Fluorescence

and
Chemiluminescence

Luminescence
Emission of radiation, which occurs during returning of
excitated molecules to ground state
Fluorescence, phosphorescence excitation is caused by
absorption of radiation

Chemiluminiscence excitation is caused by chemical reaction

Singlet state - spins of two

electrons are paired

Triplet state - spins of two

electrons are unpaired
S0

S1

T1

Fluorescence and fosforescence

Energy level diagram for photoluminescent

molecules

Radiationless transitions:
VR vibrational relaxation
IC- internal conversion
ISC intersystem crossing

Radiation transitions:
Fluorescence - transition to the ground state with the same multiplicity S 1S0
probability of fluorescence is higher than phosphorescence
Phosphorescence transition between states with different multiplcity T1S0

Stokes shift
Wavelength difference between absorption
(excitation) and fluorescence (emission)
maximum
Wavelength of emitted radiation
is longer because its energy is
lower

E = h . c/
Stokes shift

http://psych.lf1.cuni.cz/fluorescence/soubory/principy.htm

Quantitative fluorescent measurement

I0

sample

If

It

I a = I 0 - It

f =

intensity of fluorescence

If

intensity of absorption

Ia

Fluorescence efficiency (f ) is the fraction of the incident

radiation which is emitted as fluorescence f < 1

Fluorescence measurement
Filter fluorimeters
Spectrofluorometers
Fluorescent microscopes
Fluorescent scanners
Flow cytometry
source

excitation
monochromator

sample

emission
monochromator

detector
Read-out

Spectrofluorometer

Spectrofluorometer

Sources of interference
Inner filter effect
intensity of excitation light isnt constant because each
layer of the sample absorbs some of the incident
radiation (intensity of exciting light is higher in the
front part of cuvette and lower in the rear part of
cuvette
Quenching
excited molecule returns to the ground state by
radiationless transition (without emitting light) as a
result of a collision with quenching molecule
Quenching agents: O2, halogens (Br, I),
nitrocompounds

Methods of fluorescence
determination
Direct methods - natural fluorescence of the
fluorecent sample is measured
Indirect (derivatisation) methods - the nonfluorescent
compound is converted into a fluorescent
derivative by specific reaction or marked with
fluorescent dye by attaching dye to the studied
substance
Quenching methods - analytical signal is the
reduction in the intensity of some fluorescent dye
due to the quenching action of the measured
sample

Natural fluorophores

Polyaromatic hydrocarbons
Vitamin A, E
Coenzymes (FAD, FMN, NADH)
Carotenes
Quinine
Steroids
Aromatic aminoacids
Nucleotides
Fluorescent proteins GFP (green fluorescent
protein)

Nobel prize in chemistry in 2008

Osamu Shimomura discovered green fluorescent protein (GFP) in the small

glowing jellyfish Aequorea victoria
Martin Chalfie introduced using of green fluorescent protein as a marker for
gene expression
Roger Y. Tsien engineered different mutants of GFP with new optical properties
(increased fluorescence, photostability and a shift of the major excitation
peak ) and contributed to the explanation of mechanismus of GFP
fluorescence

Fluorescent probes
Compounds whose fluorescence doesnt change after their
interaction with biological material
acridine orange (DNA)
fluorescein (proteins)
rhodamine (proteins)
GFP
Compounds whose fluorescence change according to their
environment
ANS (1-anilinonaftalen-8- sulphonate) - polarity
Fura-2 - tracking the movement of calcium within cells

Some applications of fluorescence

detection

Protein conformation
Membrane potential
Membrane transport
Membrane viscosity
Enzymatic reactions
DNA analysis
Genetic engineering (manipulations)
Immunochemical methods
Cell proliferation and apoptosis

Chemiluminiscence

Luminol and
peroxidase before
adding H2O2
Chemiluminiscence
after addition H2O2

Chemiluminescence
Excitation of electrons is caused by chemical
reaction
Return to ground state is accompanied by light
emission
Bioluminescence
firefly

Noctiluca scintillans

ATP + luciferin + O2

luciferase

AMP + PPi + CO2 + H2O + oxyluciferin + light

Application of
chemiluminescence detection
NO assay
NO + O3 NO2* + O2
NO2* NO2 + light
H2O2 assay, peroxidase activity assay, immunochemical assays
Luminol + H2O2

3-aminoftalate + light

peroxidase