Fluorescence is the emission of light by a substance that has absorbed light or other . The fluorescence quantum yield gives the efficiency of the fluorescence The fluorescence lifetime refers to the average time the molecule stays in its excited . This phenomenon, known as Stokes shift, is due to energy loss between the. Here is the paradox! rotational correlation time is independent of lifetime and . + is+the+relationship+between+fluorescence+lifetime+and+anisotropy%3F&btnG= &lr= Why, the fluorescence quantum yield decreases but average lifetime of. In practice, the Stokes shift is measured as the difference between the maximum Extinction Coefficient, Quantum Yield, and Fluorescence Lifetime.
The phenomenon appears to have evolved multiple times in multiple taxa such as in the anguilliformes eelsgobioidei gobies and cardinalfishesand tetradontiformes triggerfishesalong 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 is one of the most common uses of fluorescence. Therefore, warm colors from the visual light spectrum appear less vibrant at increasing depths. Water scatters light of shorter wavelengths, meaning cooler colors dominate the visual field in the photic zone. Because the water filters out the wavelengths and intensity of water reaching certain depths, different proteins, because of the wavelengths and intensities of light they are capable of absorbing, are better suited to different depths.
Theoretically, some fish eyes can detect light as deep as m. At these depths of the aphotic zone, the only sources of light are organisms themselves, giving off light through chemical reactions in a process called bioluminescence. Fluorescence is simply defined as the absorption of electromagnetic radiation at one wavelength and its reemission at another, lower energy wavelength.
Biologically functional fluorescence is found in the photic zone, where there is not only enough light to cause biofluorescence, but enough light for other organisms to detect it. The visual field in the photic zone is naturally blue, so colors of fluorescence can be detected as bright reds, oranges, yellows, and greens.
Green is the most commonly found color in the biofluorescent spectrum, yellow the second most, orange the third, and red is the rarest. However, some cases of functional and adaptive significance of biofluorescence in the aphotic zone of the deep ocean is an active area of research. Thus, in shallow-water fishes, red, orange, and green fluorescence most likely serves as a means of communication with conspecifics, especially given the great phenotypic variance of the phenomenon.
Biofluorescent patterning was especially prominent in cryptically patterned fishes possessing complex camouflage, and that many of these lineages also possess yellow long-pass intraocular filters that could enable visualization of such patterns.
Red light can only be seen across short distances due to attenuation of red light wavelengths by water. This patterning is caused by fluorescent tissue and is visible to other members of the species, however the patterning is invisible at other visual spectra. These intraspecific fluorescent patterns also coincide with intra-species signaling. Fish such as the fairy wrasse that have developed visual sensitivity to longer wavelengths are able to display red fluorescent signals that give a high contrast to the blue environment and are conspicuous to conspecifics in short ranges, yet are relatively invisible to other common fish that have reduced sensitivities to long wavelengths.
Thus, fluorescence can be used as adaptive signaling and intra-species communication in reef fish. These spots reflect incident light, which may serve as a means of camouflage, but also for signaling to other squids for schooling purposes.
This jellyfish lives in the photic zone off the west coast of North America and was identified as a carrier of green fluorescent protein GFP by Osamu Shimomura. The gene for these green fluorescent proteins has been isolated and is scientifically significant because it is widely used in genetic studies to indicate the expression of other genes. The display involves raising the head and thorax, spreading the striking appendages and other maxillipeds, and extending the prominent, oval antennal scales laterally, which makes the animal appear larger and accentuates its yellow fluorescent markings.
Furthermore, as depth increases, mantis shrimp fluorescence accounts for a greater part of the visible light available.
During mating rituals, mantis shrimp actively fluoresce, and the wavelength of this fluorescence matches the wavelengths detected by their eye pigments. Some siphonophores, including the genus Erenna that live in the aphotic zone between depths of m and m, exhibit yellow to red fluorescence in the photophores of their tentacle-like tentilla. This fluorescence occurs as a by-product of bioluminescence from these same photophores. The siphonophores exhibit the fluorescence in a flicking pattern that is used as a lure to attract prey.
