Optical Spectroscopy 2018

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1.Fluorescent dye probes (KM), 26 Sept 2.Photochromic materials (BP), 3 Oct

3.Luminescent metal complexes (BP), 10 Oct 4.Photodynamic therapy (VT), 17 Oct

5.Fluorescence imaging (VT), 24 Oct

6.Near Infrared Spectroscopy (GSz) 31 Oct

7.Spectroscopy of chiral compounds (KM), 7 Nov

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ALAPISMERETEK

(vizsgára, doktori szigorlatra átismételni)

Kémiai anyagszerkezettan

V. OPTIKAI SPEKTROSZKÓPIA (Optsp05)

VI. A MOLEKULÁK FORGÓMOZGÁSA (Forgo05) VII. A MOLEKULÁK REZGŐMOZGÁSA (Rezgo05)

VIII. A MOLEKULÁK ELEKTRONSZERKEZETE (Molel05)

X. LÉZEREK, LÉZERSPEKTROSZKÓPIAI MÓDSZEREK (Lezer05)

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Nobel prize in Chemistry 2008

Martin Chalfie

Osamu Simamura

Roger Y. Tsien

GFP = Green

Fluorescent Protein

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Nobel prize in Chemistry 2014

Eric

Betzig Stefan W.

Hell

William E.

Moerner

STED = Stimulated Emission Depletion

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Jablonski-diagram

V R

S ₀ S ₁

T ₁ T ₂ S ₂

s z i n g u l e t t a b s z o r b c i ó

I S C

I C

f lu o r e s z c e n c i a

t r ip l e t t a b s z o r b c i ó

f o s z f o r e s z c e n c i a I C

V R : I S C : I C : S : T :

r e z g é s i r e l a x á c i ó

S p i n v á l t ó á t m e n e t ( I n t e r S y s t e m C r o s s i n g ) b e l s ő k o n v e r z i ó ( I n t e r n a l C o n v e r s i o n )

s z i n g u l e t t t r ip l e t t

v = 0 v = n

s u g á r z á s n é l k ü l i á t m e n e t s u g á r z á s o s á t m e n e t

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Advantages of detection of fluorescence over detection of absorbtion

1. Sample may be non-transparent 2. Higher sensitivity

3. Triple selectivity

- excitation wavelength - emission wavelength - delay time

Disadvantage: only a few type compounds are fluorescent

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Fluoreszcent dye probes

Function: provide information on local environment

J. R. LAKOWICZ, Principles of Fluorescence Spectroscopy, 2nd Edition, Kluwer Academic, London, 1999

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Main points

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 Instruments

stationary fluorescence spectrometer time-correlated single photon counting

 Molecular chemosensors: detection of ions, molecules

 Polarity sensors

Viscosity sensors

 Fluorescence of proteins / triptophan

 Distance measurement : FRET

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Spectrofluorimeters

-stationary

- time-resolved (measures _F, time-correlated single photon counting)

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Stacionárius

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Excitation and emission flurescence spectra

Excitation sp: similar to absorption sp, bands of S₀ →S₁, S₀ →S₂, ∙∙∙

transitions

Emission sp: only S₁ →S₀,

I_F is relative, depends on instrument! a.u.!



^nm





exc

em λ

λ 

âÎ^.û^F^.

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Fluorescence quantum yield

 



^absorbed ^photons



N

photons emitted

N

F 



Determination of _F

- integrating sphere - standard

2 2 R X X

R R

R X

X n

n A

A I

 I





I_X,I_R integrated intensities of fluorescence bands A_X, A_R absorbances at excitation wavelength

n_X, n_R indeces of refraction

X: sample R: standard

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Time-correlated single photon counting

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Fluorescence decay curve

IRF

  _



 







F F

A t t

I ^exp 

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Chemosensors: fluorescent detection of non- fluorescent ions, molecules

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CoroNa Green Chemosensor for fluorescent detection of Na⁺ions

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Distribution of Na⁺ ions eloszlása neurons,

