La fluorescencia es la emisión de luz por una sustancia que ha absorbido luz u otra radiación electromagnética . Es una forma de luminiscencia . En la mayoría de los casos, la luz emitida tiene una longitud de onda más larga y, por lo tanto, una energía más baja que la radiación absorbida. El ejemplo más sorprendente de fluorescencia ocurre cuando la radiación absorbida está en la región ultravioleta del espectro y, por lo tanto, invisible para el ojo humano, mientras que la luz emitida está en la región visible, lo que le da a la sustancia fluorescente un color distintivo que se puede ver. solo cuando se expone a la luz ultravioleta. Los materiales fluorescentes dejan de brillar casi inmediatamente cuando la fuente de radiación se detiene, a diferencia de los materiales fosforescentes , que continúan emitiendo luz durante algún tiempo después.
La fluorescencia tiene muchas aplicaciones prácticas, que incluyen mineralogía , gemología , medicina , sensores químicos ( espectroscopia de fluorescencia ), etiquetado fluorescente , tintes , detectores biológicos, detección de rayos cósmicos, pantallas fluorescentes al vacío y tubos de rayos catódicos . Su aplicación diaria más común es en lámparas fluorescentes y lámparas LED de bajo consumo , donde se utilizan revestimientos fluorescentes para convertir la luz ultravioleta de longitud de onda corta o la luz azul en luz amarilla de longitud de onda más larga, imitando así la luz cálida de las lámparas incandescentes de bajo consumo energético .
La fluorescencia también ocurre con frecuencia en la naturaleza en algunos minerales y en muchas formas biológicas en todos los reinos de la vida. Esto a veces se denomina biofluorescencia para indicar que el fluoróforo se deriva de un organismo vivo (en contraposición a la adición artificial de un tinte o tinte ). Sin embargo, en muchos casos, la sustancia puede ser fluorescente incluso si el organismo está muerto, por lo que la fluorescencia sigue siendo el término preferido.
Historia
Una observación temprana de fluorescencia fue descrita en 1560 por Bernardino de Sahagún y en 1565 por Nicolás Monardes en la infusión conocida como lignum nephriticum (en latín "madera de riñón"). Se deriva de la madera de dos especies de árboles, Pterocarpus indicus y Eysenhardtia polystachya . [1] [2] [3] [4] El compuesto químico responsable de esta fluorescencia es la matlalina, que es el producto de oxidación de uno de los flavonoides que se encuentran en esta madera. [1]
En 1819, Edward D. Clarke [5] y en 1822 René Just Haüy [6] describieron la fluorescencia en las fluoritas , Sir David Brewster describió el fenómeno de la clorofila en 1833 [7] y Sir John Herschel hizo lo mismo con la quinina en 1845. [ 8] [9]
En su artículo de 1852 sobre la "Refrangibilidad" ( cambio de longitud de onda ) de la luz, George Gabriel Stokes describió la capacidad de la fluorita y el vidrio de uranio para cambiar la luz invisible más allá del extremo violeta del espectro visible en luz azul. Llamó a este fenómeno fluorescencia : "Casi me inclino a acuñar una palabra y llamar a la apariencia fluorescencia , de fluor-spar [es decir, fluorita], ya que el término análogo opalescencia se deriva del nombre de un mineral". [10] El nombre se deriva del mineral fluorita (difluoruro de calcio), algunos ejemplos del cual contienen trazas de europio divalente , que sirve como activador fluorescente para emitir luz azul. En un experimento clave, usó un prisma para aislar la radiación ultravioleta de la luz solar y observó la luz azul emitida por una solución de etanol de quinina expuesta a ella. [11]
Principios fisicos
Mecanismo
La fluorescencia se produce cuando una molécula, un átomo o una nanoestructura excitados se relaja a un estado de menor energía (posiblemente el estado fundamental ) a través de la emisión de un fotón . Puede haber sido excitado directamente desde el estado fundamental S 0 a un estado singlete [ dudoso ] [12] S 2 desde el estado fundamental por absorción de un fotón de energía y posteriormente emite un fotón de menor energía a medida que se relaja al estado S 1 :
- Excitación:
- Fluorescencia (emisión):
En cada caso, la energía de los fotones es proporcional a su frecuencia de acuerdo a , dónde es la constante de Planck . El estado final S 1 , si no el estado fundamental, puede perder su energía restante a través de una emisión fluorescente adicional y / o relajación no radiativa en la que la energía se disipa como calor ( fonones ). Cuando el estado excitado es un estado metaestable (de larga duración), entonces esa transición fluorescente se denomina fosforescencia . La relajación de un estado excitado también puede ocurrir mediante la transferencia de parte o toda su energía a una segunda molécula a través de una interacción conocida como extinción de la fluorescencia . El oxígeno molecular (O 2 ) es un extintor de la fluorescencia extremadamente eficiente solo por su inusual estado fundamental triplete. En todos los casos, la luz emitida tiene menor energía (menor frecuencia, mayor longitud de onda) que la radiación absorbida; la diferencia en estas energías se conoce como el cambio de Stokes . En algunos casos, bajo iluminación intensa, es posible que un electrón absorba dos fotones, lo que permite la emisión de radiación de una energía de fotón más alta (longitud de onda más corta) que la radiación absorbida; tal absorción de dos fotones no se denomina fluorescencia. Una molécula que se excita a través de la absorción de luz u otro proceso (por ejemplo, de una reacción química) puede transferir su energía a una segunda molécula 'sensibilizada' [ clarificación necesaria ] , elevándola a un estado excitado desde el cual emitirá fluorescencia.