This red fluorescence is invisible to other animals, which allows these dragonfish extra light at dark ocean depths without attracting or signaling predators. The frog is pale green with dots in white, yellow or light red. The fluorescence of the frog was discovered unintentionally in Buenos Aires, Argentina.
The fluorescence was traced to a new compound found in the lymph and skin glads. Scientists behind the discovery say that the fluorescence can be used for communication.
They also think that about or species of frogs are likely to be fluorescent.15_Fluorescence Lifetime Imaging Microscopy_HJeon
Their wings contain pigment-infused crystals that provide directed fluorescent light. The wavelengths of light that the butterflies see the best correspond to the absorbance of the crystals in the butterfly's wings. This likely functions to enhance the capacity for signaling. A study using mate-choice experiments on budgerigars Melopsittacus undulates found compelling support for fluorescent sexual signaling, with both males and females significantly preferring birds with the fluorescent experimental stimulus.
This study suggests that the fluorescent plumage of parrots is not simply a by-product of pigmentationbut instead an adapted sexual signal. Considering the intricacies of the pathways that produce fluorescent pigments, there may be significant costs involved. The closely spaced vibrational energy levels of the ground state, when coupled with normal thermal motion, produce a wide range of photon energies during emission.
As a result, fluorescence is normally observed as emission intensity over a band of wavelengths rather than a sharp line. Most fluorophores can repeat the excitation and emission cycle many hundreds to thousands of times before the highly reactive excited state molecule is photobleached, resulting in the destruction of fluorescence.
For example, the well-studied probe fluorescein isothiocyanate FITC can undergo excitation and relaxation for approximately 30, cycles before the molecule no longer responds to incident illumination.
Several other relaxation pathways that have varying degrees of probability compete with the fluorescence emission process. The excited state energy can be dissipated non-radiatively as heat illustrated by the cyan wavy arrow in Figure 1the excited fluorophore can collide with another molecule to transfer energy in a second type of non-radiative process for example, quenching, as indicated by the purple wavy arrow in Figure 1or a phenomenon known as intersystem crossing to the lowest excited triplet state can occur the blue wavy arrow in Figure 1.
The latter event is relatively rare, but ultimately results either in emission of a photon through phosphorescence or a transition back to the excited singlet state that yields delayed fluorescence. Transitions from the triplet excited state to the singlet ground state are forbidden, which results in rate constants for triplet emission that are several orders of magnitude lower than those for fluorescence.
Fluorescence lifetime and quantum yield | Kurt's Microscopy Blog
Both of the triplet state transitions are diagrammed on the right-hand side of the Jablonski energy profile illustrated in Figure 1. The low probability of intersystem crossing arises from the fact that molecules must first undergo spin conversion to produce unpaired electrons, an unfavorable process. The primary importance of the triplet state is the high degree of chemical reactivity exhibited by molecules in this state, which often results in photobleaching and the production of damaging free radicals.
In biological specimens, dissolved oxygen is a very effective quenching agent for fluorophores in the triplet state. The ground state oxygen molecule, which is normally a triplet, can be excited to a reactive singlet state, leading to reactions that bleach the fluorophore or exhibit a phototoxic effect on living cells.
Fluorophores in the triplet state can also react directly with other biological molecules, often resulting in deactivation of both species. Molecules containing heavy atoms, such as the halogens and many transition metals, often facilitate intersystem crossing and are frequently phosphorescent.
The probability of a transition occurring from the ground state S 0 to the excited singlet state S 1 depends on the degree of similarity between the vibrational and rotational energy states when an electron resides in the ground state versus those present in the excited state, as outlined in Figure 2.
The Franck-Condon energy diagram illustrated in Figure 2 presents the vibrational energy probability distribution among the various levels in the ground S 0 and first excited S 1 states for a hypothetical molecule. Excitation transitions red lines from the ground to the excited state occur in such a short timeframe femtoseconds that the internuclear distance associated with the bonding orbitals does not have sufficient time to change, and thus the transitions are represented as vertical lines.
This concept is referred to as the Franck-Condon Principle. The wavelength of maximum absorption red line in the center represents the most probable internuclear separation in the ground state to an allowed vibrational level in the excited state. At room temperature, thermal energy is not adequate to significantly populate excited energy states and the most likely state for an electron is the ground state S Owhich contains a number of distinct vibrational energy states, each with differing energy levels.
