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).

7. 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

8. 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.

9. 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.

10. 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.

11. 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

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

13. FLUORESCENCE

14. 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.

15. 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.
• .

16. •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.

17. 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.

18. Perrin- Jablonski diagram

19. 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) .

20. 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

21. INTERNAL CONVERSION

22. 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.

23. 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

24. 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.

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

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

27. TYPES OF FLUORSCENCE

28. TYPES OF FLUORSCENCE
• B) Based upon the phenomenon:
I. Prompt fluorescence:
II. 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.

29. 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

30. 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

31. 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.

32. 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

33. 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.

34. 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.

35. 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.

36. 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

37. 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.

38. 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

39. 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".

40. 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.

41. 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

42. 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.

43. PHOSPHORESCENCE

44. 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.

45. 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).

46. 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.

48. 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.

49. 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.

50. 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.

51. 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.

52. 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.

53. 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.

54. THE END
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