Rendimiento cuántico
El rendimiento cuántico de fluorescencia da la eficiencia del proceso de fluorescencia. Se define como la relación entre el número de fotones emitidos y el número de fotones absorbidos. [13] [14]
El rendimiento cuántico de fluorescencia máximo posible es 1,0 (100%); cada fotón absorbido da como resultado un fotón emitido. Los compuestos con rendimientos cuánticos de 0,10 todavía se consideran bastante fluorescentes. Otra forma de definir el rendimiento cuántico de fluorescencia es mediante la tasa de desintegración del estado excitado:
dónde es la tasa constante de emisión espontánea de radiación y
es la suma de todas las tasas de deterioro del estado excitado. Otras tasas de deterioro del estado excitado son causadas por mecanismos distintos a la emisión de fotones y, por lo tanto, a menudo se denominan "tasas no radiativas", que pueden incluir: extinción dinámica de colisiones, interacción dipolo-dipolo de campo cercano (o transferencia de energía de resonancia ), conversión interna y cruce entre sistemas . Por lo tanto, si cambia la velocidad de cualquier ruta, se verán afectados tanto la vida útil del estado excitado como el rendimiento cuántico de fluorescencia.
Los rendimientos cuánticos de fluorescencia se miden por comparación con un estándar. La quinina sal sulfato de quinina en un ácido sulfúrico solución se considera como el estándar más común de fluorescencia, [15] Sin embargo, un estudio reciente reveló que el rendimiento cuántico de fluorescencia de esta solución es fuertemente afectada por la temperatura, y ya no debe ser utilizado como la solución estándar. La quinina en ácido perclórico 0,1 M (Φ = 0,60) no muestra dependencia de la temperatura hasta 45 ° C, por lo que puede considerarse como una solución estándar fiable. [dieciséis]
Toda la vida
La vida útil de la fluorescencia se refiere al tiempo promedio que la molécula permanece en su estado excitado antes de emitir un fotón. La fluorescencia sigue típicamente una cinética de primer orden :
dónde es la concentración de moléculas en estado excitado en el tiempo , es la concentración inicial y Γ {\ Displaystyle \ Gamma} es la tasa de desintegración o la inversa de la vida útil de la fluorescencia. Este es un caso de decaimiento exponencial . Varios procesos radiativos y no radiativos pueden despoblar el estado excitado. En tal caso, la tasa de desintegración total es la suma de todas las tasas:
dónde es la tasa de desintegración total, la tasa de desintegración radiativa y la tasa de desintegración no radiativa. Es similar a una reacción química de primer orden en la que la constante de velocidad de primer orden es la suma de todas las velocidades (un modelo cinético paralelo). Si la tasa de emisión espontánea, o cualquiera de las otras tasas, es rápida, la vida útil es corta. Para los compuestos fluorescentes de uso común, los tiempos de decaimiento del estado excitado típicos para las emisiones de fotones con energías desde el UV al infrarrojo cercano están dentro del rango de 0,5 a 20 nanosegundos . La vida útil de la fluorescencia es un parámetro importante para las aplicaciones prácticas de la fluorescencia, como la transferencia de energía por resonancia de fluorescencia y la microscopía de imágenes de vida útil de la fluorescencia .
Diagrama de Jablonski
El diagrama de Jablonski describe la mayoría de los mecanismos de relajación de las moléculas en estado excitado. El diagrama adjunto muestra cómo se produce la fluorescencia debido a la relajación de ciertos electrones excitados de una molécula. [17]
Anisotropía de fluorescencia
Es más probable que los fluoróforos sean excitados por fotones si el momento de transición del fluoróforo es paralelo al vector eléctrico del fotón. [18] La polarización de la luz emitida también dependerá del momento de transición. El momento de transición depende de la orientación física de la molécula de fluoróforo. Para los fluoróforos en solución, esto significa que la intensidad y la polarización de la luz emitida dependen de la difusión rotacional. Por lo tanto, las mediciones de anisotropía pueden usarse para investigar con qué libertad se mueve una molécula fluorescente en un entorno particular.