The most favored transitions will be the ones where the rotational and vibrational electron density probabilities maximally overlap in both the ground and excited states see Figure 2. However, incident photons of varying wavelength and quanta may have sufficient energy to be absorbed and often produce transitions from other internuclear separation distances and vibrational energy levels.
This effect gives rise to an absorption spectrum containing multiple peaks Figure 3. The wide range of photon energies associated with absorption transitions in fluorophores causes the resulting spectra to appear as broad bands rather than discrete lines.
The hypothetical absorption spectrum illustrated in Figure 3 blue band results from several favored electronic transitions from the ground state to the lowest excited energy state labeled S 0 and S 1respectively. Superimposed over the absorption spectrum are vertical lines yellow representing the transitions from the lowest vibrational level in the ground state to higher vibrational energy levels in the excited state.
Note that transitions to the highest excited vibrational levels are those occurring at higher photon energies lower wavelength or higher wavenumber.
The approximate energies associated with the transitions are denoted in electron-volts eV along the upper abscissa of Figure 3. Vibrational levels associated with the ground and excited states are also included along the right-hand ordinate. Scanning through the absorption spectrum of a fluorophore while recording the emission intensity at a single wavelength usually the wavelength of maximum emission intensity will generate the excitation spectrum.
Likewise, exciting the fluorophore at a single wavelength again, preferably the wavelength of maximum absorption while scanning through the emission wavelengths will reveal the emission spectral profile. The excitation and emission spectra may be considered as probability distribution functions that a photon of given quantum energy will be absorbed and ultimately enable the fluorophore to emit a second photon in the form of fluorescence radiation.
Stokes Shift and the Mirror Image Rule If the fluorescence emission spectrum of a fluorophore is carefully scrutinized, several important features become readily apparent. The emission spectrum is independent of the excitation energy wavelength as a consequence of rapid internal conversion from higher initial excited states to the lowest vibrational energy level of the S 1 excited state.
For many of the common fluorophores, the vibrational energy level spacing is similar for the ground and excited states, which results in a fluorescence spectrum that strongly resembles the mirror image of the absorption spectrum. This is due to the fact that the same transitions are most favorable for both absorption and emission. Finally, in solution where fluorophores are generally studied the detailed vibrational structure is generally lost and the emission spectrum appears as a broad band.
As previously discussed, following photon absorption, an excited fluorophore will quickly undergo relaxation to the lowest vibrational energy level of the excited state. An important consequence of this rapid internal conversion is that all subsequent relaxation pathways fluorescence, non-radiative relaxation, intersystem crossing, etc.
As with absorption, the probability that an electron in the excited state will return to a particular vibrational energy level in the ground state is proportional to the overlap between the energy levels in the respective states Figure 2. Return transitions to the ground state S 0 usually occur to a higher vibrational level see Figure 3which subsequently reaches thermal equilibrium vibrational relaxation. Because emission of a photon often leaves the fluorophore in a higher vibrational ground state, the emission spectrum is typically a mirror image of the absorption spectrum resulting from the ground to first excited state transition.
In effect, the probability of an electron returning to a particular vibrational energy level in the ground state is similar to the probability of that electron's position in the ground state before excitation.
This concept, known as the Mirror Image Rule, is illustrated in Figure 3 for the emission transitions blue lines from the lowest vibrational energy level of the excited state back to various vibrational levels in ground state.
The resulting emission spectrum red band is a mirror image of the absorption spectrum displayed by the hypothetical chromophore.
In many cases, excitation by high energy photons leads to the population of higher electronic and vibrational levels S 2S 3etc. Because of this rapid relaxation process, emission spectra are generally independent of the excitation wavelength some fluorophores emit from higher energy states, but such activity is rare. For this reason, emission is the mirror image of the ground state to lowest excited state transitions, but not of the entire absorption spectrum, which may include transitions to higher energy levels.
An excellent test of the mirror image rule is to examine absorption and emission spectra in a linear plot of the wavenumber the reciprocal of wavelength or the number of waves per centimeterwhich is directly proportional to the frequency and quantum energy.
