Bioluminescence

1. Bioluminescence By GC “Some things which are neither fire nor form of fire seem to produce light by nature” ~Aristotle

2. What is Bioluminescence? • Defined : the emission of light as a result of a chemical reaction during which chemical energy is converted into light energy • From a living organism that functions for its survival or mating • Comes from Greek word “bios” for ‘living’ and Latin word “lumen” for ‘light’, so it literally means living light • “Cold light” resulting from a specific biochemical mechanism involving chemical processes • Specific for that organism • Found all over biosphere, phosphorescence in sea water observed in all oceans • Bioluminescence of visible light is found in a majority of marine organisms but is rare in terrestrial organism

3. How does Bioluminescence Occur?

4. How does Bioluminescence Occur? • All involve an oxygen oxidation of organic molecule (luciferin) • Catalyzed by an enzyme called luciferase • These proteins are called “photoproteins” -> oxygen is already bound to the luciferin • A photoprotein is a protein with a luciferin bound to it • “Antenna proteins” -> adjust the colour of bioluminescence likeness to proteins on similar function in photosynthesis except they act in reverse • 5 known distinct chemical classes of luciferins: • Aldehydes, benzothiazoles, imidazolopyrazines, tetrapyrroles and flavins • Key organ -> photophore (light producing organ) • Seen in many luminous fish and vividly in cephalopods • Make up of complex photogenic (light emitting) cells • Bioluminescent reaction components detected in stomach, secretory organs and liver of some organisms

5. Bioluminescence, Fluorescence, Phosphorescence • Bioluminescence: light is created through a chemical reaction. Bioluminescence is a subset of chemiluminescence • Fluorescence: a particular atom or molecule absorbs light of one length, but emits one of a longer length. The light going in is usually UV photons, and comes out as visible light • Phosphorescence: requires more time to remit radiation absorbed than fluorescence. This is because the sub atomic reactions required to remit light occur less often than in fluorescence. Fluorescence

6. History of Bioluminescence • Stories of mysterious light or fires seen over fields or mountains were often said to be dragons or the gods • Greeks and romans first to report luminous organisms • Reports of fireflies found in early religious writing of India and China • Earlier recordings believed to have come from these and ancient eastern civilizations and refered to firelies and glow worms • Aristotle (384-322BC) (first to discover) decribed more than 180 marine species, first to recognize “cold light” • Later, complete and extensive descriptions of luminous organisms published by Pliny the Elder (23-79 CE) • 16th century references to bioluminescence found in literature such as Shakespeare, specifically in hamlet who talked about “effectual fire of the glow worm” • First book devoted to bioluminescence and chemiluminescence published in 1555 by Conrad Gesner • 1667 – Robert Boyle documented oxygen was needed for luminescence

7. History of Bioluminescence • Raphael Dubois performed experiment, he extracted the two key components of bioluminescence reaction and was able to create light and that there 2 key components= “luciferine” and heat labile “luciferase”* • One of most eminent scientists of 20th century was “Princeton Professor” E. Newton Harvey* • He was looking for existence of lucifernin- luciferase system in all luminous organisms • First luciferin isolated in 1956 • First photo protein isolated was the calcium activated photoprotienaequorin in the 1960’s

8. History of Bioluminescence • Calcium dependant photoprotein cloned in 1985 • Intensity of luminescence varies with calcium concentration • Therefore aequorin has been used in monitoring of cell calcium • 1985 firefly luciferase was cloned Firefly Luciferin chemical structure Tomopteris

9. Light Emission • All colours of the visible light spectrum; red, orange, yellow, green, blue, indigo, violet • Different colours dependant on role the light plays and which organism it is produced in • Many only produce one colour, however some are capable of many colours • Eg. Jamaican click beetle; this is due to the same luciferin substrate as the firefly, but different luciferase structures • Bioluminescence releases a large amount of energy, not heat • Visible light radiation is equal to light wavelengths of 400-700 nm • Bioluminescence max of most marine species is 460-510 nm • Terrestrial organisms mostly yellow-green BL, more yellow • Marine mostly blue-green (400-500nm) luminescence because it travels the best through water

10. Uses of Bioluminescence in Nature • Plays an important role in nature due to the darkness 200m underwater • Location on body of bioluminescence gives clues to the functional role of the luminescence • Attracting a mate • Eg. The wave lengths attract mates, but do not draw attention to themselves • Attracting prey • Eg. Angler fish has a luminescent lure • Finding food • eg. Loose jaw fish, bioluminescent organs in cheeks to see in dark water • Communication • eg. Fireflies flash certain patterns to either attract mates or communicate • Camouflage • eg. Bobtail squid blends into background • Defense against predators • eg. Deep sea shrip vomits bioluminescent material in direction of attacker Deep- sea shrimp vomits bioluminescent material