La anisotropía de fluorescencia se puede definir cuantitativamente como
dónde es la intensidad emitida paralela a la polarización de la luz de excitación y es la intensidad emitida perpendicular a la polarización de la luz de excitación. [19]
Fluorencia
Los pigmentos fuertemente fluorescentes a menudo tienen una apariencia inusual que a menudo se describe coloquialmente como un "color neón" (originalmente "day-glo" a fines de la década de 1960, principios de la de 1970). Este fenómeno fue denominado "Farbenglut" por Hermann von Helmholtz y "fluorence" por Ralph M. Evans. En general, se cree que está relacionado con el alto brillo del color en relación con lo que sería como componente del blanco. La fluorescencia desplaza la energía en la iluminación incidente de longitudes de onda más cortas a más largas (como el azul al amarillo) y, por lo tanto, puede hacer que el color fluorescente parezca más brillante (más saturado) de lo que podría ser por reflejo solo. [20]
Reglas
Hay varias reglas generales que tratan con la fluorescencia. Cada una de las siguientes reglas tiene excepciones, pero son pautas útiles para comprender la fluorescencia (estas reglas no se aplican necesariamente a la absorción de dos fotones ).
La regla de Kasha
La regla de Kasha dicta que el rendimiento cuántico de luminiscencia es independiente de la longitud de onda de la radiación excitante. [21] Esto ocurre porque las moléculas excitadas suelen decaer al nivel vibratorio más bajo del estado excitado antes de que se produzca la emisión de fluorescencia. La regla de Kasha-Vavilov no siempre se aplica y se viola severamente en muchas moléculas simples. Una afirmación algo más fiable, aunque todavía con excepciones, sería que el espectro de fluorescencia muestra muy poca dependencia de la longitud de onda de la radiación excitante. [22]
Regla de imagen reflejada
Para muchos fluoróforos, el espectro de absorción es una imagen especular del espectro de emisión. [23] Esto se conoce como la regla de la imagen especular y está relacionada con el principio de Franck-Condon que establece que las transiciones electrónicas son verticales, es decir, cambios de energía sin cambio de distancia, como se puede representar con una línea vertical en el diagrama de Jablonski. Esto significa que el núcleo no se mueve y los niveles de vibración del estado excitado se asemejan a los niveles de vibración del estado fundamental.
Cambio de Stokes
En general, la luz de fluorescencia emitida tiene una longitud de onda más larga y una energía más baja que la luz absorbida. [24] Este fenómeno, conocido como desplazamiento de Stokes , se debe a la pérdida de energía entre el momento en que se absorbe un fotón y cuando se emite uno nuevo. Las causas y la magnitud del cambio de Stokes pueden ser complejas y dependen del fluoróforo y su entorno. Sin embargo, existen algunas causas comunes. Con frecuencia se debe a la desintegración no radiativa al nivel de energía vibratoria más bajo del estado excitado. Otro factor es que la emisión de fluorescencia deja con frecuencia un fluoróforo en un nivel vibratorio más alto del estado fundamental.
En naturaleza
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.
Compared to bioluminescence and biophosphorescence
Fluorescence
Fluorescence is the temporary absorption of electromagnetic wavelengths from the visible light spectrum by fluorescent molecules, and the subsequent emission of light at a lower energy level. When it occurs in a living organism, it is sometimes called biofluorescence. This causes the light that is emitted to be a different color than the light that is absorbed. Stimulating light excites an electron, raising energy to an unstable level. This instability is unfavorable, so the energized electron is returned to a stable state almost as immediately as it becomes unstable. This return to stability corresponds with the release of excess energy in the form of fluorescence light. This emission of light is only observable when the stimulant light is still providing light to the organism/object and is typically yellow, pink, orange, red, green, or purple. Fluorescence is often confused with the following forms of biotic light, bioluminescence and biophosphorescence.[25] Pumpkin toadlets that live in the Brazilian Atlantic forest are fluorescent.[26]
Bioluminescence
Bioluminescence differs from fluorescence in that it is the natural production of light by chemical reactions within an organism, whereas fluorescence is the absorption and reemission of light from the environment.[25] Fireflies and anglerfish are two examples of bioluminescent organisms.[27] To add to the potential confusion, some organisms are both bioluminescent and fluorescent, like the sea pansy Renilla reniformis, where bioluminescence serves as the light source for fluorescence.[28]
Phosphorescence
Phosphorescence is similar to fluorescence in its requirement of light wavelengths as a provider of excitation energy. The difference here lies in the relative stability of the energized 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.[25] For example, glow-in-the-dark stickers are phosphorescent, but there are no truly biophosphorescent animals known.[29]
Mechanisms
Epidermal chromatophores
Pigment cells that exhibit fluorescence are called fluorescent chromatophores, and function somatically similar to regular chromatophores. These cells are dendritic, and contain pigments called fluorosomes. These pigments contain fluorescent proteins which are activated by K+ (potassium) ions, and it is their movement, aggregation, and dispersion within the fluorescent chromatophore that cause directed fluorescence patterning.[30][31] Fluorescent cells are innervated the same as other chromatophores, like melanophores, pigment cells that contain melanin. Short term fluorescent patterning and signaling is controlled by the nervous system.[30] Fluorescent chromatophores can be found in the skin (e.g. in fish) just below the epidermis, amongst other chromatophores.
