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cone within the crystal and is focused to a point on top the green light is absorbed and spontaneously remitted as red light not all of the light is absorbed and a small portion of the 520 nm laser light transmits through the top unaltered by the ruby s red color when an excited molecule atom or nanostructure emits a photon and the lower energy state usually the ground state has the same electronic spin multiplicity as the excited state the process is called fluorescence when the initial and final states have different multiplicity spin the phenomenon is termed phosphorescence 12 when a molecule in its ground state called s 0 is photoexcited it may end up in any one of a number of excited electronic states s 1 s 2 s 3 it can occupy different vibrational states within the electronic state these vibrational states are populated in proportion to their overlap with the ground state according to the franck condon principle 13 31 these vibrational excited states typically decay rapidly to the ground vibrational state of the excited electronic state s 1 followed by radiative transition to the ground state or to vibrational states close to the ground state this transition is called fluorescence all of these states are singlet states 14 225 a different pathway for deexcitation is intersystem crossing from the s 1 to a triplet state t 1 decay from t 1 to s 0 is typically slower and less intense and is called phosphorescence 14 225 absorption of a photon of energy h ν e x displaystyle h nu _ ex results in an excited state of the same multiplicity spin of the ground state usually a singlet s n with n 0 in solution states with n 1 relax rapidly to the lowest vibrational level of the first excited state s 1 by transferring energy to the solvent molecules through non radiative processes including internal conversion followed by vibrational relaxation in which the energy is dissipated as heat thus the fluorescence energy is typically less than the photoexcitation energy 13 38 the excited state s 1 can relax by other mechanisms that do not involve the emission of light these processes called non radiative processes compete with fluorescence emission and decrease its efficiency 13 examples include internal conversion intersystem crossing to the triplet state and energy transfer to another molecule an example of energy transfer is förster resonance energy transfer relaxation from an excited state can also occur through collisional quenching a process where a molecule the quencher collides with the fluorescent molecule during its excited state lifetime molecular oxygen o 2 is an extremely efficient quencher of fluorescence because of its unusual triplet ground state quantum yield edit the fluorescence quantum yield gives the efficiency of the fluorescence process it is defined as the ratio of the number of photons emitted to the number of photons absorbed 15 p10 13 φ number of photons emitted number of photons absorbed displaystyle phi frac text number of photons emitted text number of photons absorbed the maximum possible fluorescence quantum yield is 1 0 100 each photon absorbed results in a photon emitted compounds with quantum yields of 0 10 are still considered quite fluorescent another way to define the quantum yield of fluorescence is by the rate of excited state decay φ k f i k i displaystyle phi frac k _ f sum _ i k _ i where k f displaystyle k _ f is the rate constant of spontaneous emission of radiation and i k i displaystyle sum _ i k _ i is the sum of all rates of excited state decay other rates of excited state decay are caused by mechanisms other than photon emission and are therefore often called non radiative rates which can include dynamic collisional quenching near field dipole dipole interaction or resonance energy transfer internal conversion intersystem crossing thus if the rate of any pathway changes both the excited state lifetime and the fluorescence quantum yield will be affected fluorescence quantum yields are measured by comparison to a standard 16 the quinine salt quinine sulfate in a sulfuric acid solution was regarded as the most common fluorescence standard 17 however a recent study revealed that the fluorescence quantum yield of this solution is strongly affected by the temperature and should no longer be used as the standard solution the quinine in 0 1 m perchloric acid φ 0 60 shows no temperature dependence up to 45 c therefore it can be considered as a reliable standard solution 18 lifetime edit jablonski diagram after an electron absorbs a high energy photon the system is excited electronically and vibrationally the system relaxes vibrationally and eventually fluoresces at a longer wavelength than the original high energy photon had the fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon fluorescence typically follows first order kinetics s 1 s 1 0 e γ t displaystyle left s_ 1 right left s_ 1 right _ 0 e gamma t where s 1 displaystyle left