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135-476: The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses fluorescence in order to analyze multiple analytes, meaning it can detect the secretion of more than one type of protein. Cecil Czerkinsky first described ELISpot in 1983 as a new way to quantify the production of an antigen-specific immunoglobulin by hybridoma cells . In 1988, Czerkinsky developed an ELISA spot assay that quantified

270-402: A Hermitian operator . In 1924, Satyendra Nath Bose derived Planck's law of black-body radiation without using any electromagnetism, but rather by using a modification of coarse-grained counting of phase space . Einstein showed that this modification is equivalent to assuming that photons are rigorously identical and that it implied a "mysterious non-local interaction", now understood as

405-521: A point-like particle since it is absorbed or emitted as a whole by arbitrarily small systems, including systems much smaller than its wavelength, such as an atomic nucleus (≈10 m across) or even the point-like electron . While many introductory texts treat photons using the mathematical techniques of non-relativistic quantum mechanics, this is in some ways an awkward oversimplification, as photons are by nature intrinsically relativistic. Because photons have zero rest mass , no wave function defined for

540-744: A certain symmetry at every point in spacetime . The intrinsic properties of particles, such as charge , mass , and spin , are determined by gauge symmetry . The photon concept has led to momentous advances in experimental and theoretical physics, including lasers , Bose–Einstein condensation , quantum field theory , and the probabilistic interpretation of quantum mechanics. It has been applied to photochemistry , high-resolution microscopy , and measurements of molecular distances . Moreover, photons have been studied as elements of quantum computers , and for applications in optical imaging and optical communication such as quantum cryptography . The word quanta (singular quantum, Latin for how much )

675-588: A certain threshold; light of frequency lower than the threshold, no matter how intense, does not initiate the reaction. Similarly, electrons can be ejected from a metal plate by shining light of sufficiently high frequency on it (the photoelectric effect ); the energy of the ejected electron is related only to the light's frequency, not to its intensity. At the same time, investigations of black-body radiation carried out over four decades (1860–1900) by various researchers culminated in Max Planck 's hypothesis that

810-513: A charge is accelerated it emits synchrotron radiation . During a molecular , atomic or nuclear transition to a lower energy level , photons of various energy will be emitted, ranging from radio waves to gamma rays . Photons can also be emitted when a particle and its corresponding antiparticle are annihilated (for example, electron–positron annihilation ). In empty space, the photon moves at c (the speed of light ) and its energy and momentum are related by E = pc , where p

945-467: A choice of measuring either one of two "canonically conjugate" quantities, like the position and the momentum of a particle. According to the uncertainty principle, no matter how the particle is prepared, it is not possible to make a precise prediction for both of the two alternative measurements: if the outcome of the position measurement is made more certain, the outcome of the momentum measurement becomes less so, and vice versa. A coherent state minimizes

1080-414: A different frequency , which is the fluorescent glow. Fluorescence is a form of luminescence . In nearly all cases, the emitted light has a longer wavelength , and therefore a lower photon energy , than the absorbed radiation. The most common example occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light

1215-510: A dimmer afterglow for a short time after the excitation was removed, which became labeled "phosphorescence" or "triplet phosphorescence". The typical decay times ranged from a few microseconds to one second, which are still fast enough by human-eye standards to be colloquially referred to as fluorescent. Common examples include fluorescent lamps, organic dyes, and even fluorspar. Longer emitters, commonly referred to as glow-in-the-dark substances, ranged from one second to many hours, and this mechanism

1350-495: A gauge boson , below.) Einstein's 1905 predictions were verified experimentally in several ways in the first two decades of the 20th century, as recounted in Robert Millikan 's Nobel lecture. However, before Compton's experiment showed that photons carried momentum proportional to their wave number (1922), most physicists were reluctant to believe that electromagnetic radiation itself might be particulate. (See, for example,

1485-512: A geometric sum. However, Debye's approach failed to give the correct formula for the energy fluctuations of black-body radiation, which were derived by Einstein in 1909. In 1925, Born , Heisenberg and Jordan reinterpreted Debye's concept in a key way. As may be shown classically, the Fourier modes of the electromagnetic field —a complete set of electromagnetic plane waves indexed by their wave vector k and polarization state—are equivalent to

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1620-424: 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. Additionally, it is suggested that fluorescent tissues that surround an organism's eyes are used to convert blue light from

1755-475: A higher energy E i {\displaystyle E_{i}} is proportional to the number N j {\displaystyle N_{j}} of atoms with energy E j {\displaystyle E_{j}} and to the energy density ρ ( ν ) {\displaystyle \rho (\nu )} of ambient photons of that frequency, where B j i {\displaystyle B_{ji}}

1890-504: A higher energy E i {\displaystyle E_{i}} to a lower energy E j {\displaystyle E_{j}} is where A i j {\displaystyle A_{ij}} is the rate constant for emitting a photon spontaneously , and B i j {\displaystyle B_{ij}} is the rate constant for emissions in response to ambient photons ( induced or stimulated emission ). In thermodynamic equilibrium,

2025-408: A higher vibrational level of the ground state. 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 is the phenomenon of absorption of electromagnetic radiation, typically from ultraviolet or visible light , by a molecule and the subsequent emission of a photon of