When presented in this manner see Figure 3symmetry between extinction coefficients and intensity of the excitation and emission spectra as a function of energy yield mirrored spectra when reciprocal transitions are involved.
Presented in Figure 4 are the absorption and emission spectra for quinine, the naturally occurring antimalarial agent and first known fluorophore whose fluorescent properties were originally described by Sir John Fredrick William Hershel in Quinine does not adhere to the mirror image rule as is evident by inspecting the single peak in the emission spectrum at nanometerswhich does not mirror the two peaks at and nanometers featured in the bimodal absorption spectrum.
The shorter wavelength ultraviolet absorption peak nanometers is due to an excitation transition to the second excited state from S 0 to S 2 that quickly relaxes to the lowest excited state S 1.
As a consequence, fluorescence emission occurs exclusively from the lowest excited singlet state S 1resulting in a spectrum that mirrors the ground to first excited state transition nanometer peak in quinine and not the entire absorption spectrum. Because the energy associated with fluorescence emission transitions see Figures is typically less than that of absorption, the resulting emitted photons have less energy and are shifted to longer wavelengths.
This phenomenon is generally known as Stokes Shift and occurs for virtually all fluorophores commonly employed in solution investigations. The primary origin of the Stokes shift is the rapid decay of excited electrons to the lowest vibrational energy level of the S 1 excited state.
In addition, fluorescence emission is usually accompanied by transitions to higher vibrational energy levels of the ground state, resulting in further loss of excitation energy to thermal equilibration of the excess vibrational energy.
Other events, such as solvent orientation effects, excited-state reactions, complex formation, and resonance energy transfer can also contribute to longer emission wavelengths. In practice, the Stokes shift is measured as the difference between the maximum wavelengths in the excitation and emission spectra of a particular fluorochrome or fluorophore. The size of the shift varies with molecular structure, but can range from just a few nanometers to over several hundred nanometers.
For example, the Stokes shift for fluorescein is approximately 20 nanometers, while the shift for quinine is nanometers see Figure 4 and that for the porphyrins is over nanometers. The existence of Stokes shift is critical to the extremely high sensitivity of fluorescence imaging measurements.
The red emission shift enables the use of precision bandwidth optical filters to effectively block excitation light from reaching the detector so the relatively faint fluorescence signal having a low number of emitted photons can be observed against a low-noise background. Molar extinction coefficients are widely employed in the fields of spectroscopy, microscopy, and fluorescence in order to convert units of absorbance into units of molar concentration for a variety of chemical substances.
The extinction coefficient is determined by measuring the absorbance at a reference wavelength characteristic of the absorbing molecule for a one molar M concentration one mole per liter of the target chemical in a cuvette having a one-centimeter path length. The reference wavelength is usually the wavelength of maximum absorption in the ultraviolet or visible light spectrum. Extinction coefficients are a direct measure of the ability of a fluorophore to absorb light, and those chromophores having a high extinction coefficient also have a high probability of fluorescence emission.
Also, because the intrinsic lifetime discussed below of a fluorophore is inversely proportional to the extinction coefficient, molecules exhibiting a high extinction coefficient have an excited state with a short intrinsic lifetime. Quantum yield sometimes incorrectly termed quantum efficiency is a gauge for measuring the efficiency of fluorescence emission relative to all of the possible pathways for relaxation and is generally expressed as the dimensionless ratio of photons emitted to the number of photons absorbed.
In other words, the quantum yield represents the probability that a given excited fluorochrome will produce an emitted photon fluorescence. Quantum yields typically range between a value of zero and one, and fluorescent molecules commonly employed as probes in microscopy have quantum yields ranging from very low 0. In general, a high quantum yield is desirable in most imaging applications.
The quantum yield of a given fluorophore varies, sometimes to large extremes, with environmental factors such as pH, concentration, and solvent polarity. The fluorescence lifetime is the characteristic time that a molecule remains in an excited state prior to returning to the ground state and is an indicator of the time available for information to be gathered from the emission profile. During the excited state lifetime, a fluorophore can undergo conformational changes as well as interact with other molecules and diffuse through the local environment.
