Bioluminescence,fluorescence and phosphorescence

In this slide, I gave full definition, mechanism, application and uses about bioluminescence and photoluminescence for biophysics.

1. FLuoreSCENCE AND PHOSPhOreSCENCE

2. BIOLUMINESCENCE

3. INTRODUCTION Bioluminescence is the production and emission of light by a living organism. It is a form of chemiluminescence . Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some bioluminescent bacteria, and terrestrial arthropods such as fireflies. In some animals, the light is bacteriogenic , produced by symbiotic bacteria such as those from the genus Vibrio ; in others, it is autogenic, produced by the animals themselves .

4. Bio means 'living' in Greek while lumen means 'light' in Latin. During the process, chemical energy is converted into light energy.The process is caused by an enzyme-catalyzed chemoluminescence reaction. The light production from bioluminescence is “cold light” emission, wherein less than 20% of the light is thermal radiation. Bioluminescence on land and in freshwater is rare compared to its occurrence in the ocean. In the deep ocean 90% of the animals are luminescent Higher in deep-living and planktonic organisms.

5. HOW DOES BIOLUMINESCENCE WORKS? Bioluminescence is a product of a chemical reaction in an organism. In a general sense, the principal chemical reaction in bioluminescence involves a light-emitting molecule and an enzyme, generally called luciferin and luciferase , respectively. Because these are generic names, luciferins and luciferases are often distinguished by including the species or group, e.g. firefly luciferin . It involves a class of chemicals called luciferins (light bringers). The luciferin oxidizes in the presence of a catalytic enzyme ( luciferase ) to create light and an ineffective compound ( oxyluciferin ).

6. DIFFERENT ORGANISMS SHOWS BIOLUMINESCENCE 1. Bacteria Family Vibrionaceae contains most bioluminescent bacteria Typically found as symbionts with deep sea animals, gram negative, one or more flagella. It uses bacterial luciferin for bioluminescence. They create the phenomenon of a "milky sea" known to sailors for centuries

7. 2. Mushrooms and other Fungi “Foxfire” referred to the green glow light emitted by wood decaying mushrooms and other fungi. It was used as a light source for the early wooden submarine. They use luciferin illudin for bioluminescence, which is toxic to ingest.

8. 3. Worms Both marine and terrestrial worms that exhibit bioluminescence. Earthworm luminescence is produced by the coelomic fluid, and ranges from blue to orange depending on the specie.

9. 4 . Insects Firefly is the most common terrestrial bioluminescence organism. Variety of firefly species are found in the temperate to tropical regions of the Americas and parts of S.E. Asia.

10. 5. Jelly Fishes : It is estimated that about 50% of jellyfish are bioluminescent. Most jellyfish bioluminescence is used for defense against predators. Jellyfish such as comb jellies produce bright flashes to startle a predator, Some jellyfish can release their tentacles as glowing decoys. Others produce a glowing slime that can stick to a potential predator an make it vulnerable to its predators .

11. USES OF BIOLUMINESCENCE 1. In nature Counter- illumination camouflage Attraction Defense Warning Communication Mimicry Illumination 2. Biotechnology Biology and medicine Light production

12. FLUORESCENCE

13. INTRODUCTION Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after. Fluorescence has many practical applications, including mineralogy, gemology, medicine, chemical sensors (fluorescence spectroscopy), fluorescent labeling, dyes, biological detectors, and cosmic-ray detection.

14. HISTORY An early observation of fluorescence was described in 1560 by Bernardino de Sahagun and in 1565 by Nicolas Monardes in the infusion known as lignum nephriticum (In Latin "kidney wood"). It was derived from the wood of two tree species, Pterocarpus indicus and Eysenhardtia polystachya . The chemical compound responsible for this fluorescence is matlaline , which is the oxidation product of one of the flavonoids found in this wood. Later in 1819 , Edward D. Clarke and in 1822 Rene Just Hauy described fluorescence in fluorites, Sir David Brewster described the phenomenon for chlorophyll in 1833 and Sir John Herschel did the same for quinine in 1845. .

15. In his 1852 paper on the " Refrangibility " (wavelength change) of light, George Gabriel Stokes described the ability of fluorspar and uranium glass to change invisible light beyond the violet end of the visible spectrum into blue light. He named this phenomenon fluorescence.