2160-455: A light beam may have mixtures of these two values; a linearly polarized light beam will act as if it were composed of equal numbers of the two possible angular momenta. The spin angular momentum of light does not depend on its frequency, and was experimentally verified by C. V. Raman and S. Bhagavantam in 1931. The collision of a particle with its antiparticle can create photons. In free space at least two photons must be created since, in

2295-406: A lower energy (smaller frequency, longer wavelength). This causes the light that is emitted to be a different color than the light that is absorbed. Stimulating light excites an electron to an excited state. When the molecule returns to the ground state, it releases a photon, which is the fluorescent emission. The excited state lifetime is short, so emission of light is typically only observable when

2430-611: A means of communication with conspecifics , especially given the great phenotypic variance of the phenomenon. Many fish that exhibit fluorescence, such as sharks , lizardfish , scorpionfish , wrasses , and flatfishes , also possess yellow intraocular filters. Yellow intraocular filters in the lenses and cornea of certain fishes function as long-pass filters. These filters enable the species to visualize and potentially exploit fluorescence, in order to enhance visual contrast and patterns that are unseen to other fishes and predators that lack this visual specialization. Fish that possess

2565-590: A paper in which he proposed that many light-related phenomena—including black-body radiation and the photoelectric effect —would be better explained by modelling electromagnetic waves as consisting of spatially localized, discrete energy quanta. He called these a light quantum (German: ein Lichtquant ). The name photon derives from the Greek word for light, φῶς (transliterated phôs ). Arthur Compton used photon in 1928, referring to Gilbert N. Lewis , who coined

2700-459: A photon can have all the properties familiar from wave functions in non-relativistic quantum mechanics. In order to avoid these difficulties, physicists employ the second-quantized theory of photons described below, quantum electrodynamics , in which photons are quantized excitations of electromagnetic modes. Another difficulty is finding the proper analogue for the uncertainty principle , an idea frequently attributed to Heisenberg, who introduced

2835-399: A photon is calculated by equations that describe waves. This combination of aspects is known as wave–particle duality . For example, the probability distribution for the location at which a photon might be detected displays clearly wave-like phenomena such as diffraction and interference . A single photon passing through a double slit has its energy received at a point on the screen with

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2970-411: A predator or engaging in a mating ritual. 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

3105-402: A probability distribution given by its interference pattern determined by Maxwell's wave equations . However, experiments confirm that the photon is not a short pulse of electromagnetic radiation; a photon's Maxwell waves will diffract, but photon energy does not spread out as it propagates, nor does this energy divide when it encounters a beam splitter . Rather, the received photon acts like

3240-472: 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. Thus any type of fluorescence depends on the presence of external sources of light. Biologically functional fluorescence is found in the photic zone, where there is not only enough light to cause fluorescence, but enough light for other organisms to detect it. The visual field in

3375-410: A relatively simple assumption. He decomposed the electromagnetic field in a cavity into its Fourier modes , and assumed that the energy in any mode was an integer multiple of h ν {\displaystyle h\nu } , where ν {\displaystyle \nu } is the frequency of the electromagnetic mode. Planck's law of black-body radiation follows immediately as

3510-506: A reliable standard solution. 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 : 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}}

3645-497: A semi-classical, statistical treatment of photons and atoms, which implies a link between the rates at which atoms emit and absorb photons. The condition follows from the assumption that functions of the emission and absorption of radiation by the atoms are independent of each other, and that thermal equilibrium is made by way of the radiation's interaction with the atoms. Consider a cavity in thermal equilibrium with all parts of itself and filled with electromagnetic radiation and that

3780-450: A semiclassical approach, and, in 1927, succeeded in deriving all the rate constants from first principles within the framework of quantum theory. Dirac's work was the foundation of quantum electrodynamics, i.e., the quantization of the electromagnetic field itself. Dirac's approach is also called second quantization or quantum field theory ; earlier quantum mechanical treatments only treat material particles as quantum mechanical, not

3915-460: A set of uncoupled simple harmonic oscillators . Treated quantum mechanically, the energy levels of such oscillators are known to be E = n h ν {\displaystyle E=nh\nu } , where ν {\displaystyle \nu } is the oscillator frequency. The key new step was to identify an electromagnetic mode with energy E = n h ν {\displaystyle E=nh\nu } as

4050-444: A similar effect in chlorophyll which he also considered a form of opalescence. Sir John Herschel studied quinine in 1845 and came to a different incorrect conclusion. In 1842, A.E. Becquerel observed that calcium sulfide emits light after being exposed to solar ultraviolet , making him the first to state that the emitted light is of longer wavelength than the incident light. While his observation of photoluminescence

4185-458: A standard. The quinine salt quinine sulfate in a sulfuric acid solution was regarded as the most common fluorescence standard, 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

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4320-399: A state with n {\displaystyle n} photons, each of energy h ν {\displaystyle h\nu } . This approach gives the correct energy fluctuation formula. Dirac took this one step further. He treated the interaction between a charge and an electromagnetic field as a small perturbation that induces transitions in the photon states, changing

4455-414: A unit related to the illumination of the eye and the resulting sensation of light and was used later in a physiological context. Although Wolfers's and Lewis's theories were contradicted by many experiments and never accepted, the new name was adopted by most physicists very soon after Compton used it. In physics, a photon is usually denoted by the symbol γ (the Greek letter gamma ). This symbol for

