An intrinsic fluorophore is a ion, molecule or macromolecule that fluoresces strongly in it native form while an extrinsic fluorophore is a species that has been made to fluoresce strongly through reaction with a fluorometric reagent. Among organic molecules only a small fraction are intrinsic fluorophores.
What are intrinsic and extrinsic fluorophores?
Intrinsic fluorophores, such as aromatic amino acids, neurotransmitters, porphyrins, and green fluorescent protein, are those that occur naturally. Extrinsic fluorophores are synthetic dyes or modified biochemicals that are added to a specimen to produce fluorescence with specific spectral properties.
What causes intrinsic fluorescence of proteins?
The intrinsic fluorescence of proteins is caused by three amino acid residues with aromatic side chains: phenylalanine, tyrosine and tryptophan. Out of these three, the latter plays the most important role due to its excitation and emission spectra having the longest wavelength (near the UV range) and longest lifetime.
What is meant by the term'fluorescence'?
Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime.
What is fluorescence emission intensity and how is it monitored?
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.
What is intrinsic fluorescence?
An intrinsic fluorophore is a ion, molecule or macromolecule that fluoresces strongly in it native form while an extrinsic fluorophore is a species that has been made to fluoresce strongly through reaction with a fluorometric reagent. Among organic molecules only a small fraction are intrinsic fluorophores.
What are the two types of fluorescence?
Luminescence – The Simple Explanation 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.
What is fluorescence and its types?
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation.
What are the three stages of fluorescence?
In short, the 3 steps of fluorescence are absorption (or excitation), non-radiative transition (or excited-state lifetime), and fluorescence emission.
What is difference between fluorescence and luminescence?
The main difference between fluorescence and luminescence is that luminescence describes any process where photons are emitted without heat being the cause, whereas fluorescence is, in fact, a type of luminescence where a photon is initially absorbed, which causes the atom to be in an excited singlet state.
What are the two types of quenching?
There are two different ways of quenching: static and dynamic quenching.
What are examples of fluorescence?
Examples of Fluorescence For instance, minerals and gemstones often emit visible colors when UV rays fall on them. Diamond, rubies, emeralds, calcite, amber, etc. show the same phenomenon when UV rays or X-rays fall on them. One of the best fluorescence examples in nature is bioluminescence.
What factors affect fluorescence?
Three important factors influencing the intensity of fluorescence emission were theoretical analyzed, including the absorption ability of excitation photons, fluorescence quantum yield, and fluorescence saturation & fluorescence quenching.
What is difference between fluorescence and phosphorescence?
Difference Between Fluorescence and Phosphorescence Fluorescent emission of radiation or light suddenly stops on the removal of the soucre of excitation. On the other hand, phosphorescence emission of radiation remains for some time even after the removal of the source of excitation.
What is the process of fluorescence?
Some molecules are capable of being excited, via absorption of light energy, to a higher energy state, also called an excited state. The energy of the excited state—which cannot be sustained for long— “decays” or decreases, resulting in the emission of light energy. This process is called fluorescence.
What is the difference between fluorescence emission and excitation spectrum?
The excitation spectrum shows at what wavelengths the solution uses to produce its fluorescence. The emission spectrum shows what wavelengths are given off from the solution.
What is excitation in fluorescence?
The fluorescence excitation spectrum characterizes the electron distribution of the molecule in the ground state. Excitation is equivalent to absorption since upon absorption, the molecule reaches the excited state Sn.
What are the examples of fluorescence?
Examples of Fluorescence Diamond, rubies, emeralds, calcite, amber, etc. show the same phenomenon when UV rays or X-rays fall on them.
What is difference between fluorometer and spectrofluorometer?
A fluorometer is a filter based, fixed wavelength, instrument suitable for established quantitative fluorescence methods. A spectrofluorometer is equipped with two scanning monochromoators permitting variation in excitation wavelength, the emission wavelength or both (constant energy difference mode).
What are some examples of fluorescent light?
There are several different types of fluorescent lighting including linear fluorescent tubes, fluorescent bent tubes, fluorescent circline tubes, and CFLs (compact fluorescent lamps).
What are fluorophores?
What are Fluorophores? Fluorophores are microscopic molecules, which may be proteins, small organic compounds, or synthetic polymers that absorb light of specific wavelengths and emit light of longer wavelengths.
How does fluorescence work?