Epidermal fluorescent cells in fish also respond to hormonal stimuli by the α–MSH and MCH hormones much the same as melanophores. This suggests that fluorescent cells may have color changes throughout the day that coincide with their circadian rhythm.[32] Fish may also be sensitive to cortisol induced stress responses to environmental stimuli, such as interaction with a predator or engaging in a mating ritual.[30]
Phylogenetics
Evolutionary origins
The incidence of fluorescence across the tree of life is widespread, and has been studied most extensively in cnidarians and fish. The phenomenon appears to have evolved multiple times in multiple taxa such as in the anguilliformes (eels), gobioidei (gobies and cardinalfishes), and tetradontiformes (triggerfishes), along with the other taxa discussed later in the article. Fluorescence is highly genotypically and phenotypically variable even within ecosystems, in regards to the wavelengths emitted, the patterns displayed, and the intensity of the fluorescence. Generally, the species relying upon camouflage exhibit the greatest diversity in fluorescence, likely because camouflage may be one of the uses of fluorescence.[33]
It is suspected by some scientists that GFPs and GFP-like proteins began as electron donors activated by light. These electrons were then used for reactions requiring light energy. Functions of fluorescent proteins, such as protection from the sun, conversion of light into different wavelengths, or for signaling are thought to have evolved secondarily.[34]
Adaptive functions
Currently, relatively little is known about the functional significance of fluorescence and fluorescent proteins.[34] 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.[35]
Aquatic
Water absorbs light of long wavelengths, so less light from these wavelengths reflects back to reach 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 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 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.[33] 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.[36] The visual field in the photic zone is naturally blue, so colors of fluorescence can be detected as bright reds, oranges, yellows, and greens. Green is the most commonly found color in the 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 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.[37]
Photic zone
Fish
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 a means of communication with conspecifics, especially given the great phenotypic variance of the phenomenon.[33]
Many fish that exhibit fluorescence, such as sharks, lizardfish, scorpionfish, wrasses, and flatfishes, also possess yellow intraocular filters.[38] Yellow intraocular filters in the lenses and cornea of certain fishes function as long-pass filters. These filters enable the species that 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.[33] Fish that possess 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.[38]
Another adaptive use of fluorescence is to generate orange and red light from 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.[39] 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.[39] Current research suspects that this red fluorescence is used for private communication between members of the same species.[30][33][39] 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 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.[39][40]
Additionally, it is suggested that fluorescent tissues that surround an organism's eyes are used to convert blue light from the photic zone or green bioluminescence in the aphotic zone into red light to aid vision.[39]
Sharks
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.[41]
Coral
Fluorescence serves a wide variety of functions in coral. Fluorescent proteins in corals may contribute to photosynthesis by converting otherwise unusable wavelengths of light into ones for which the coral's symbiotic algae are able to conduct photosynthesis.[42] Also, the proteins may fluctuate in number as more or less light becomes available as a means of photoacclimation.[43] Similarly, these fluorescent proteins may possess antioxidant capacities to eliminate oxygen radicals produced by photosynthesis.[44] Finally, through modulating photosynthesis, the fluorescent proteins may also serve as a means of regulating the activity of the coral's photosynthetic algal symbionts.[45]
Cephalopods
Alloteuthis subulata and Loligo vulgaris, two types of nearly transparent squid, have fluorescent spots above their eyes. These spots reflect incident light, which may serve as a means of camouflage, but also for signaling to other squids for schooling purposes.[46]
Jellyfish
Another, well-studied example of fluorescence in the ocean is the hydrozoan Aequorea victoria. This jellyfish lives in the photic zone off the west coast of North America and was identified as a carrier of green fluorescent protein (GFP) by Osamu Shimomura. The gene for these green fluorescent proteins has been isolated and is scientifically significant because it is widely used in genetic studies to indicate the expression of other genes.[47]
Mantis shrimp
Several species of mantis shrimp, which are stomatopod crustaceans, including Lysiosquillina glabriuscula, have yellow fluorescent markings along their antennal scales and carapace (shell) that males present during threat displays to predators and other males. The display involves raising the head and thorax, spreading the striking appendages and other maxillipeds, and extending the prominent, oval antennal scales laterally, which makes the animal appear larger and accentuates its yellow fluorescent markings. Furthermore, as depth increases, mantis shrimp fluorescence accounts for a greater part of the visible light available. During mating rituals, mantis shrimp actively fluoresce, and the wavelength of this fluorescence matches the wavelengths detected by their eye pigments.[48]
Aphotic zone
Siphonophores