4590-563: Is a quantum of the electromagnetic field , including electromagnetic radiation such as light and radio waves , and the force carrier for the electromagnetic force . Photons are massless particles that always move at the speed of light measured in vacuum. The photon belongs to the class of boson particles. As with other elementary particles, photons are best explained by quantum mechanics and exhibit wave–particle duality , their behavior featuring properties of both waves and particles . The modern photon concept originated during

4725-459: 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 where I ∥ {\displaystyle I_{\parallel }}

4860-475: 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. In general, emitted fluorescence light has a longer wavelength and lower energy than the absorbed light. This phenomenon, known as Stokes shift , is due to energy loss between

4995-485: Is in the visible region . This gives the fluorescent substance a distinct color that is best seen when it has been exposed to UV light , making it appear to glow in the dark. However, any light of a shorter wavelength may cause a material to fluoresce at a longer wavelength. Fluorescent materials may also be excited by certain wavelengths of visible light, which masks the glow, yet their colors may appear bright and intensified. Other fluorescent materials emit their light in

5130-404: Is matlaline, which is the oxidation product of one of the flavonoids found in this wood. In 1819, E.D. Clarke and in 1822 René Just Haüy described some varieties of fluorites that had a different color depending if the light was reflected or (apparently) transmitted; Haüy's incorrectly viewed the effect as light scattering similar to opalescence . In 1833 Sir David Brewster described

5265-452: Is merely a byproduct of the organism's tissue biochemistry and does not have a functional purpose. However, some cases of functional and adaptive significance of fluorescence in the aphotic zone of the deep ocean is an active area of research. Bony fishes living in shallow water generally have good color vision due to their living in a colorful environment. Thus, in shallow-water fishes, red, orange, and green fluorescence most likely serves as

5400-483: Is not quantized, but matter appears to obey the laws of quantum mechanics . Although the evidence from chemical and physical experiments for the existence of photons was overwhelming by the 1970s, this evidence could not be considered as absolutely definitive; since it relied on the interaction of light with matter, and a sufficiently complete theory of matter could in principle account for the evidence. Nevertheless, all semiclassical theories were refuted definitively in

5535-456: Is on the surface. 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 1000 m. At these depths of the aphotic zone, the only sources of light are organisms themselves, giving off light through chemical reactions in

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5670-440: Is one of two kinds of emission of light by a substance that has absorbed light or other electromagnetic radiation . When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colored visible light. The color of the light emitted depends on the chemical composition of the substance. Fluorescent materials generally cease to glow nearly immediately when the radiation source stops. This distinguishes them from

5805-402: Is possible that the absorption of one analyte can affect the secretion of another analyte; this is called capture effects. The effect an analyte has on another analyte could be positive or negative (the production of the second analyte can either increase or decrease). To counteract capture effects, it is possible to use co-stimulation in order to bypass the decreased production of an analyte. This

5940-586: Is proportional to its frequency ν {\displaystyle \nu } according to E = h ν {\displaystyle E=h\nu } , where h {\displaystyle h} is the Planck constant . 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. Examples include internal conversion , intersystem crossing to

6075-575: Is sufficient to describe the substance itself as fluorescent . Fluorescence was observed long before it was named and understood. An early observation of fluorescence was known to the Aztecs and described in 1560 by Bernardino de Sahagún and in 1565 by Nicolás Monardes in the infusion known as lignum nephriticum ( Latin for "kidney wood"). It was derived from the wood of two tree species, Pterocarpus indicus and Eysenhardtia polystachya . The chemical compound responsible for this fluorescence

6210-479: Is termed phosphorescence . The ground state of most molecules is a singlet state , denoted as S 0 . A notable exception is molecular oxygen , which has a triplet ground state. 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

6345-505: Is the gauge boson for electromagnetism , and therefore all other quantum numbers of the photon (such as lepton number , baryon number , and flavour quantum numbers ) are zero. Also, the photon obeys Bose–Einstein statistics , and not Fermi–Dirac statistics . That is, they do not obey the Pauli exclusion principle and more than one can occupy the same bound quantum state. Photons are emitted in many natural processes. For example, when

6480-414: Is the magnitude of the momentum vector p . This derives from the following relativistic relation, with m = 0 : The energy and momentum of a photon depend only on its frequency ( ν {\displaystyle \nu } ) or inversely, its wavelength ( λ ): where k is the wave vector , where Since p {\displaystyle {\boldsymbol {p}}} points in

6615-512: Is the rate constant for absorption. For the reverse process, there are two possibilities: spontaneous emission of a photon, or the emission of a photon initiated by the interaction of the atom with a passing photon and the return of the atom to the lower-energy state. Following Einstein's approach, the corresponding rate R i j {\displaystyle R_{ij}} for the emission of photons of frequency ν {\displaystyle \nu } and transition from

6750-399: 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. Anisotropy is independent of the intensity of the absorbed or emitted light, it is the property of the light, so photobleaching of the dye will not affect

6885-432: 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: where Γ t o t {\displaystyle \Gamma _{tot}}