The decay of fluorescence intensity as a function of time in a uniform population of molecules excited with a brief pulse of light is described by an exponential function: This quantity is the reciprocal of the rate constant for fluorescence decay from the excited state to the ground state. Because the level of fluorescence is directly proportional to the number of molecules in the excited singlet state, lifetime measurements can be conducted by measuring fluorescence decay after a brief pulse of excitation.
In a uniform solvent, fluorescence decay is usually a monoexponential function, as illustrated by the plots of fluorescence intensity as a function of time in Figures 5 a and 5 b. More complex systems, such as viable tissues and living cells, contain a mixed set of environments that often yield multiexponential values Figure 5 c when fluorescence decay is measured.
In addition, several other processes can compete with fluorescence emission for return of excited state electrons to the ground state, including internal conversion, phosphorescence intersystem crossingand quenching. Aside from fluorescence and phosphorescence, non-radiative processes are the primary mechanism responsible for relaxation of excited state electrons.
All non-fluorescent processes that compete for deactivation of excited state electrons can be conveniently combined into a single rate constant, termed the non-radiative rate constant and denoted by the variable k nr. The non-radiative rate constant usually ignores any contribution from vibrational relaxation because the rapid speeds picoseconds of these conversions are several orders of magnitude faster than slower deactivation nanoseconds transitions.
Thus, the quantum yield can now be expressed in terms of rate constants: Because the measured lifetime is always less than the intrinsic lifetime, the quantum yield never exceeds a value of unity. Many of the common probes employed in optical microscopy have fluorescence lifetimes measured in nanoseconds, but these can vary over a wide range depending on molecular structure, the solvent, and environmental conditions. Quantitative fluorescence lifetime measurements enable investigators to distinguish between fluorophores that have similar spectral characteristics but different lifetimes, and can also yield clues to the local environment.
Specifically, the pH and concentration of ions in the vicinity of the probe can be determined without knowing the localized fluorophore concentration, which is of significant benefit when used with living cells and tissues where the probe concentration may not be uniform. In addition, lifetime measurements are less sensitive to photobleaching artifacts than are intensity measurements. Quenching and Photobleaching The consequences of quenching and photobleaching are an effective reduction in the amount of emission and should be of primary consideration when designing and executing fluorescence investigations.
The two phenomena are distinct in that quenching is often reversible whereas photobleaching is not. Quenching arises from a variety of competing processes that induce non-radiative relaxation without photon emission of excited state electrons to the ground state, which may be either intramolecular or intermolecular in nature. Because non-radiative transition pathways compete with the fluorescence relaxation, they usually dramatically lower or, in some cases, completely eliminate emission.
Most quenching processes act to reduce the excited state lifetime and the quantum yield of the affected fluorophore. A common example of quenching is observed with the collision of an excited state fluorophore and another non-fluorescent molecule in solution, resulting in deactivation of the fluorophore and return to the ground state. In most cases, neither of the molecules is chemically altered in the collisional quenching process. A wide variety of simple elements and compounds behave as collisional quenching agents, including oxygen, halogens, amines, and many electron-deficient organic molecules.
Collisional quenching can reveal the presence of localized quencher molecules or moieties, which via diffusion or conformational change, may collide with the fluorophore during the excited state lifetime. The mechanisms for collisional quenching include electron transfer, spin-orbit coupling, and intersystem crossing to the excited triplet state. Other terms that are often utilized interchangeably with collisional quenching are internal conversion and dynamic quenching.
A second type of quenching mechanism, termed static or complex quenching, arises from non-fluorescent complexes formed between the quencher and fluorophore that serve to limit absorption by reducing the population of active, excitable molecules. This effect occurs when the fluorescent species forms a reversible complex with the quencher molecule in the ground state, and does not rely on diffusion or molecular collisions.
In static quenching, fluorescence emission is reduced without altering the excited state lifetime. A fluorophore in the excited state can also be quenched by a dipolar resonance energy transfer mechanism when in close proximity with an acceptor molecule to which the excited state energy can be transferred non-radiatively.
In some cases, quenching can occur through non-molecular mechanisms, such as attenuation of incident light by an absorbing species including the chromophore itself. In contrast to quenching, photobleaching also termed fading occurs when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification.