The fluorescence process is governed by three important events, all of which occur on timescales that are separated by several orders of magnitude (see Table 1). Excitation of a susceptible molecule by an incoming photon happens in femtoseconds (10E-15 seconds), while vibrational relaxation of excited state electrons to the lowest energy level is much slower and can be measured in picoseconds (10E-12 seconds). The final process, emission of a longer wavelength photon and return of the molecule to the ground state, occurs in the relatively long time period of nanoseconds (10E-9 seconds). Although the entire molecular fluorescence lifetime, from excitation to emission, is measured in only billionths of a second, the phenomenon is a stunning manifestation of the interaction between light and matter that forms the basis for the expansive fields of steady state and time-resolved fluorescence spectroscopy and microscopy. Because of the tremendously sensitive emission profiles, spatial resolution, and high specificity of fluorescence investigations, the technique is rapidly becoming an important tool in genetics and cell biology.
When was fluorescence first discovered?
Fluorescence was first encountered in optical microscopy during the early part of the twentieth century by several notable scientists, including August Köhler and Carl Reichert, who initially reported that fluorescence was a nuisance in ultraviolet microscopy.
Why does fluorescence depolarize?
Because the emission transition dipole moment is displaced from the excitation transition moment due to spatial orientation (see Figure 8) , some degree of depolarization will occur even if the fluorophores are fixed in place. Thus, the absorption process itself is the first source of fluorescence depolarization. Rotational motion of the fluorophore, which invariably occurs during the lifetime of the excited state, results in additional displacement of the emission transition dipole from the original orientation, and will further lower the observed emission anisotropy. The rate of rotation is dependent both on the size of the fluorophore (or the macromolecule to which it is bound) and the viscosity of its localized environment. The fluorescence anisotropy of a solution can be related to the rotational mobility of the fluorophore by the Perrin equation:
Why should fluorescent probes be monitored?
Quantitative fluorescence investigations should be constantly monitored to scan for potential shifts in emission profiles, even when they are not intended nor expected. In simple systems where a homogeneous concentration can be established, a progressive emission intensity increase should be observed as a function of increasing fluorophore concentration, and vice versa. However, in complex biological systems, fluorescent probe concentration may vary locally over a wide range, and intensity fluctuations or spectral shifts are often the result of changes in pH, calcium ion concentration, energy transfer, or the presence of a quenching agent rather than fluorophore stoichiometry. The possibility of unexpected solvent or other environmental effects should always be considered in evaluating the results of experimental procedures.
How does fluorophore absorption work?
The various energy levels involved in the absorption and emission of light by a fluorophore are classically presented by a Jablonski energy diagram (see Figure 1), named in honor of the Polish physicist Professor Alexander Jablonski. A typical Jablonski diagram illustrates the singlet ground ( S (0)) state, as well as the first ( S (1)) and second ( S (2)) excited singlet states as a stack of horizontal lines. In Figure 1, the thicker lines represent electronic energy levels, while the thinner lines denote the various vibrational energy states (rotational energy states are ignored). Transitions between the states are illustrated as straight or wavy arrows, depending upon whether the transition is associated with absorption or emission of a photon (straight arrow) or results from a molecular internal conversion or non-radiative relaxation process (wavy arrows). Vertical upward arrows are utilized to indicate the instantaneous nature of excitation processes, while the wavy arrows are reserved for those events that occur on a much longer timescale.
Why do fluorescence emission wavelengths change?
Because the energy associated with fluorescence emission transitions (see Figures 1-4) 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.
What is the process of generating luminescence?
Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which is formally divided into two categories, fluorescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway.
What is the integrity of the fluorescent carbon dot?
Integrity of the fluorescent carbon dot (FCD) emission deserves its highest appreciation when sample purification is performed with extreme care. Several controversial phenomena of FCD fluorescence including excitation dependent emission, spectral migration with time and thereby violation of Kasha-Vavilov rule, those sparked intense debate during recent reports, disappeared when we rigorously purified the as-synthesized FCD sample. Purification was performed by first visual silica column chromatography (observing the emissions under UV-illumination) and subsequently a prolonged membrane dialysis. Most of the surprising phenomena of FCD fluorescence reported earlier apparently arose from a ground state spectral heterogeneity of FCD sample containing a large amount of fluorescent impurities (mostly polymeric or oligomeric in nature). Observation of our ensemble spectroscopic measurements albeit nicely matched with recent reports based on single-particle experiments, but differed largely from other ensemble measurements. Our results reconciled a number of long-standing controversies on FCD emission mostly by emphasizing the urgency of sample purification with more scientific rigor.
What is chemical sensing?