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7020-402: Is the natural production of light by chemical reactions within an organism, whereas fluorescence is the absorption and reemission of light from the environment. Fireflies and anglerfish are two examples of bioluminescent organisms. To add to the potential confusion, some organisms are both bioluminescent and fluorescent, like the sea pansy Renilla reniformis , where bioluminescence serves as

7155-427: Is the photon's frequency . The photon has no electric charge , is generally considered to have zero rest mass and is a stable particle . The experimental upper limit on the photon mass is very small, on the order of 10 kg; its lifetime would be more than 10 years. For comparison the age of the universe is about 1.38 × 10 years. In a vacuum, a photon has two possible polarization states. The photon

7290-464: Is the rate constant of spontaneous emission of radiation and 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: 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

7425-417: 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

7560-418: Is their movement, aggregation, and dispersion within the fluorescent chromatophore that cause directed fluorescence patterning. 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. Fluorescent chromatophores can be found in the skin (e.g. in fish) just below

7695-417: Is used for private communication between members of the same species. Due to the prominence of blue light at ocean depths, red light and light of longer wavelengths are muddled, and many predatory reef fish have little to no sensitivity for light at these wavelengths. Fish such as the fairy wrasse that have developed visual sensitivity to longer wavelengths are able to display red fluorescent signals that give

7830-536: Is violated by simple molecules, such an example is azulene. 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. For many fluorophores the absorption spectrum is a mirror image of the emission spectrum. This is known as the mirror image rule and is related to the Franck–Condon principle which states that electronic transitions are vertical, that

7965-686: Is when a second antibody that stimulates the production of the same analyte is added to the wells. The ELISpot and FluoroSpot assays can be used in many research fields: vaccine development, cancer, allergies, monocytes/macrophages/dendritic cells characterization, apolipoproteins analysis, and veterinary research. With the ELISpot, you can study antigen-specific cytokine responses, antibody specific secreting cells, tumor antigens, granzyme B and Perforin release by T cells, vaccine efficacy, epitope mapping, cytotoxic T-cell activity, detection of IL-4, IL-5, and IL-13, vaccine-induced antibody responses, antigen-specific memory B cells, and much more. More specifically,

8100-499: The center of momentum frame , the colliding antiparticles have no net momentum, whereas a single photon always has momentum (determined by the photon's frequency or wavelength, which cannot be zero). Hence, conservation of momentum (or equivalently, translational invariance ) requires that at least two photons are created, with zero net momentum. The energy of the two photons, or, equivalently, their frequency, may be determined from conservation of four-momentum . Seen another way,

8235-458: The degeneracy of the state i {\displaystyle i} and that of j {\displaystyle j} , respectively, E i {\displaystyle E_{i}} and E j {\displaystyle E_{j}} their energies, k {\displaystyle k} the Boltzmann constant and T {\displaystyle T}

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8370-423: The energy of a Maxwellian light wave were localized into point-like quanta that move independently of one another, even if the wave itself is spread continuously over space. In 1909 and 1916, Einstein showed that, if Planck's law regarding black-body radiation is accepted, the energy quanta must also carry momentum p = ⁠ h  / λ  ⁠ , making them full-fledged particles. This photon momentum

8505-476: The photoelectric effect , Einstein introduced the idea that light itself is made of discrete units of energy. In 1926, Gilbert N. Lewis popularized the term photon for these energy units. Subsequently, many other experiments validated Einstein's approach. In the Standard Model of particle physics , photons and other elementary particles are described as a necessary consequence of physical laws having

8640-407: The probability amplitude of observable events is calculated by summing over all possible intermediate steps, even ones that are unphysical; hence, virtual photons are not constrained to satisfy E = p c {\displaystyle E=pc} , and may have extra polarization states; depending on the gauge used, virtual photons may have three or four polarization states, instead of

8775-415: The 1970s and 1980s by photon-correlation experiments. Hence, Einstein's hypothesis that quantization is a property of light itself is considered to be proven. Photons obey the laws of quantum mechanics, and so their behavior has both wave-like and particle-like aspects. When a photon is detected by a measuring instrument, it is registered as a single, particulate unit. However, the probability of detecting

8910-543: The IFN-y cytokine and granzyme B secretion in order to assess cytotoxic T-cell responses. Both of these are used for cancer research. With B-cell FluoroSpot, vaccine efficacy can also be observed by quantifying the secretion of IgG, IgA, and IgM before and after a vaccination. This analysis of multiple immunoglobulins is made possible because of the fluorescence method used in the FluoroSpot. Fluorescence Fluorescence

9045-543: The Maxwell theory of light allows for all possible energies of electromagnetic radiation, most physicists assumed initially that the energy quantization resulted from some unknown constraint on the matter that absorbs or emits the radiation. In 1905, Einstein was the first to propose that energy quantization was a property of electromagnetic radiation itself. Although he accepted the validity of Maxwell's theory, Einstein pointed out that many anomalous experiments could be explained if

9180-466: The Nobel lectures of Wien , Planck and Millikan.) Instead, there was a widespread belief that energy quantization resulted from some unknown constraint on the matter that absorbed or emitted radiation. Attitudes changed over time. In part, the change can be traced to experiments such as those revealing Compton scattering , where it was much more difficult not to ascribe quantization to light itself to explain