Upon transition from an excited singlet state to the excited triplet state, fluorophores may interact with another molecule to produce irreversible covalent modifications. The triplet state is relatively long-lived with respect to the singlet state, thus allowing excited molecules a much longer timeframe to undergo chemical reactions with components in the environment.
The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment. Some fluorophores bleach quickly after emitting only a few photons, while others that are more robust can undergo thousands or millions of cycles before bleaching.
Presented in Figure 6 is a typical example of photobleaching fading observed in a series of digital images captured at different time points for a multiply-stained culture of bovine pulmonary artery epithelial cells. The nuclei were stained with 4,6-diamidinophenylindole DAPI; blue fluorescencewhile the mitochondria and actin cytoskeleton were stained with MitoTracker Red red fluorescence and a phalloidin derivative green fluorescencerespectively. Time points were taken in two-minute intervals using a fluorescence filter combination with bandwidths tuned to excite the three fluorophores simultaneously while also recording the combined emission signals.
Note that all three fluorophores have a relatively high intensity in Figure 6 abut the DAPI blue intensity starts to drop rapidly at two minutes and is almost completely gone at six minutes. The mitochondrial and actin stains are more resistant to photobleaching, but the intensity of both drops over the course of the timed sequence 10 minutes.
An important class of photobleaching events are photodynamic, meaning they involve the interaction of the fluorophore with a combination of light and oxygen. Reactions between fluorophores and molecular oxygen permanently destroy fluorescence and yield a free radical singlet oxygen species that can chemically modify other molecules in living cells.
The amount of photobleaching due to photodynamic events is a function of the molecular oxygen concentration and the proximal distance between the fluorophore, oxygen molecules, and other cellular components.
Fluorescence - Wikipedia
Photobleaching can be reduced by limiting the exposure time of fluorophores to illumination or by lowering the excitation energy. However, these techniques also reduce the measurable fluorescence signal.
In many cases, solutions of fluorophores or cell suspensions can be deoxygenated, but this is not feasible for living cells and tissues. Perhaps the best protection against photobleaching is to limit exposure of the fluorochrome to intense illumination using neutral density filters coupled with the judicious use of commercially available antifade reagents that can be added to the mounting solution or cell culture medium. Under certain circumstances, the photobleaching effect can also be utilized to obtain specific information that would not otherwise be available.
For example, in fluorescence recovery after photobleaching FRAP experiments, fluorophores within a target region are intentionally bleached with excessive levels of irradiation. As new fluorophore molecules diffuse into the bleached region of the specimen recoverythe fluorescence emission intensity is monitored to determine the lateral diffusion rates of the target fluorophore.
In this manner, the translational mobility of fluorescently labeled molecules can be ascertained within a very small 2 to 5 micrometer region of a single cell or section of living tissue. Solvent Effects on Fluorescence Emission A variety of environmental factors affect fluorescence emission, including interactions between the fluorophore and surrounding solvent molecules dictated by solvent polarityother dissolved inorganic and organic compounds, temperature, pH, and the localized concentration of the fluorescent species.
The effects of these parameters vary widely from one fluorophore to another, but the absorption and emission spectra, as well as quantum yields, can be heavily influenced by environmental variables.
In fact, the high degree of sensitivity in fluorescence is primarily due to interactions that occur in the local environment during the excited state lifetime.
A fluorophore can be considered an entirely different molecule in the excited state than in the ground stateand thus will display an alternate set of properties in regard to interactions with the environment in the excited state relative to the ground state. In solution, solvent molecules surrounding the ground state fluorophore also have dipole moments that can interact with the dipole moment of the fluorophore to yield an ordered distribution of solvent molecules around the fluorophore.
Energy level differences between the ground and excited states in the fluorophore produce a change in the molecular dipole moment, which ultimately induces a rearrangement of surrounding solvent molecules.
However, the Franck-Condon principle dictates that, upon excitation of a fluorophore, the molecule is excited to a higher electronic energy level in a far shorter timeframe than it takes for the fluorophore and solvent molecules to re-orient themselves within the solvent-solute interactive environment. As a result, there is a time delay between the excitation event and the re-ordering of solvent molecules around the solvated fluorophore as illustrated in Figure 7which generally has a much larger dipole moment in the excited state than in the ground state.