s_ 1 right is the concentration of excited state molecules at time t displaystyle t s 1 0 displaystyle left s_ 1 right _ 0 is the initial concentration and γ displaystyle gamma is the decay rate or the inverse of the fluorescence lifetime this is an instance of exponential decay various radiative and non radiative processes can de populate the excited state in such case the total decay rate is the sum over all rates γ t o t γ r a d γ n r a d displaystyle gamma _ tot gamma _ rad gamma _ nrad where γ t o t displaystyle gamma _ tot is the total decay rate γ r a d displaystyle gamma _ rad the radiative decay rate and γ n r a d displaystyle gamma _ nrad the non radiative decay rate it is similar to a first order chemical reaction in which the first order rate constant is the sum of all of the rates a parallel kinetic model if the rate of spontaneous emission or any of the other rates are fast the lifetime is short for commonly used fluorescent compounds typical excited state decay times for photon emissions with energies from the uv to near infrared are within the range of 0 5 to 20 nanoseconds the fluorescence lifetime is an important parameter for practical applications of fluorescence such as fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy jablonski diagram edit the jablonski diagram describes most of the relaxation mechanisms for excited state molecules the diagram alongside shows how fluorescence occurs due to the relaxation of certain excited electrons of a molecule 19 fluorescence anisotropy edit fluorophores are more likely to be excited by photons if the transition moment of the fluorophore is parallel to the electric vector of the photon 15 pp12 13 the polarization of the emitted light will also depend on the transition moment the transition moment is dependent on the physical orientation of the fluorophore molecule for fluorophores in solution the intensity and polarization of the emitted light is dependent on rotational diffusion therefore anisotropy measurements can be used to investigate how freely a fluorescent molecule moves in a particular environment fluorescence anisotropy can be defined quantitatively as r i i i 2 i displaystyle r i_ parallel i_ perp over i_ parallel 2i_ perp where i displaystyle i_ parallel is the emitted intensity parallel to the polarization of the excitation light and i displaystyle i_ perp is the emitted intensity perpendicular to the polarization of the excitation light 13 anisotropy is independent of the intensity of the absorbed or emitted light it is a property of the light so photobleaching of the dye will not affect the anisotropy value as long as the signal is detectable fluorescence edit fluorescent security strip in a us twenty dollar bill under uv light strongly fluorescent pigments often have an unusual appearance which is often described colloquially as a neon color originally day glo in the late 1960s early 1970s this phenomenon was termed farbenglut by hermann von helmholtz and fluorence by ralph m evans it is generally thought to be related to the high brightness of the color relative to what it would be as a component of white fluorescence shifts energy in the incident illumination from shorter wavelengths to longer such as blue to yellow and thus can make the fluorescent color appear brighter more saturated than it could possibly be by reflection alone 20 rules edit there are several general rules that deal with fluorescence each of the following rules have exceptions but they are useful guidelines for understanding fluorescence these rules do not necessarily apply to two photon absorption kasha s rule edit kasha s rule states that the luminesce fluorescence or phosphorescence of a molecule will be emitted only from the lowest excited state of its given multiplicity 21 vavilov s rule a logical extension of kasha s rule thusly called kasha vavilov rule dictates that the quantum yield of luminescence is independent of the wavelength of exciting radiation and is proportional to the absorbance of the excited wavelength 22 kasha s rule does not always apply and is violated by simple molecules such an example is azulene 23 a somewhat more reliable statement although still with exceptions would be that the fluorescence spectrum shows very little dependence on the wavelength of exciting radiation 24 mirror image rule edit the fluorescent dye rhodamine 6g is commonly used in applications such as highlighter pens dye lasers and automotive leak detection the absorption profile is a mirror of the emission profile for many fluorophores the absorption spectrum is a mirror image of the emission spectrum 15 pp6 8 this is known as the mirror image rule and is related to the franck condon principle which states that electronic transitions are vertical that is energy changes without distance changing as can be represented with a vertical line in jablonski diagram this means the nucleus does not move and the vibration levels of the excited state resemble the vibration levels