9315-534: The T-cell ELISpot assay is used to characterize T-cell subsets. This is because the assay can detect the production of cytokines IFN-y, IL-2, TNF-alpha, IL-4, IL-5, and IL-13. The first three cytokines are produced by Th1 cells, while the last three are produced by Th2 cells. Measuring T-cell responses through cytokine production also makes it possible to study vaccine efficacy. With T-cell FluoroSpot, you can monitor tumor-infiltrating lymphocytes. You can also analyze

9450-499: The absorbing light is on. Fluorescence can be of any wavelength but is often more significant when emitted photons are in the visible spectrum. When it occurs in a living organism, it is sometimes called biofluorescence. Fluorescence should not be confused with bioluminescence and biophosphorescence. Pumpkin toadlets that live in the Brazilian Atlantic forest are fluorescent. Bioluminescence differs from fluorescence in that it

9585-730: The ambient blue light of the photic zone to aid vision. Red light can only be seen across short distances due to attenuation of red light wavelengths by water. Many fish species that fluoresce are small, group-living, or benthic/aphotic, and have conspicuous patterning. 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. The patterns present in ocular rings to indicate directionality of an individual's gaze, and along fins to indicate directionality of an individual's movement. Current research suspects that this red fluorescence

9720-399: The anisotropy value as long as the signal is detectable. 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

9855-609: 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. 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

9990-438: The atoms can emit and absorb that radiation. Thermal equilibrium requires that the energy density ρ ( ν ) {\displaystyle \rho (\nu )} of photons with frequency ν {\displaystyle \nu } (which is proportional to their number density ) is, on average, constant in time; hence, the rate at which photons of any particular frequency are emitted must equal

10125-527: The average across many interactions between matter and radiation. However, refined Compton experiments showed that the conservation laws hold for individual interactions. Accordingly, Bohr and his co-workers gave their model "as honorable a funeral as possible". Nevertheless, the failures of the BKS model inspired Werner Heisenberg in his development of matrix mechanics . A few physicists persisted in developing semiclassical models in which electromagnetic radiation

10260-486: The coefficients A i j {\displaystyle A_{ij}} , B j i {\displaystyle B_{ji}} and B i j {\displaystyle B_{ij}} once physicists had obtained "mechanics and electrodynamics modified to accommodate the quantum hypothesis". Not long thereafter, in 1926, Paul Dirac derived the B i j {\displaystyle B_{ij}} rate constants by using

10395-567: 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. 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 states that

10530-418: The concept in analyzing a thought experiment involving an electron and a high-energy photon . However, Heisenberg did not give precise mathematical definitions of what the "uncertainty" in these measurements meant. The precise mathematical statement of the position–momentum uncertainty principle is due to Kennard , Pauli , and Weyl . The uncertainty principle applies to situations where an experimenter has

10665-466: The direction of the photon's propagation, the magnitude of its momentum is The photon also carries spin angular momentum , which is related to photon polarization . (Beams of light also exhibit properties described as orbital angular momentum of light ). The angular momentum of the photon has two possible values, either +ħ or −ħ . These two possible values correspond to the two possible pure states of circular polarization . Collections of photons in

10800-500: The efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the 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: where k f {\displaystyle {k}_{f}}

10935-484: The electric field of an atomic nucleus. The classical formulae for the energy and momentum of electromagnetic radiation can be re-expressed in terms of photon events. For example, the pressure of electromagnetic radiation on an object derives from the transfer of photon momentum per unit time and unit area to that object, since pressure is force per unit area and force is the change in momentum per unit time. Current commonly accepted physical theories imply or assume

11070-450: The electromagnetic field. Einstein was troubled by the fact that his theory seemed incomplete, since it did not determine the direction of a spontaneously emitted photon. A probabilistic nature of light-particle motion was first considered by Newton in his treatment of birefringence and, more generally, of the splitting of light beams at interfaces into a transmitted beam and a reflected beam. Newton hypothesized that hidden variables in

11205-487: The electromagnetic wave, Δ N {\displaystyle \Delta N} , and the uncertainty in the phase of the wave, Δ ϕ {\displaystyle \Delta \phi } . However, this cannot be an uncertainty relation of the Kennard–Pauli–Weyl type, since unlike position and momentum, the phase ϕ {\displaystyle \phi } cannot be represented by

11340-441: The energy of any system that absorbs or emits electromagnetic radiation of frequency ν is an integer multiple of an energy quantum E = hν . As shown by Albert Einstein , some form of energy quantization must be assumed to account for the thermal equilibrium observed between matter and electromagnetic radiation ; for this explanation of the photoelectric effect, Einstein received the 1921 Nobel Prize in physics. Since

11475-420: 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 . Fish may also be sensitive to cortisol induced stress responses to environmental stimuli, such as interaction with

11610-404: The eye. Therefore, warm colors from the visual light spectrum appear less vibrant at increasing depths. Water scatters light of shorter wavelengths above violet, meaning cooler colors dominate the visual field in the photic zone . Light intensity decreases 10 fold with every 75 m of depth, so at depths of 75 m, light is 10% as intense as it is on the surface, and is only 1% as intense at 150 m as it