11. Notable Figures Robert Boyle • British philosopher and scientist • In 1667 performed experiment and found that bioluminescence didn’t occur when a known bioluminescent type of fungus was not in the presence of air • 1672 he discovers that bioluminescence reactions require air, when he later discovered oxygen, we realize is the oxygen component of air that is needed Robert Boyle Oxygen Atom

12. Notable Figures Raphael Dubois • 1887 he discovered luciferin and luciferase through experiments with clams, beetles, and other species • He did an experiment using a clam called the common paddock • Ground tissue of the clam up in cold water, and light was produced for several minutes. This showed he has extracted the light producing chemical • Then made a hot water extract from another clam and added it to the cold water, which reactivated the light reaction • A hot water extraction alone produced no light reaction • Called the hot water extraction luciferin • Called cold water extraction luciferase • Noted that luciferin could only glow in the presence of luciferase Raphael Dubois

13. Notable Figures Edmund Newton Harvey • Greatly popularized the study of bioluminescence • Discovered that luciferins and luciferase from different animals are not interchangeable • Evidence of evolution of bioluminescence to fit various needs of different species • Now shown that evolution of bioluminescent systems has occurred over 30 times, which accounts for the differences in colours and uses in different species Humpback Angler Fish Jamaican Click Beetle

14. Modern Applications and Where We are Going • The discovery and study on bioluminescent organisms is the goal of many expeditions of ocean going research vessels and marine submersibles Commercial Applications • Glowing trees to save electricity bills • Agricultural crops that luminesce when in need of water • Detection of bacteria in contaminated foods • Novelty pets • Bio-indentifies for convicts, mentally ill • Glowing toys, glowing greeting cards • Luminescent beer and champange “Glofish”

15. Modern Applications and Where We are Going MedicalApplications • Reporter genes • Bioluminescent Imaging* • In vivo analysis • Detection of bacteria • Observations of protein to protein interacting • Testing for genetically modified organisms* • Water quality testing* • Green Florescent Protein*

16. Bioluminescent Imaging (BLI) • Used on small animals • Real time monitoring of the progression of infections in the same animal • Typically 2D imagery, with lower resolution • Can mark progress at different time points without euthanizing the animal, uses animals mice than conventional methods of progress tracking • Quicker and relatively inexpensive • Allowed specific molecular and cellular events such as cell migration and signal transduction to be investigated in a living, intact animal

17. Bioluminescent Imaging (BLI) • Bioluminescence is decreased by pigmentation of organs such and liver, spleen and the fur • To get around this they can shave the mice or use ones with the albino gene • Used to study bacteria distribution, distinguish between more and less virulent strains and moniter antibiotic therapy • Also used to study viruses, but because of low resolution, it can be difficult to distinguish between viral infections in adjacent tissues • Used to investigate parasite infections • High correlation between light intensity and amount of parasites in spleen Mouse and BLI

18. Microtox VibrioFischeri • For water quality/ toxicity testing • Uses the bioluminescent marine bacteria ‘vibriofischeri’ • When the organism is challenged by a toxin, its breathing pathway is disrupted, resulted in decreased bioluminescence intensity Green Fluorescent Protein (GFP) • Now a well established and excelled genetag and protein • It can be fused to a protein of interest and fluorescence and can be tracked within a cell to study its localization and behavior • Outstanding structural stability • Excellent for studying the cell and sub cellular processes • Cloned in 1992, expressed in various organism in 1994

19. Lumora and Improved GMO Testing • People want to know, “Does this food contain genetically modified organisms?” and they have the right to know • Genetically Modified Organisms (GMO): genetic material (DNA or RNA) that has been altered in ways that would not occur under natural conditions or natural processes • Most common technique used to test for GMOS is polymerase chain reaction (PCR) • PCR requires complex DNA extraction techniques, rapid thermocycling, expensive equipment, and is a lengthy process • Company called Lumora came up with a breakthrough technology to test for GMO • It’s a combination of 2 technologies; Bioluminescence and isothermal DNA amplification • Bioluminescence used is bioluminescence real time reporter (BART) Organic food symbol