of the ground state stokes shift edit main article stokes shift in general emitted fluorescence light has a longer wavelength and lower energy than the absorbed light 15 pp6 7 this phenomenon known as stokes shift is due to energy loss between the time a photon is absorbed and when a new one is emitted the causes and magnitude of stokes shift can be complex and are dependent on the fluorophore and its environment however there are some common causes it is frequently due to non radiative decay to the lowest vibrational energy level of the excited state another factor is that the emission of fluorescence frequently leaves a fluorophore in a higher vibrational level of the ground state in nature edit fluorescent coral main article fluorescence in the life sciences there are many natural compounds that exhibit fluorescence and they have a number of applications some deep sea animals such as the greeneye have fluorescent structures fluorescence edit when fluorescence occurs in a living organism it is sometimes called biofluorescence fluorescence is distinct from bioluminescence and biophosphorescence 25 pumpkin toadlets that live in the brazilian atlantic forest are fluorescent 26 bioluminescence edit main article bioluminescence bioluminescence differs from fluorescence in that it is the natural production of light by chemical reactions within an organism whereas fluorescence is the absorption and reemission of light from the environment 25 fireflies and anglerfish are two examples of bioluminescent organisms 27 some organisms are both bioluminescent and fluorescent like the sea pansy renilla reniformis where bioluminescence serves as the light source for fluorescence 28 phosphorescence edit main article phosphorescence phosphorescence is similar to fluorescence in its requirement of light wavelengths as a provider of excitation energy the difference here lies in the relative stability of the energized molecule unlike with fluorescence in phosphorescence the energized molecule is metastable emitting light that continues to glow in the dark even after the stimulating light source has been removed 25 for example glow in the dark stickers are phosphorescent citation needed biophosphorescence in living organisms is exceptionally rare only reported in a few millipedes 29 mechanisms edit epidermal chromatophores edit pigment cells that exhibit fluorescence are called fluorescent chromatophores and function somatically similar to regular chromatophores these cells are dendritic and contain pigments called fluorosomes these pigments contain fluorescent proteins which are activated by k potassium ions and it is their movement aggregation and dispersion within the fluorescent chromatophore that cause directed fluorescence patterning 30 31 fluorescent cells are innervated the same as other chromatophores like melanophores pigment cells that contain melanin short term fluorescent patterning and signaling is controlled by the nervous system 30 fluorescent chromatophores can be found in the skin e g in fish just below the epidermis amongst other chromatophores epidermal fluorescent cells in fish also respond to hormonal stimuli by the α msh and mch hormones much the same as melanophores this suggests that fluorescent cells may have color changes throughout the day that coincide with their circadian rhythm 32 fish may also be sensitive to cortisol induced stress responses to environmental stimuli such as interaction with a predator or engaging in a mating ritual 30 phylogenetics edit evolutionary origins edit the incidence of fluorescence across the tree of life is widespread and has been studied most extensively in cnidarians and fish the phenomenon appears to have evolved multiple times in multiple taxa such as in the anguilliformes eels gobioidei gobies and cardinalfishes and tetradontiformes triggerfishes along with the other taxa discussed later in the article fluorescence is highly genotypically and phenotypically variable even within ecosystems in regards to the wavelengths emitted the patterns displayed and the intensity of the fluorescence generally the species relying upon camouflage exhibit the greatest diversity in fluorescence likely because camouflage may be one of the uses of fluorescence 33 fluorescence has multiple origins in the tree of life this diagram displays the origins within actinopterygians ray finned fish it is suspected by some scientists that gfps and gfp like proteins began as electron donors activated by light these electrons were then used for reactions requiring light energy functions of fluorescent proteins such as protection from the sun conversion of light into different wavelengths or for signaling are thought to have evolved secondarily 34 adaptive functions edit currently relatively little is known about the functional significance of fluorescence and fluorescent proteins 34 however it is suspected that fluorescence may serve important fu...
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