achieved by nick translation in combination with our SEEBRIGHT® fluorescent dye-dUTPs. We also offer a variety of immune- and biochemical assays utilizing luminescent quantification. For further insight into these methods, please have a look at our TechNotes on successful research tips, molecular biology and drug discovery. Or contact our Technical Support Team for further assistance.

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