Siphonophorae is an order of marine animals from the phylum Hydrozoa that consist of a specialized medusoid and polyp zooid. Some siphonophores, including the genus Erenna that live in the aphotic zone between depths of 1600 m and 2300 m, exhibit yellow to red fluorescence in the photophores of their tentacle-like tentilla. This fluorescence occurs as a by-product of bioluminescence from these same photophores. The siphonophores exhibit the fluorescence in a flicking pattern that is used as a lure to attract prey.[49]
Dragonfish
The predatory deep-sea dragonfish Malacosteus niger, the closely related genus Aristostomias and the species Pachystomias microdon use fluorescent red accessory pigments to convert the blue light emitted from their own bioluminescence to red light from suborbital photophores. This red luminescence is invisible to other animals, which allows these dragonfish extra light at dark ocean depths without attracting or signaling predators.[50]
Terrestrial
Amphibians
Fluorescence is widespread among amphibians and has been documented in several families of frogs, salamanders and caecilians, but the extent of it varies greatly.[51]
The polka-dot tree frog (Hypsiboas punctatus), widely found in South America, was unintentionally discovered to be the first fluorescent amphibian in 2017. The fluorescence was traced to a new compound found in the lymph and skin glands.[52] The main fluorescent compound is Hyloin-L1 and it gives a blue-green glow when exposed to violet or ultraviolet light. The scientists behind the discovery suggested that the fluorescence can be used for communication. They speculated that fluorescence possibly is relatively widespread among frogs.[53] Only a few months later, fluorescence was discovered in the closely related Hypsiboas atlanticus. Because it is linked to secretions from skin glands, they can also leave fluorescent markings on surfaces where they have been.[54]
In 2019, two other frogs, the tiny pumpkin toadlet (Brachycephalus ephippium) and red pumpkin toadlet (B. pitanga) of southeastern Brazil, were found to be have naturally fluorescent skeletons, which is visible through their skin when exposed to ultraviolet light.[55][56] It was initially speculated that the fluorescence supplemented their already aposematic colours (they are toxic) or that it was related to mate choice (species recognition or determining fitness of a potential partner),[55] but later studies indicate that the former explanation is unlikely, as predation attempts on the toadlets appear to be unaffected by the presence/absence of fluorescence.[57]
In 2020 it was confirmed that green or yellow fluorescence is widespread not only in adult frogs that are exposed to blue or ultraviolet light, but also among tadpoles, salamanders and caecilians. The extent varies greatly depending on species; in some it is highly distinct and in others it is barely noticeable. It can be based on their skin pigmentation, their mucous or their bones.[51]
Butterflies
Swallowtail (Papilio) butterflies have complex systems for emitting fluorescent light. Their wings contain pigment-infused crystals that provide directed fluorescent light. These crystals function to produce fluorescent light best when they absorb radiance from sky-blue light (wavelength about 420 nm). The wavelengths of light that the butterflies see the best correspond to the absorbance of the crystals in the butterfly's wings. This likely functions to enhance the capacity for signaling.[58]
Parrots
Parrots have fluorescent plumage that may be used in mate signaling. A study using mate-choice experiments on budgerigars (Melopsittacus undulates) found compelling support for fluorescent sexual signaling, with both males and females significantly preferring birds with the fluorescent experimental stimulus. This study suggests that the fluorescent plumage of parrots is not simply a by-product of pigmentation, but instead an adapted sexual signal. Considering the intricacies of the pathways that produce fluorescent pigments, there may be significant costs involved. Therefore, individuals exhibiting strong fluorescence may be honest indicators of high individual quality, since they can deal with the associated costs.[59]
Arachnids
Spiders fluoresce under UV light and possess a huge diversity of fluorophores. Remarkably, spiders are the only known group in which fluorescence is "taxonomically widespread, variably expressed, evolutionarily labile, and probably under selection and potentially of ecological importance for intraspecific and interspecific signaling". A study by Andrews et al. (2007) reveals that fluorescence has evolved multiple times across spider taxa, with novel fluorophores evolving during spider diversification. In some spiders, ultraviolet cues are important for predator-prey interactions, intraspecific communication, and camouflaging with matching fluorescent flowers. Differing ecological contexts could favor inhibition or enhancement of fluorescence expression, depending upon whether fluorescence helps spiders be cryptic or makes them more conspicuous to predators. Therefore, natural selection could be acting on expression of fluorescence across spider species.[60]
Scorpions also fluorescent due to the presence of beta carboline in their cuticles.[61]
Platypus
In 2020 fluorescence was reported for several specimen of platypus.[62]
Plants
Many plants are fluorescent due to the presence of chlorophyll, which is probably the most widely-distributed fluorescent molecule, producing red emission under a range of excitation wavelengths.[63] This attribute of chlorophyll is commonly used by ecologists to measure photosynthetic efficiency.[64]
The Mirabilis jalapa flower contains violet, fluorescent betacyanins and yellow, fluorescent betaxanthins. Under white light, parts of the flower containing only betaxanthins appear yellow, but in areas where both betaxanthins and betacyanins are present, the visible fluorescence of the flower is faded due to internal light-filtering mechanisms. Fluorescence was previously suggested to play a role in pollinator attraction, however, it was later found that the visual signal by fluorescence is negligible compared to the visual signal of light reflected by the flower.[65]
Abiotic
Gemology, mineralogy and geology
Gemstones, minerals, may have a distinctive fluorescence or may fluoresce differently under short-wave ultraviolet, long-wave ultraviolet, visible light, or X-rays.