11745-501: The final blow to particle models of light. The Maxwell wave theory , however, does not account for all properties of light. The Maxwell theory predicts that the energy of a light wave depends only on its intensity , not on its frequency ; nevertheless, several independent types of experiments show that the energy imparted by light to atoms depends only on the light's frequency, not on its intensity. For example, some chemical reactions are provoked only by light of frequency higher than

11880-411: The first two decades of the 20th century with the work of Albert Einstein , who built upon the research of Max Planck . While Planck was trying to explain how matter and electromagnetic radiation could be in thermal equilibrium with one another, he proposed that the energy stored within a material object should be regarded as composed of an integer number of discrete, equal-sized parts. To explain

12015-418: The galactic vector potential . Although the galactic vector potential is large because the galactic magnetic field exists on great length scales, only the magnetic field would be observable if the photon is massless. In the case that the photon has mass, the mass term ⁠ 1 / 2 ⁠ m A μ A would affect the galactic plasma. The fact that no such effects are seen implies an upper bound on

12150-657: The galactic vector potential have been shown to be model-dependent. If the photon mass is generated via the Higgs mechanism then the upper limit of m ≲ 10  eV/ c from the test of Coulomb's law is valid. In most theories up to the eighteenth century, light was pictured as being made of particles. Since particle models cannot easily account for the refraction , diffraction and birefringence of light, wave theories of light were proposed by René Descartes (1637), Robert Hooke (1665), and Christiaan Huygens (1678); however, particle models remained dominant, chiefly due to

12285-404: The influence of Isaac Newton . In the early 19th century, Thomas Young and August Fresnel clearly demonstrated the interference and diffraction of light, and by 1850 wave models were generally accepted. James Clerk Maxwell 's 1865 prediction that light was an electromagnetic wave – which was confirmed experimentally in 1888 by Heinrich Hertz 's detection of radio waves – seemed to be

12420-595: The infrared or even the ultraviolet regions of the spectrum. Fluorescence has many practical applications, including mineralogy , gemology , medicine , chemical sensors ( fluorescence spectroscopy ), fluorescent labelling , dyes , biological detectors, cosmic-ray detection, vacuum fluorescent displays , and cathode-ray tubes . Its most common everyday application is in ( gas-discharge ) fluorescent lamps and LED lamps , in which fluorescent coatings convert UV or blue light into longer-wavelengths resulting in white light which can even appear indistinguishable from that of

12555-455: The light particle determined which of the two paths a single photon would take. Similarly, Einstein hoped for a more complete theory that would leave nothing to chance, beginning his separation from quantum mechanics. Ironically, Max Born 's probabilistic interpretation of the wave function was inspired by Einstein's later work searching for a more complete theory. In 1910, Peter Debye derived Planck's law of black-body radiation from

12690-850: The light source for fluorescence. 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 electron. Unlike with fluorescence, in phosphorescence the electron retains stability, emitting light that continues to "glow in the dark" even after the stimulating light source has been removed. For example, glow-in-the-dark stickers are phosphorescent, but there are no truly biophosphorescent animals known. 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

12825-404: 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 . Therefore, most commonly, fluorescence occurs from the first singlet excited state, S 1 . Fluorescence is the emission of a photon accompanying

12960-434: The luminesce (fluorescence or phosphorescence) of a molecule will be emitted only from the lowest excited state of its given multiplicity. 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. Kasha's rule does not always apply and

13095-400: The nanosecond (billionth of a second) range. In physics, this first mechanism was termed "fluorescence" or "singlet emission", and is common in many laser mediums such as ruby. Other fluorescent materials were discovered to have much longer decay times, because some of the atoms would change their spin to a triplet state , thus would glow brightly with fluorescence under excitation but produce

13230-439: The necessary yellow intraocular filters for visualizing fluorescence potentially exploit a light signal from members of it. Fluorescent patterning was especially prominent in cryptically patterned fishes possessing complex camouflage. Many of these lineages also possess yellow long-pass intraocular filters that could enable visualization of such patterns. Another adaptive use of fluorescence is to generate orange and red light from

13365-418: The number of atoms in state i {\displaystyle i} and those in state j {\displaystyle j} must, on average, be constant; hence, the rates R j i {\displaystyle R_{ji}} and R i j {\displaystyle R_{ij}} must be equal. Also, by arguments analogous to the derivation of Boltzmann statistics ,

13500-509: The numbers of photons in the modes, while conserving energy and momentum overall. Dirac was able to derive Einstein's A i j {\displaystyle A_{ij}} and B i j {\displaystyle B_{ij}} coefficients from first principles, and showed that the Bose–Einstein statistics of photons is a natural consequence of quantizing the electromagnetic field correctly (Bose's reasoning went in

13635-470: The observed results. Even after Compton's experiment, Niels Bohr , Hendrik Kramers and John Slater made one last attempt to preserve the Maxwellian continuous electromagnetic field model of light, the so-called BKS theory . An important feature of the BKS theory is how it treated the conservation of energy and the conservation of momentum . In the BKS theory, energy and momentum are only conserved on

13770-472: The opposite direction; he derived Planck's law of black-body radiation by assuming B–E statistics). In Dirac's time, it was not yet known that all bosons, including photons, must obey Bose–Einstein statistics. Dirac's second-order perturbation theory can involve virtual photons , transient intermediate states of the electromagnetic field; the static electric and magnetic interactions are mediated by such virtual photons. In such quantum field theories ,