20. Lumora and Improved GMO Testing • Loop mediated isothermal amplification (LAMP) • BART uses luciferase and detects DNA and It lights up when it find specific DNA and RNA sequences linked to genetic modifications • GMO testing can be done out in the field or in a food processing center • LAMP-BART technique requires only basic equipment for DNA extraction, a constant temperature and simple light detection • Quicker than PCR • Good for farmers to have a cheap, reliable way to test products for GMO’s • Lumora’s GMO detector can also be used to test for things like salmonella in foods

21. Questions? • What are the biological advantage of light emission to the animal? • Evolution history? • Metabolic/ dietary source of the luciferins? • What are the control mechanisms for light flashing? • Discovering- luminous mollusc, a roman delicacy, the bioluminescence mechanism of it is still not completely solved

22. Picture References • Deep-sea Shrimp • http://www.nytimes.com/imagepages/2011/12/20/science/20JPBIO1_SPAN.html • Chemical equations and fluorescence animations: • http://www.lifesci.ucsb.edu/~biolum/chem/ • Tomopteris • http://discovermagazine.com/photos/1-8-marine-creatures-that-light-up-the-sea • Chemical Structure drawing of Luciferin • http://www.biotium.com/product/applications/Enzyme_substrates/price_and_info.asp?item=10100&layer1=D;&layer2=D01; • Robert Boyle • http://www.friedpost.com/sciencetech/greatest-science-discoveris-boyle%E2%80%99s-law-1977.html • Raphael Dubois • http://en.wikipedia.org/wiki/File:Raphael_Dubois_(1849-1929).jpg • Oxygen Atom • http://www.historyforkids.org/scienceforkids/chemistry/atoms/oxygen.htm • Bioluminescent Imaging mouse • http://www.caliperls.com/products/preclinical-imaging/?gclid=CMiigb2T_q8CFWwDQAodmVZCzg • Glofish • http://fish-blog.co.uk/News/manufacturer_supports_glofish_industry/ • VibrioFischeri • http://microbewiki.kenyon.edu/index.php/Vibrio_fischeri • Bioluminescent Imaging mouse • http://www.caliperls.com/products/preclinical-imaging/?gclid=CMiigb2T_q8CFWwDQAodmVZCzg • Organic food stamp • http://www.google.ca/imgres?um=1&hl=en&sa=N&biw=1366&bih=569&tbm=isch&tbnid=pcYC93zfdGquhM:&imgrefurl=http://www.omafra.gov.on.ca/english/crops/organic/certification.htm&docid=MuinqDkP2Ld1-M&imgurl=http://www.omafra.gov.on.ca/english/crops/organic/certificationf10.jpg&w=600&h=616&ei=JSGwT6XnFarE6QG1rqCvCQ&zoom=1 • Humpback Angler Fish • http://written-4u.blogspot.ca/2007/01/allah-creator-sustainer-surely-it-is.html • Jamaican Click Beetle • http://www.asknature.org/media/image/6970

23. References • Beecher, C. (2012, May 7). Breakthrough Offers Promise of Improved GMO Testing. In Food Safety News. Retrieved May 9, 2012, from http://www.foodsafetynews.com/2012/05/breakthrough-offers-promise-of-improved-gmo-testing/ • Binger, J. M. (2007, March 14). Bioluminescence. In Center for Biophotonics Science and Technology. Retrieved May 6, 2012, from cbst.ucdavis.edu/education/courses/spring-2007./bingerfinaldraft.doc • Hutchens, M., & Luker, G. D. (2007, June 24). Applications of bioluminescence imaging to the study of infectious diseases. Retrieved May 7, 2012, from Wiley Online Library (10.1111/j.1462-5822.2007.00995.x). •  John, L. (08). Basic Bioluminescence. In Photobiological Sciences Online. Retrieved May 7, 2012, from http://www.photobiology.info/LeeBasicBiolum.html • Lee, J. (08). A History of Bioluminescence. In Photobiological Sciences Online. Retrieved May 6, 2012, from http://www.photobiology.info/HistBiolum.html • Lumora welcomes expressions of interest for GMO test commercialisation after BART technology demonstrates ability to detect genetically modified contamination of crops rapidly and at very low levels o. (2012, April 30). In Lumora . Retrieved May 9, 2012, from http://79.170.44.87/lumora.co.uk/index.php?option=com_content&view=article&id=100:lumora-welcomes-expressions-of-interest-for-gmo-test-commercialisation-after-bart-technology-demonstrates-ability-to-d