fluorescence aj
Post on 01-Jun-2018
231 Views
Preview:
TRANSCRIPT
-
8/9/2019 Fluorescence Aj
1/18
FluorescenceFluorescence
andandChemiluminescenceChemiluminescence
-
8/9/2019 Fluorescence Aj
2/18
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
-
8/9/2019 Fluorescence Aj
3/18
Fluorescence and fosforescence
-
8/9/2019 Fluorescence Aj
4/18
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
-
8/9/2019 Fluorescence Aj
5/18
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
-
8/9/2019 Fluorescence Aj
6/18
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
-
8/9/2019 Fluorescence Aj
7/18
Fluorescence measurement
sample
emissionmonochromator
detector
sourceexcitation
monochromator
"ead!out
Filter fluorimeters
Spectrofluorometers
Fluorescent microscopes
Fluorescent scanners
Flow cytometry
-
8/9/2019 Fluorescence Aj
8/18
Spectrofluorometer
-
8/9/2019 Fluorescence Aj
9/18
Spectrofluorometer
-
8/9/2019 Fluorescence Aj
10/18
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
-
8/9/2019 Fluorescence Aj
11/18
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
-
8/9/2019 Fluorescence Aj
12/18
9atural fluorophores
: 'olyaromatic hydrocarbons
: $itamin ;, E
: Coenymes .F;8, F79, 9;8
-
8/9/2019 Fluorescence Aj
13/18
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
-
8/9/2019 Fluorescence Aj
14/18
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
-
8/9/2019 Fluorescence Aj
15/18
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
-
8/9/2019 Fluorescence Aj
16/18
Chemiluminiscence
Luminol and
peroxidase beforeadding
-
8/9/2019 Fluorescence Aj
17/18
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
-
8/9/2019 Fluorescence Aj
18/18
;pplication of
chemiluminescence detection: 94 assay
94 A 4B945A 45
945945A light
:
top related