13905-417: The other type of light emission, phosphorescence . Phosphorescent materials continue to emit light for some time after the radiation stops. Fluorescence occurs when a photon of the incoming radiation is absorbed by an orbital electron in a molecule of the material, exciting it to a higher energy level . The electron then returns to its former energy level by losing energy, emitting another photon of

14040-405: The overall uncertainty as far as quantum mechanics allows. Quantum optics makes use of coherent states for modes of the electromagnetic field. There is a tradeoff, reminiscent of the position–momentum uncertainty relation, between measurements of an electromagnetic wave's amplitude and its phase. This is sometimes informally expressed in terms of the uncertainty in the number of photons present in

14175-400: 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 marine spectrum, yellow the second most, orange the third, and red is the rarest. Fluorescence can occur in organisms in the aphotic zone as a byproduct of that same organism's bioluminescence. Some fluorescence in the aphotic zone

14310-413: The photic zone or green bioluminescence in the aphotic zone into red light to aid vision. A new fluorophore was described in two species of sharks, wherein it was due to an undescribed group of brominated tryptophane-kynurenine small molecule metabolites. Photon A photon (from Ancient Greek φῶς , φωτός ( phôs, phōtós )  'light') is an elementary particle that

14445-400: The photon can be considered as its own antiparticle (thus an "antiphoton" is simply a normal photon with opposite momentum, equal polarization, and 180° out of phase). The reverse process, pair production , is the dominant mechanism by which high-energy photons such as gamma rays lose energy while passing through matter. That process is the reverse of "annihilation to one photon" allowed in

14580-463: The photon mass of m < 3 × 10  eV/ c . The galactic vector potential can also be probed directly by measuring the torque exerted on a magnetized ring. Such methods were used to obtain the sharper upper limit of 1.07 × 10  eV/ c (the equivalent of 10   daltons ) given by the Particle Data Group . These sharp limits from the non-observation of the effects caused by

14715-415: The photon probably derives from gamma rays , which were discovered in 1900 by Paul Villard , named by Ernest Rutherford in 1903, and shown to be a form of electromagnetic radiation in 1914 by Rutherford and Edward Andrade . In chemistry and optical engineering , photons are usually symbolized by hν , which is the photon energy , where h is the Planck constant and the Greek letter ν ( nu )

14850-473: The photon to be strictly massless. If photons were not purely massless, their speeds would vary with frequency, with lower-energy (redder) photons moving slightly slower than higher-energy photons. Relativity would be unaffected by this; the so-called speed of light, c , would then not be the actual speed at which light moves, but a constant of nature which is the upper bound on speed that any object could theoretically attain in spacetime. Thus, it would still be

14985-400: The presence of multiple analytes on one plate of wells, whereas the ELISpot assay can only analyze one analyte at a time. The FluoroSpot assay accomplishes this by using fluorescence rather than an enzymatic reaction for detection. The steps for a FluoroSpot assay are also similar, with a few differences. Since the FluoroSpot assay identifies and quantifies the presence of multiple analytes, it

15120-444: The rate at which they are absorbed . Einstein began by postulating simple proportionality relations for the different reaction rates involved. In his model, the rate R j i {\displaystyle R_{ji}} for a system to absorb a photon of frequency ν {\displaystyle \nu } and transition from a lower energy E j {\displaystyle E_{j}} to

15255-564: 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 . The Jablonski diagram describes most of

15390-524: The ratio of N i {\displaystyle N_{i}} and N j {\displaystyle N_{j}} is g i / g j exp ⁡ ( E j − E i ) / ( k T ) , {\displaystyle g_{i}/g_{j}\exp {(E_{j}-E_{i})/(kT)},} where g i {\displaystyle g_{i}} and g j {\displaystyle g_{j}} are

15525-428: 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. 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. The polarization of the emitted light will also depend on the transition moment. The transition moment

15660-417: The relaxation of the excited state to the ground state. Fluorescence photons are lower in energy ( h ν e m {\displaystyle h\nu _{em}} ) compared to the energy of the photons used to generate the excited state ( h ν e x {\displaystyle h\nu _{ex}} ) In each case the photon energy E {\displaystyle E}

15795-405: The requirement for a symmetric quantum mechanical state . This work led to the concept of coherent states and the development of the laser. In the same papers, Einstein extended Bose's formalism to material particles (bosons) and predicted that they would condense into their lowest quantum state at low enough temperatures; this Bose–Einstein condensation was observed experimentally in 1995. It

15930-411: The secretion of a lymphokine by T cells . In the same year, dual-color ELISpot was combined with computer imaging for the first time, which allowed for the enumeration and analysis of spots. 1988 also marked the first use of membrane-bottomed plates for performing these assays. The FluoroSpot assay is very similar to the ELISpot assay. The main difference is that the FluoroSpot assay is able to analyze

16065-448: The source's temperature. Advances in spectroscopy and quantum electronics between the 1950s and 1970s provided a way to distinguish between the three different mechanisms that produce the light, as well as narrowing down the typical timescales those mechanisms take to decay after absorption. In modern science, this distinction became important because some items, such as lasers, required the fastest decay times, which typically occur in