Many types of calcite and amber will fluoresce under shortwave UV, longwave UV and visible light. Rubies, emeralds, and diamonds exhibit red fluorescence under long-wave UV, blue and sometimes green light; diamonds also emit light under X-ray radiation.
Fluorescence in minerals is caused by a wide range of activators. In some cases, the concentration of the activator must be restricted to below a certain level, to prevent quenching of the fluorescent emission. Furthermore, the mineral must be free of impurities such as iron or copper, to prevent quenching of possible fluorescence. Divalent manganese, in concentrations of up to several percent, is responsible for the red or orange fluorescence of calcite, the green fluorescence of willemite, the yellow fluorescence of esperite, and the orange fluorescence of wollastonite and clinohedrite. Hexavalent uranium, in the form of the uranyl cation, fluoresces at all concentrations in a yellow green, and is the cause of fluorescence of minerals such as autunite or andersonite, and, at low concentration, is the cause of the fluorescence of such materials as some samples of hyalite opal. Trivalent chromium at low concentration is the source of the red fluorescence of ruby. Divalent europium is the source of the blue fluorescence, when seen in the mineral fluorite. Trivalent lanthanides such as terbium and dysprosium are the principal activators of the creamy yellow fluorescence exhibited by the yttrofluorite variety of the mineral fluorite, and contribute to the orange fluorescence of zircon. Powellite (calcium molybdate) and scheelite (calcium tungstate) fluoresce intrinsically in yellow and blue, respectively. When present together in solid solution, energy is transferred from the higher-energy tungsten to the lower-energy molybdenum, such that fairly low levels of molybdenum are sufficient to cause a yellow emission for scheelite, instead of blue. Low-iron sphalerite (zinc sulfide), fluoresces and phosphoresces in a range of colors, influenced by the presence of various trace impurities.
Crude oil (petroleum) fluoresces in a range of colors, from dull-brown for heavy oils and tars through to bright-yellowish and bluish-white for very light oils and condensates. This phenomenon is used in oil exploration drilling to identify very small amounts of oil in drill cuttings and core samples.
Organic liquids
Organic solutions such anthracene or stilbene, dissolved in benzene or toluene, fluoresce with ultraviolet or gamma ray irradiation. The decay times of this fluorescence are on the order of nanoseconds, since the duration of the light depends on the lifetime of the excited states of the fluorescent material, in this case anthracene or stilbene.[66]
Scintillation is defined a flash of light produced in a transparent material by the passage of a particle (an electron, an alpha particle, an ion, or a high-energy photon). Stilbene and derivatives are used in scintillation counters to detect such particles. Stilbene is also one of the gain mediums used in dye lasers.
Atmosphere
Fluorescence is observed in the atmosphere when the air is under energetic electron bombardment. In cases such as the natural aurora, high-altitude nuclear explosions, and rocket-borne electron gun experiments, the molecules and ions formed have a fluorescent response to light.[67]
Common materials that fluoresce
- Vitamin B2 fluoresces yellow.
- Tonic water fluoresces blue due to the presence of quinine.
- Highlighter ink is often fluorescent due to the presence of pyranine.
- Banknotes, postage stamps and credit cards often have fluorescent security features.
