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FluorescenceFluorescence

andandChemiluminescenceChemiluminescence

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LuminescenceEmission of radiation, which occurs during returning of

excitated molecules to ground stateFluorescence, phosphorescence excitation is caused by

absorption of radiation

Chemiluminiscence excitation is caused by chemical reaction

S0 S1 1

E

Singlet state! spins of two

electrons are paired

riplet state! spins of two

electrons are unpaired

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Fluorescence and fosforescence

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Energy level diagram for photoluminescentmolecules

"adiationless transitions#

$" %ibrational relaxation

&C! internal con%ersion

&SC intersystem crossing

E

"adiation transitions#

Fluorescence ! transition to the ground state with the same multiplicity S1S0

probability of fluorescence is higher than phosphorescence

'hosphorescence'hosphorescence transition between states with different multiplcity transition between states with different multiplcity 11

SS00

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Sto(es shift

)a%elength of emitted radiation

is longer because its energy is

lower

E * h + c

Sto(es- shift

)a%elength difference between absorption

.excitation/ and fluorescence .emission/

maximum

http#psych+lf1+cuni+cfluorescencesouboryprincipy+htm

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uantitati%e fluorescent measurement

&0 &t

&f

intensity of fluorescence &f

intensity of absorption &af* *

&a * &0 ! &t

sample

Fluorescence efficiency .f/is the fraction of the incident

radiation which is emitted as fluorescence f 2 1

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Fluorescence measurement

sample

emissionmonochromator

detector

sourceexcitation

monochromator

"ead!out

Filter fluorimeters

Spectrofluorometers

Fluorescent microscopes

Fluorescent scanners

Flow cytometry

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Spectrofluorometer

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Spectrofluorometer

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Sources of interference&nner filter effect

intensity of excitation light isn-t constant because eachlayer of the sample absorbs some of the incidentradiation .intensity of exciting light is higher in thefront part of cu%ette and lower in the rear part of

cu%ette

uenching

excited molecule returns to the ground state byradiationless transition .without emitting light/ as aresult of a collision with 3uenching molecule

uenching agents# 45, halogens .6r, &/,

nitrocompounds

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7ethods of fluorescence

determination

8irect methods ! natural fluorescence of thefluorecent sample is measured

&ndirect .deri%atisation/ methods ! the nonfluorescentcompound is con%erted into a fluorescentderi%ati%e by specific reaction or mar(ed withfluorescent dye by attaching dye to the studiedsubstance

uenching methods ! analytical signal is thereduction in the intensity of some fluorescent dyedue to the 3uenching action of the measured

sample

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9atural fluorophores

: 'olyaromatic hydrocarbons

: $itamin ;, E

: Coenymes .F;8, F79, 9;8

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9obel prie in chemistry in 500>

4samu Shimomura disco%ered green fluorescent protein .=F'/ in the small

glowing ?ellyfish ;e3uorea %ictoria

7artin Chalfie introduced using of green fluorescent protein as a mar(er for

gene expression

"oger @+ sien engineered different mutants of =F' with new optical properties

.increased fluorescence, photostability and a shift of the ma?or excitation

pea( / and contributed to the explanation of mechanismus of =F'

fluorescence

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Fluorescent probes

Compounds whose fluorescence doesn-t change after theirinteraction with biological material

acridine orange .89;/

fluorescein .proteins/

rhodamine .proteins/

=F'

Compounds whose fluorescence change according to their

en%ironment

;9S .1!anilinonaftalen!>! sulphonate/ ! polarity

Fura!5 ! trac(ing the mo%ement of calcium within cells

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Some applications of fluorescence

detection: 'rotein conformation

: 7embrane potential

: 7embrane transport

: 7embrane %iscosity

: Enymatic reactions

: 89; analysis

: =enetic engineering .manipulations/

: &mmunochemical methods: Cell proliferation and apoptosis

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Chemiluminiscence

Luminol and

peroxidase beforeadding

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Chemiluminescence: Excitation of electrons is caused by chemical

reaction: "eturn to ground state is accompanied by light

emission

6ioluminescencefirefly

9octiluca scintillans

;' A luciferin A 45 ;7' A ''i A C45A

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;pplication of

chemiluminescence detection: 94 assay

94 A 4B945A 45

945945A light

: