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. 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.
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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 3 , which 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 2 , S 3 , etc.
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 centimeter , which is directly proportional to the frequency and quantum energy. When presented in this manner see Figure 3 , symmetry 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 nanometers , which 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 1 , resulting 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.
Three fundamental parameters commonly used in describing and comparing fluorophores are the extinction coefficient e , quantum yield F , and fluorescence lifetime t. 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.
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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 crossing , and 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:.
The reciprocal of the decay rate constant equals the intrinsic lifetime t o , which is defined as the lifetime of the excited state in the absence of all processes that compete for excited state deactivation. In practice, the fluorescence excited state lifetime is shortened by non-radiative processes, resulting in a measured lifetime t f that is a combination of the intrinsic lifetime and competing non-fluorescent relaxation mechanisms. 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.
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.
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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 fluorescence , while the mitochondria and actin cytoskeleton were stained with MitoTracker Red red fluorescence and a phalloidin derivative green fluorescence , respectively.
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 a , but 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.
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 recovery , the 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. A variety of environmental factors affect fluorescence emission, including interactions between the fluorophore and surrounding solvent molecules dictated by solvent polarity , other 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 state , and 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 7 , which generally has a much larger dipole moment in the excited state than in the ground state. After the fluorophore has been excited to higher vibrational levels of the first excited singlet state S 1 , excess vibrational energy is rapidly lost to surrounding solvent molecules as the fluorophore slowly relaxes to the lowest vibrational energy level occurring in the picosecond time scale.
Solvent molecules assist in stabilizing and further lowering the energy level of the excited state by re-orienting termed solvent relaxation around the excited fluorophore in a slower process that requires between 10 and picoseconds.
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This has the effect of reducing the energy separation between the ground and excited states, which results in a red shift to longer wavelengths of the fluorescence emission. Finally, we provide two examples of how these various preparative and spectroscopic methods are employed in the study of RNA folding and RNP assembly processes.
Nature Methods Single - molecule fluorescence experiments with microsecond time resolution are made possible using a photoprotection cocktail that reduces dye blinking and bleaching with a combination of dissolved oxygen, a triplet quencher and a free-radical. Single - molecule fluorescence experiments with microsecond time resolution are made possible using a photoprotection cocktail that reduces dye blinking and bleaching with a combination of dissolved oxygen, a triplet quencher and a free-radical scavenger.
The structural and dynamic details of protein folding are still widely unexplored due to the enormous level of heterogeneity intrinsic to this process. The unfolded polypeptide chain can assume a vast number of possible conformations, and many. The unfolded polypeptide chain can assume a vast number of possible conformations, and many complex pathways lead from the ensemble of unfolded conformations to the ensemble of native conformations in an overall funnel-shaped energy landscape.
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Classical experimental methods involve measurements on bulk samples and usually yield only average values characteristic of the entire molecular ensemble under study. The observation of individual molecules avoids this averaging and allows, in principle, microscopic distributions of conformations and folding trajectories to be revealed. This chapter focuses on practical aspects and different experimental realizations for protein folding investigations by using single - molecule fluorescence.
Protein-protein and protein-DNA interactions play critical roles in biological systems, and these interactions often involve complex mechanisms and inhomogeneous dynamics. Single - molecule spectroscopy is a powerful and complimentary approach to. Single - molecule spectroscopy is a powerful and complimentary approach to decipher such spatially and temporally inhomogeneous protein interaction systems, providing new information that are not obtainable from static structure analyses, thermodynamics characterization, and ensemble-averaged measurements. To illustrate the single - molecule spectroscopy and imaging technology and their applications on studying protein-ligand interactions, this chapter focuses on discussing two recent single - molecule spectroscopy studies on protein-protein interaction in cell signaling process and on protein-DNA interactions in DNA damage recognition process.
Key Words: Single - molecule spectroscopy and imaging; single - molecule protein conformational dynamics; protein-protein interaction dynamics in cell signaling; protein-DNA recognition dynamics in DNA damage recognition and repair. Several manual and scanning spectro-fluorimeters enable researchers to sense fluorescently stained samples in gels. Scanning spectrofluorimeters use laser beams to induce fluorescence.
Stamford, Conn. These molecules could be fragments of DNA generated by sequencing reactions or proteins labeled with a fluorescently tagged antibody. Instruments such as the Laser Trak generally allow investigators to measure fluorescence emissions at specific wavelengths, depending on the type of fluorochrome chosen. Instead of labeling samples with radioactivity and detecting them by autoradiography, fluorescence techniques permit sensitive detection of samples directly in gels. Additional benefits are less exposure to hazardous radioisotopes for the user and a much lower cost of disposal.
The optical system of the FluorImager SI focuses a scanning laser beam on the samples in a gel or a plate, which results in fluorescence, termed laser-induced fluorescence LIF. The emitted light then is measured to produce a computerized image of the scanned sample. An integrated LIF detector permits a researcher to analyze the separated fragment, which may consist of oligonucleotides, DNA amplified by the polymerase chain reaction PCR , and restriction fragments.
Since each nucleotide in DNA will appear as a distinct color, all four sequencing reactions can be run in a single capillary tube. When the desired target is a cell rather than a molecule, flow cytometry may be a better tool. This technology permits investigators to rapidly identify, count, and even sort cells into homogeneous populations.
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In flow cytometry, particles or cells are first labeled with fluorochrome-tagged antibodies or other fluorescent tracers such as propidium iodide, specific for DNA. Once labeled, cells are placed in the flow cytometer, which provides a focused beam of laser light to excite the fluorochrome. The laser studies each cell as it passes single-file through a flow cell, at rates of up to thousands per second.
A computer then electronically selects specific cells for analysis, or to separate them into a homogeneous cell population. Miami-based Coulter Corp. These systems are appropriate for cell sorting and other types of cellular analysis, including immunophenotyping and DNA analysis. For applications typically performed in medical laboratories, Coulter's EPICS XL system has been equipped with new software that allows the instrument to sense cells labeled with up to four fluorescent colors. Multicolor labeling allows researchers to study several cellular features at once by attaching different fluorochromes to the same cell and measuring the light emitted at specific wavelengths.
An area in which fluorescence-based flow cytometry may have an impact in the future is FISH, or fluorescence in situ hybridization. FISH techniques use fluorescently labeled nucleic acid probes, which bind to similar target sequences directly in specimens mounted on slides or the surfaces of microplate wells. FISH by flow cytometry-known as fluorescence in situ hybridization en solution, or FISHES-may eventually be applied to studies of gene expression at the level of individual cells.
As opposed to intensity-based determinations of fluorescence, time-resolved spectroscopy measures changes in fluorescence emissions by a molecule over time.