What is fluorescence?

Fluorescence is the property of emitting electromagnetic

radiation in the form of light as a result of the absorption
of light from another source.

Important characteristics of fluorescence:

It is the result of the absorption of light.
It occurs during absorption only.
It involves the emission of light.
An outside source of energy is required.

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Definition of fluorescence

Fluorescence is a member of the luminescence family of

processes in which;
susceptible molecules emit light from electronically
excited states created by either a physical (for example,
absorption of light), mechanical (friction), or chemical
mechanism. Generation of luminescence through
excitation of a molecule by ultraviolet or visible light
photons is a phenomenon termed photoluminescence,
which is formally divided into two categories,
fluorescence and phosphorescence,

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Fluorescence

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Luminescence

Bio-luminescence Chemi-luminescence

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Energetic Transitions of Electrons
During Fluorescence
Perrin-Jablonski diagram

Lifetime of electrons in excited state is short: 10-13 (absorption) and 10-9 s (emission).

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Singlet and Triplet states

A singlet or a triplet can form when one electron

is excited to a higher energy level. In an excited
singlet state, the electron is promoted in the
same spin orientation as it was in the ground
state (paired).
In a triplet excited stated, the electron that is
promoted has the same spin orientation (parallel)
to the other unpaired electron.

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About the energy state

The difference between the spins of

ground singlet, excited singlet, and excited
triplet state

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Vibrational relaxaiton and internal
conversion

Vibrational relaxation is where the energy deposited by the

photon into the electron is given away to other vibrational modes
as kinetic energy.

This kinetic energy may stay within the same molecule, or it may
be transferred to other molecules around the excited molecule,
largely depending on the phase of the probed sample. This
process is also very fast, between 10-14 and 10-11 seconds.

However, if vibrational energy levels strongly overlap electronic

energy levels, a possibility exists that the excited electron can
transition from a vibration level in one electronic state to another
vibration level in a lower electronic state. This process is called
internal conversion.

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Timescale Range for Fluorescence Processes

Timescale

Timescale
Transition Process Rate Constant
(Seconds)

S(0) => S(1) or Absorption

Instantaneous 10-15
S(n) (Excitation)

Internal
S(n) => S(1) k(ic) 10-14 to 10-10
Conversion

Vibrational
S(1) => S(1) k(vr) 10-12 to 10-10
Relaxation

S(1) => S(0) Fluorescence k(f) or 10-9 to 10-7

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 The category of molecules capable of undergoing electronic
transitions that ultimately result in fluorescence are known as
fluorescent probes, fluorochromes, or simply dyes.
Fluorochromes that are conjugated to a larger macromolecule
(such as a nucleic acid, lipid, enzyme, or protein) through
adsorption or covalent bonds are termed fluorophores.
In general, fluorophores are divided into two broad classes,
termed intrinsic and extrinsic.
Intrinsic fluorophores, such as aromatic amino acids,
neurotransmitters, porphyrins, and green fluorescent protein, are
those that occur naturally. Extrinsic fluorophores are synthetic
dyes or modified biochemicals

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Fluorescein A Typical Fluorescent Probe
120

100

80

Intensity
60

40

20

0
380 480 nm 580 680

FITC

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FITC vs GFP !!

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Franck Condon Principle

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The FranckCondon principle explains the intensity
of vibronic transitions.

Vibronic transitions are the simultaneous changes in

electronic and vibrational energy levels of a molecule
due to the absorption or emission of a photon of the
appropriate energy.

The principle states that during an electronic

transition, a change from one vibrational energy
level to another will be more likely to happen if the two
vibrational wave functions overlap more significantly.

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BITS Pilani, K K Birla Goa Campus