16200-465: The speed of light. If Coulomb's law is not exactly valid, then that would allow the presence of an electric field to exist within a hollow conductor when it is subjected to an external electric field. This provides a means for precision tests of Coulomb's law . A null result of such an experiment has set a limit of m ≲ 10  eV/ c . Sharper upper limits on the mass of light have been obtained in experiments designed to detect effects caused by

16335-412: The speed of spacetime ripples ( gravitational waves and gravitons ), but it would not be the speed of photons. If a photon did have non-zero mass, there would be other effects as well. Coulomb's law would be modified and the electromagnetic field would have an extra physical degree of freedom . These effects yield more sensitive experimental probes of the photon mass than the frequency dependence of

16470-489: The summation as well; for example, two photons may interact indirectly through virtual electron – positron pairs . Such photon–photon scattering (see two-photon physics ), as well as electron–photon scattering, is meant to be one of the modes of operations of the planned particle accelerator, the International Linear Collider . In modern physics notation, the quantum state of the electromagnetic field

16605-575: The sun, conversion of light into different wavelengths, or for signaling are thought to have evolved secondarily. Currently, relatively little is known about the functional significance of fluorescence and fluorescent proteins. However, it is suspected that fluorescence may serve important functions in signaling and communication, mating , lures, camouflage , UV protection and antioxidation, photoacclimation, dinoflagellate regulation, and in coral health. Water absorbs light of long wavelengths, so less light from these wavelengths reflects back to reach

16740-582: The system's temperature . From this, it is readily derived that g i B i j = g j B j i {\displaystyle g_{i}B_{ij}=g_{j}B_{ji}} and The A i j {\displaystyle A_{ij}} and B i j {\displaystyle B_{ij}} are collectively known as the Einstein coefficients . Einstein could not fully justify his rate equations, but claimed that it should be possible to calculate

16875-565: The term in a letter to Nature on 18 December 1926. The same name was used earlier but was never widely adopted before Lewis: in 1916 by the American physicist and psychologist Leonard T. Troland , in 1921 by the Irish physicist John Joly , in 1924 by the French physiologist René Wurmser (1890–1993), and in 1926 by the French physicist Frithiof Wolfers (1891–1971). The name was suggested initially as

17010-405: 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

17145-477: The traditional but energy-inefficient incandescent lamp . Fluorescence also occurs frequently in nature in some minerals and in many biological forms across all kingdoms of life. The latter may be referred to as biofluorescence , indicating that the fluorophore is part of or is extracted from a living organism (rather than an inorganic dye or stain ). But since fluorescence is due to a specific chemical, which can also be synthesized artificially in most cases, it

17280-496: 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 just because of its unusual triplet ground state. The fluorescence quantum yield gives

17415-423: The two states of real photons. Although these transient virtual photons can never be observed, they contribute measurably to the probabilities of observable events. Indeed, such second-order and higher-order perturbation calculations can give apparently infinite contributions to the sum. Such unphysical results are corrected for using the technique of renormalization . Other virtual particles may contribute to

17550-442: The violet end of the visible spectrum into visible light. He named this phenomenon fluorescence Neither Becquerel nor Stokes understood one key aspect of photoluminescence: the critical difference from incandescence , the emission of light by heated material. To distinguish it from incandescence, in the late 1800s, Gustav Wiedemann proposed the term luminescence to designate any emission of light more intense than expected from

17685-406: Was called persistent phosphorescence or persistent luminescence , to distinguish it from the other two mechanisms. Fluorescence occurs when an excited molecule, atom, or nanostructure , relaxes to a lower energy state (usually the ground state ) through emission of a photon without a change in electron spin . When the initial and final states have different multiplicity (spin), the phenomenon

17820-441: Was later used by Lene Hau to slow, and then completely stop, light in 1999 and 2001. The modern view on this is that photons are, by virtue of their integer spin, bosons (as opposed to fermions with half-integer spin). By the spin-statistics theorem , all bosons obey Bose–Einstein statistics (whereas all fermions obey Fermi–Dirac statistics ). In 1916, Albert Einstein showed that Planck's radiation law could be derived from

17955-493: Was observed experimentally by Arthur Compton , for which he received the Nobel Prize in 1927. The pivotal question then, was how to unify Maxwell's wave theory of light with its experimentally observed particle nature. The answer to this question occupied Albert Einstein for the rest of his life, and was solved in quantum electrodynamics and its successor, the Standard Model . (See § Quantum field theory and § As

18090-419: Was similar to that described 10 years later by Stokes, who observed a fluorescence of a solution of quinine , the phenomenon that Becquerel described with calcium sulfide is now called phosphorescence . In his 1852 paper on the "Refrangibility" ( wavelength change) of light, George Gabriel Stokes described the ability of fluorspar , uranium glass and many other substances to change invisible light beyond

18225-533: Was used before 1900 to mean particles or amounts of different quantities , including electricity . In 1900, the German physicist Max Planck was studying black-body radiation , and he suggested that the experimental observations, specifically at shorter wavelengths , would be explained if the energy stored within a molecule was a "discrete quantity composed of an integral number of finite equal parts", which he called "energy elements". In 1905, Albert Einstein published

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