En tecnología novedosa
In August 2020 researchers reported the creation of the brightest fluorescent solid optical materials so far by enabling the transfer of properties of highly fluorescent dyes via spatial and electronic isolation of the dyes by mixing cationic dyes with anion-binding cyanostar macrocycles. According to a co-author these materials may have applications in areas such as solar energy harvesting, bioimaging, and lasers.[68][69][70][71]
Aplicaciones
Lighting
The common fluorescent lamp relies on fluorescence. Inside the glass tube is a partial vacuum and a small amount of mercury. An electric discharge in the tube causes the mercury atoms to emit mostly ultraviolet light. The tube is lined with a coating of a fluorescent material, called the phosphor, which absorbs ultraviolet light and re-emits visible light. Fluorescent lighting is more energy-efficient than incandescent lighting elements. However, the uneven spectrum of traditional fluorescent lamps may cause certain colors to appear different than when illuminated by incandescent light or daylight. The mercury vapor emission spectrum is dominated by a short-wave UV line at 254 nm (which provides most of the energy to the phosphors), accompanied by visible light emission at 436 nm (blue), 546 nm (green) and 579 nm (yellow-orange). These three lines can be observed superimposed on the white continuum using a hand spectroscope, for light emitted by the usual white fluorescent tubes. These same visible lines, accompanied by the emission lines of trivalent europium and trivalent terbium, and further accompanied by the emission continuum of divalent europium in the blue region, comprise the more discontinuous light emission of the modern trichromatic phosphor systems used in many compact fluorescent lamp and traditional lamps where better color rendition is a goal.[72]
Fluorescent lights were first available to the public at the 1939 New York World's Fair. Improvements since then have largely been better phosphors, longer life, and more consistent internal discharge, and easier-to-use shapes (such as compact fluorescent lamps). Some high-intensity discharge (HID) lamps couple their even-greater electrical efficiency with phosphor enhancement for better color rendition.[citation needed]
White light-emitting diodes (LEDs) became available in the mid-1990s as LED lamps, in which blue light emitted from the semiconductor strikes phosphors deposited on the tiny chip. The combination of the blue light that continues through the phosphor and the green to red fluorescence from the phosphors produces a net emission of white light.[73]
Glow sticks sometimes utilize fluorescent materials to absorb light from the chemiluminescent reaction and emit light of a different color.[72]
Analytical chemistry
Many analytical procedures involve the use of a fluorometer, usually with a single exciting wavelength and single detection wavelength. Because of the sensitivity that the method affords, fluorescent molecule concentrations as low as 1 part per trillion can be measured.[74]
Fluorescence in several wavelengths can be detected by an array detector, to detect compounds from HPLC flow. Also, TLC plates can be visualized if the compounds or a coloring reagent is fluorescent. Fluorescence is most effective when there is a larger ratio of atoms at lower energy levels in a Boltzmann distribution. There is, then, a higher probability of excitement and release of photons by lower-energy atoms, making analysis more efficient.
Spectroscopy
Usually the setup of a fluorescence assay involves a light source, which may emit many different wavelengths of light. In general, a single wavelength is required for proper analysis, so, in order to selectively filter the light, it is passed through an excitation monochromator, and then that chosen wavelength is passed through the sample cell. After absorption and re-emission of the energy, many wavelengths may emerge due to Stokes shift and various electron transitions. To separate and analyze them, the fluorescent radiation is passed through an emission monochromator, and observed selectively by a detector.[75]
Biochemistry and medicine
Fluorescence in the life sciences is used generally as a non-destructive way of tracking or analysis of biological molecules by means of the fluorescent emission at a specific frequency where there is no background from the excitation light, as relatively few cellular components are naturally fluorescent (called intrinsic or autofluorescence). In fact, a protein or other component can be "labelled" with an extrinsic fluorophore, a fluorescent dye that can be a small molecule, protein, or quantum dot, finding a large use in many biological applications.[76]
The quantification of a dye is done with a spectrofluorometer and finds additional applications in:
Microscopy
- When scanning the fluorescence intensity across a plane one has fluorescence microscopy of tissues, cells, or subcellular structures, which is accomplished by labeling an antibody with a fluorophore and allowing the antibody to find its target antigen within the sample. Labelling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image (multiple channels). DNA microarrays are a variant of this.
- Immunology: An antibody is first prepared by having a fluorescent chemical group attached, and the sites (e.g., on a microscopic specimen) where the antibody has bound can be seen, and even quantified, by the fluorescence.
- FLIM (Fluorescence Lifetime Imaging Microscopy) can be used to detect certain bio-molecular interactions that manifest themselves by influencing fluorescence lifetimes.
- Cell and molecular biology: detection of colocalization using fluorescence-labelled antibodies for selective detection of the antigens of interest using specialized software such as ImageJ.
Other techniques
- FRET (Förster resonance energy transfer, also known as fluorescence resonance energy transfer) is used to study protein interactions, detect specific nucleic acid sequences and used as biosensors, while fluorescence lifetime (FLIM) can give an additional layer of information.
- Biotechnology: biosensors using fluorescence are being studied as possible Fluorescent glucose biosensors.
- Automated sequencing of DNA by the chain termination method; each of four different chain terminating bases has its own specific fluorescent tag. As the labelled DNA molecules are separated, the fluorescent label is excited by a UV source, and the identity of the base terminating the molecule is identified by the wavelength of the emitted light.
- FACS (fluorescence-activated cell sorting). One of several important cell sorting techniques used in the separation of different cell lines (especially those isolated from animal tissues).