16. PRINCIPLE OF FLUORSCENCE The electronic states of most organic molecules can be divided into singlet states and triplet states Singlet ground state : All electrons in the molecule are spin-paired. Singlet excited state : Unpaired electrons of opposite spin-paired. Triplet state : Unpaired electrons of same spin-paired.

17. Perrin- Jablonski diagram

18. PRINCILPE OF FLUORSCENCE Energy of emitted radiation is less than that of absorbed radiation because a part of energy is lost due to vibrational or collisional processes. Hence the emitted radiation has longer wavelength (less energy) than the absorbed radiation. Vibrational deactivation takes place through intermolecular collisions at a time scale of 10 -12 s (faster than that of fluorescence process) .

19. INTERNAL CONVERSION As electronic energy increases, the energy levels grow more closely spaced. It is more likely that there will be overlap between the high vibrational energy levels of S n-1 and low vibrational energy levels of S n. This overlap makes transition between states highly probable. Internal conversion is a transition occurring between states of the same multiplicity and it takes place at a time scale of 10 -12s (faster than that of fluorescence process). The energy gap between S ₁ and S ₀ is significantly larger than that between other adjacent states → S ₁ lifetime is longer → radiative emission can compete effectively with non- radiative emission .

20. INTERNAL CONVERSION

21. RULES STOKES SHIFT The difference between the max wavelength of the excitation light and the max wavelength of the emitted fluorescence lights is a constant – stokes shift.

22. MIRROR IMAGE RULE Vibrational levels in the excited states and ground states are similar. An absorption spectrum reflects the vibrational levels of the electronically excited state. An emission spectrum reflects the vibrational levels of the electronic ground state. Fluorescence emission spectrum is mirror image of absorption spectrum

23. MIRROR IMAGE RULE Mirror-image rule typically applies when only S₀ → S₁ excitation takes place. Deviations from the mirror-image rule are observed when S₀ → S₂ or transitions to even higher excited states also take place.

24. TYPES OF FLUORSCENCE Based upon the wavelength of emitted radiation when compared to absorbed radiation : Stoke’s fluorescence Anti-stock’s fluorescence Resonance fluorescence B) Based upon the phenomenon Prompt fluorescence Delayed fluorescence

25. TYPES OF FLUORSCENCE A) Based upon the wavelength of emitted radiation when compared to absorbed radiation : Stoke’s fluorescence: The wavelength of emitted radiation is longer than the Absorbed radiation e.g . Conventional fluorimetric experiments. Anti-stock’s fluorescence: The wavelength of emitted radiation is shorter than the Absorbed radiation e.g. Thermally assisted fluorescence. Resonance fluorescence: When the wavelength of emitted radiation is equal to the Absorbed radiation e.g. Mercury vapour at 254 nm .

26. TYPES OF FLUORSCENCE

27. TYPES OF FLUORSCENCE B ) Based upon the phenomenon: Prompt fluorescence: Delayed fluorescence: Delayed Fluorescencere are delayed emissions whose spectra coincide exactly with the prompt fluorescence from the lowest singlet state, the only difference is in their life time.

28. IN NATURE 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. Vs. 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 . New form of biofluorescence In a study published in the journal iScience , a new form of biofluorescence was described in two species of sharks, wherein it was due to an undescribed group of brominated tryptophane-kynurenine small molecule metabolites

29. IN NATURE 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 re-emission of light from the environment. A firefly and anglerfish are two example of bioluminescent organisms. Phosphorescence BioPhosphorescence 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. Glow-in-the-dark stickers are phosphorescent, but there are no truly phosphorescent animals known .

30. IN NATURE 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. Adaptive functions 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.

31. IN NATURE 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. It includes Photic zone, aphotic zone

32. Photic Zone- Fish 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 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. Photic Zone- 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. Similarly, these fluorescent proteins may possess antioxidant capacities to eliminate oxygen radicals produced by photosynthesis.

34. Photic Zone- 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.

35. Photic Zone- 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. Furthermore, as depth increases, mantis shrimp fluorescence accounts for a greater part of the visible light available .

36. Apohtic zone- Siphonophores Siphonophores is an order of marine animals from the phylum Hydrozoa that consist of a specialized medusoid and polyp zooid. They 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. Aphotic Zone- 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.