Chemical sensing aims to detect subtle changes in the chemical environment by transforming relevant chemical or physical properties of molecular or ionic species (i.e., analytes) into an analytically useful output. Optical arrays based on chemoresponsive colorants (dyes and nanoporous pigments) probe the chemical reactivity of analytes, rather than their physical properties (e.g., mass). The chemical specificity of the olfactory system does not come from specific receptors for specific analytes (e.g., the traditional lock-and-key model of substrate–enzyme interactions), but rather olfaction makes use of pattern recognition of the combined response of several hundred olfactory receptors. In a similar fashion, arrays of chemoresponsive colorants provide high-dimensional data from the color or fluorescence changes of the dyes in these arrays as they are exposed to analytes. This provides chemical sensing with high sensitivity (often down to parts per billion levels), impressive discrimination among very similar analytes, and exquisite fingerprinting of extremely similar mixtures over a wide range of analyte types, in both the gas and liquid phases. Design of both sensor arrays and instrumentation for their analysis are discussed. In addition, the various chemometric and statistical analyses of high-dimensional data (including hierarchical cluster analysis (HCA), principal component analysis (PCA), linear discriminant analysis (LDA), support vector machines (SVMs), and artificial neural networks (ANNs)) are presented and critiqued in reference to their use in chemical sensing. A variety of applications are also discussed, including personal dosimetry of toxic industrial chemical, detection of explosives or accelerants, quality control of foods and beverages, biosensing intracellularly, identification of bacteria and fungi, and detection of cancer and disease biomarkers.
What is density functional theory?
Density functional theory (DFT) as one of molecular simulation techniques has been widely used to become rapidly a powerful tool for research and technology development for the past three decades. In particular, the DFT-based theoretical and fundamental knowledge have shed light on our understanding of the fundamental surface science, catalysis, sensors, materials science, and biology. Oxygen, nitrogen, boron, phosphorus, and sulfur are the most common heteroatoms introduced on the functional carbon nanomaterials surface with different surface functionalities. This book chapter aims to provide a pedagogical narrative of the DFT and relevant computational methods applied for surface chemistry, homogeneous/heterogeneous catalysis, and the fluorescence-based sensing properties of carbon nanomaterials. We overview several representative case studies associated with energy and chemicals production and discuss relevant principles of computationally driven carbon nanomaterials design.
What are the parameters of fluorescence?
In addition, fluorescence possesses several important parameters such as intensity, excitation and emission spectra, polarisation, lifetime, and quantum yield. These parameters are functions of solvent nature, temperature, polarity and viscosity, and can be used to research the structure and function of various systems and for analytical ...
Which amino acid residues are responsible for the intrinsic fluorescence of proteins?
The intrinsic fluorescence of proteins is caused by three amino acid residues with aromatic side chains: phenylalanine, tyrosine and tryptophan.
Is non-TRP emission a semiquantitative?
To make this method semi-quantitative, we need to normalise the non-Trp emission against a reference signal. This signal should vary to the same extent as the emission of the fluorescent PTMs as a function of experimental conditions (intensity of excitation, geometry etc.). Since the concentration of Trp remains nearly constant in spite of the modifications (1–2%), emission of its “red-shifted” fraction ideally fits this purpose.
Can protein fluorescence be used to detect changes in the eye?
This generic basis for conformational disorders led us to conceive whether it would be possible to use protein fluorescence to detect changes in the eye lens structure. The eye lens created by nature is a perfectly transparent organ that represents an excellent experimental model for fluorescence measurements. A challenge in the use of tryptophan (Trp) fluorescence for cataract diagnostics is the incredibly high concentration of the eye lens proteins (crystallins) (200–400 mg mL –1) which makes the lens’ optical density in the spectral range of the Trp absorption band (260–300 nm) a hundred of units. Such a high optical density does not allow the excitation light to penetrate deeper than a hundred of microns into the lens body. However, this experimental challenge can be overcome by the use of the so-called red-edge excitation of Trp; meaning excitation on the long-wavelength (“red”) slope of the absorption band. 3 First, due to the steepness of this slope, the optical density drops significantly and an excitation light of 317 nm wavelength travels throughout the lens with an attenuation of just about 25% (Figure 1, left panel).
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- Fluorescence emission from a wide variety of specimens becomes polarized when the intrinsic or extrinsic fluorophores are excited with plane-polarized light. The level of polarized emission is described in terms of the anisotropy, and specimens that display some degree of anisotropy also exhibit a detectable level of polarized emission. Observation...