- DNA detection: the compound ethidium bromide, in aqueous solution, has very little fluorescence, as it is quenched by water. Ethidium bromide's fluorescence is greatly enhanced after it binds to DNA, so this compound is very useful in visualising the location of DNA fragments in agarose gel electrophoresis. Intercalated ethidium is in a hydrophobic environment when it is between the base pairs of the DNA, protected from quenching by water which is excluded from the local environment of the intercalated ethidium. Ethidium bromide may be carcinogenic – an arguably safer alternative is the dye SYBR Green.
- FIGS (Fluorescence image-guided surgery) is a medical imaging technique that uses fluorescence to detect properly labeled structures during surgery.
- Intravascular fluorescence is a catheter-based medical imaging technique that uses fluorescence to detect high-risk features of atherosclerosis and unhealed vascular stent devices.[77] Plaque autofluorescence has been used in a first-in-man study in coronary arteries in combination with optical coherence tomography.[78] Molecular agents has been also used to detect specific features, such as stent fibrin accumulation and enzymatic activity related to artery inflammation.[79]
- SAFI (species altered fluorescence imaging) an imaging technique in electrokinetics and microfluidics.[80] It uses non-electromigrating dyes whose fluorescence is easily quenched by migrating chemical species of interest. The dye(s) are usually seeded everywhere in the flow and differential quenching of their fluorescence by analytes is directly observed.
- Fluorescence-based assays for screening toxic chemicals. The optical assays consist of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns.[81] This approach can reduce the need for laboratory animals in biomedical research and pharmaceutical industry.
- Bone-margin detection: Alizarin-stained specimens and certain fossils can be lit by fluorescent lights to view anatomical structures, including bone margins.[82]
Forensics
Fingerprints can be visualized with fluorescent compounds such as ninhydrin or DFO (1,8-Diazafluoren-9-one). Blood and other substances are sometimes detected by fluorescent reagents, like fluorescein. Fibers, and other materials that may be encountered in forensics or with a relationship to various collectibles, are sometimes fluorescent.
Non-destructive testing
Fluorescent penetrant inspection is used to find cracks and other defects on the surface of a part. Dye tracing, using fluorescent dyes, is used to find leaks in liquid and gas plumbing systems.
Signage
Fluorescent colors are frequently used in signage, particularly road signs. Fluorescent colors are generally recognizable at longer ranges than their non-fluorescent counterparts, with fluorescent orange being particularly noticeable.[83] This property has led to its frequent use in safety signs and labels.
Optical brighteners
Fluorescent compounds are often used to enhance the appearance of fabric and paper, causing a "whitening" effect. A white surface treated with an optical brightener can emit more visible light than that which shines on it, making it appear brighter. The blue light emitted by the brightener compensates for the diminishing blue of the treated material and changes the hue away from yellow or brown and toward white. Optical brighteners are used in laundry detergents, high brightness paper, cosmetics, high-visibility clothing and more.
Ver también
- Absorption-re-emission atomic line filters use the phenomenon of fluorescence to filter light extremely effectively.
- Black light
- Blacklight paint
- Fluorescence-activating and absorption-shifting tag
- Fluorescence correlation spectroscopy
- Fluorescence image-guided surgery
- Fluorescence in plants
- Fluorescence spectroscopy
- Fluorescent lamp
- Fluorescent multilayer card
- Fluorescent Multilayer Disc
- Fluorometer
- High-visibility clothing
- Integrated fluorometer
- Laser-induced fluorescence
- List of light sources
- Microbial art, using fluorescent bacteria
- Mössbauer effect, resonant fluorescence of gamma rays
- Organic light-emitting diodes can be fluorescent
- Phosphorescence
- Phosphor thermometry, the use of phosphorescence to measure temperature.
- Spectroscopy
- Two-photon absorption
- Vibronic spectroscopy
- X-ray fluorescence
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Bibliografía
- Lakowicz, Joseph R. (1999). Principles of Fluorescence Spectroscopy. Kluwer Academic / Plenum Publishers. ISBN 978-0-387-31278-1.
Otras lecturas
- The Story of Fluorescence. Raytech Industries. 1965.
enlaces externos
- Fluorophores.org[permanent dead link], the database of fluorescent dyes
- FSU.edu, Basic Concepts in Fluorescence
- "A nano-history of fluorescence" lecture by David Jameson
- Excitation and emission spectra of various fluorescent dyes
- Database of fluorescent minerals with pictures, activators and spectra (fluomin.org)
- "Biofluorescent Night Dive – Dahab/Red Sea (Egypt), Masbat Bay/Mashraba, "Roman Rock"". YouTube. 9 October 2012.
- Steffen O. Beyer. "FluoPedia.org: Publications". fluopedia.org.
- Steffen O. Beyer. "FluoMedia.org: Science". fluomedia.org.