37. Terrestrial Amphibian Fluorescence is widespread among amphibians and has been documented in several families of frogs, salamanders and caecilians, but the extent of it varies greatly. The polka-dot tree frog ( Hypsiboas punctatus ), widely found in South America, was unintentionally discovered to be the first fluorescent amphibian in 2017 .

38. Terrestrial Butterflies swallowtail( Papilio ) butterflies have complex systems for emitting fluorescent light. Their wings contain pigment-infused crystals that provide directed fluorescent light. 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".

39. APPLICATIONS 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 .

40. APPLICATIONS 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 . Fluorescence in several wavelengths can be detected by an array detector, to detect compounds from HPLC flow. 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. 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 ..

41. APPLICATIONS 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. 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 pluming systems.

42. PHOSPHORESCENCE

43. INTRODUCTION Phosphorescence is a type of photoluminescence related to fluorescence. Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. The slower time scales of the re-emission are associated with "forbidden" energy state transitions in quantum mechanics. As these transitions occur very slowly in certain materials, absorbed radiation is re-emitted at a lower intensity for up to several hours after the original excitation. Everyday examples of phosphorescent materials are the glow-in-the dark toys, stickers, paint, wristwatch and clock dials that glow after being charged with a bright light such as in any normal reading or room light. Typically, the glow slowly fades out, sometimes within a few minutes or up to a few hours in a dark room.

44. HISTORICAL BACKGROUND The term ‘phosphor’ has been used since the Middle Ages. Phosphorescence was first observed in the 17th century but was not studied scientifically until the 19th century. Around 1604, Vincenzo Casciarolo discovered a "lapis solaris " near Bologna, Italy. Once heated in an oxygen-rich furnace, it thereafter absorbed sunlight and glowed in the dark. The study of phosphorescent materials led to the discovery of radioactivity in 1896. This was followed by the discovery of a number of substances which become luminous either after heating or exposure to light: Homberg’s phosphorus(obtained by heating calcium chloride) John Canton’s phosphorus(calcium sulphide ) Balduin’s phosphorus(calcium nitrate).

45. HOW PHOSPHORESCENCE WORKS A phosphorescent materials store and re-emit light because of their unusual property of trapping electrons in a higher state of movement. A Phosphorescent materials absorbs high energy light, causing the electrons to move into the higher energy state, but the transition to a lower energy state occurs much slowly and the direction of the electron spin may change. A phosphorescent materials may appear to glow for several seconds up to a couple of days after the light source has been cut off. The reason behinds this is because of excited electrons jumps to a higher energy level than for fluorescence. The electrons have more energy to loss and may spend time at different energy levels between the excited state and ground state.

46. PHOSPHOR Phosphor is a chemical compound which emits light when it is exposed to the light of a different wavelength. Sometimes this element can be confused with phosphorus but there are no similarities between them. We can find this element in fluorescent bulbs, toys or safety signs in buildings.

47. CHEMILUMINESCENCE Some examples of glow-in-the-dark materials do not glow by phosphorescence. In chemiluminescence , an excited state is created via a chemical reaction.

48. LUMINOUS PAINT Phosphorescent paint is made from phosphors such as silver-activated zinc sulfide or doped strontium aluminate . Escape paths in aircraft and decorative use such as "stars" applied to walls and ceilings. When applied as a paint or a more sophisticated coating, phosphorescence can be used for temperature detection or degradation measurements known as phosphor thermometry.

49. Tritium & luminosity in watches Tritium is a radioactive isotope of hydrogen very difficult to find on Earth, it was first discovered in 1934. This isotope can damage our health or contaminate the environment, but it is still used for nuclear weapons or controlled nuclear fusion. Also, this material is used in watches because the electrons produced by tritium create a fluorescent light that can last up to 20 years. Obviously tritium in watches is hermetically closed inside small glass tubes.

50. Phosphorescence in nature Bioluminescence is the emission and production of light by a living organism, this type of chemiluminescence is produced when a pigment and an enzyme join in a chemical reaction. Bioluminescence is used by animals for communicating, imitating other organisms, illuminating or even camouflaging. Sometimes the sea water can illuminate by some plankton with this kind of bioluminescence, this is one of the most beautiful events that bioluminescence can produce.

51. MATERIALS USED Common pigments used in phosphorescent materials include zinc sulfide and strontium aluminate . Strontium aluminate has a luminance approximately 10 times greater than zinc sulfide.

52. THE END thank you for your patience