Microscopic image with application of CoroNa Green

W. J. Tyler et al. , PlosOne 3, e3511 (2008)

Chemosensor for fluorescent detection of Na⁺ions

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MQAE

Its operation is based on dynamic quenching

Selective: nitrate, phosphate – no quenching, Br^-, I^- quenching Chemosensor for fluorescent detection of Cl^-ions

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Distribution of Cl- ions in neurons

IF image FLIM: fluorescence lifetime imaging

Chemosensor for fluorescent detection of Cl^-ions

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M + h

M + 

M + Q M^*

Dynamic quenching: Stern-Volmer equation

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Deactivation rates and fluorescence quantum yields in the absence and presence of quencher

No quencher

 

^ ^

 

^ ^

 

^

 k M k M

dt M d

nr f

With quencher

 

^k

 

^M ^k

   

^M ^k ^M

^{ }

^Q

dt M d

q nr

f



 







     

f nr f nr

f 0 f

k k

k M

k

M Φ k

 

 _  ^ _

 

^M ^k

   

^M

 

^k ^M

^{ }

^Q ^k ^k ^k ^k

^{ }

^Q

k

M Φ k

q nr

f

f q

nr f

f



 



 _  _ ^ _

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   

^Q

k k

1 k k

k

Q k

k k

Φ Φ

nr f

q nr

f

q nr

0 f

 

 



 

 

^Q

k k

1 k I

I

nr f

q F

F 0

 



Stern-Volmer equation

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Polarity sensors

Nile red

water, - methanol - ethanol - acetonitrile - dimethylformamide, 6.

acetone - ethyl acetate - dichloromethane - n-hexane - methyl-tert- butylether - cyclohexane - toluene.

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Solvatochromic dye: color depends on solvent

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The accumulation of oil droplets (golden dots). Red represents chlorophyll autofluorescence.

Confocal image of the algae stained by Nile red

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S₀ S₁

solvent polarity

„charge transfer (CT)” dyes

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Effect of polarity on spectra:

Lippert equation

+

_

- - - -

+ + + +

2a

_G v. _E

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Lippert equation



_E _G



² _VR

2 2 F 3

A

E

1 n

2 1 n

1 2

1 a

h 2

h      



 







 







 







+

_

- - - -

+ + + +

2a

_G v. _E

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Stokes shifts of naphthalane derivatives

Lakowicz, p. 191

ethanol-water solvent mixtures

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Viscosity sensors

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Orientation relaxation of dye solutes (rotational diffusion)

M 0

or τ

kT fC ηV

τ  

Stokes-Einstein-Debye equation

f shape factor (spheres f = 1) C friction factor (0<C<1)

 local viscosity

V_M molecular volume T temperaturet

k Boltzmann constant

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Fluorescence of nile blue on ion exchange resin

Habuchi et al., (Sapporo), Anal. Chem. 73, 366-372 (2001)

Resin: styrene – divinylbenzene copolymer

Cross-linking frequency (): 8 % divinylbenzene Ionexchange group: Na-sulphonate

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Determination of _or :

via measuring fluorescence depolarization

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Fluorescence of nile blue adsorbed on ion exchange resin

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Dual fluorescence:

twisted intramoleculat charge transfer = TICT

Fluoresc. spectrum of DMANCN in ethyleneglycol, the ratio of the intensities of the two bands varies with viscosity

Lakowicz, p. 201

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CH2 CH NH2

COOH

*

COOH NH2 CH2 CH

HO

*

COOH NH2 CH2 CH

NH

Fluorescent amino acids

phenyl alanine

tyrosine

triptophane

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Absorption and fluorescence spectra of triptophane

(water, pH 7) Lakowicz, p. 446

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Lakowicz p. 453

Fluorescence spectra of tryptophane in different local environments

1) Apoazurin Pfl 2) T1 ribonuclease 3) staphillococcus

nuclease 4) glucagon

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Lakowicz, p. 461

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Lakowicz, p. 461

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Resonance energy transfer

(Förster resonance energy transfer = FRET)

Molecular ruler to measure distances!

Resolution of optical microscope: max. ~ 200 nm, depends on

FRET: detection of 2-10 nm distances

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Donor dye – acceptor dye, fluorescence band of D overlaps with absorption band of A

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If D and A are close, FRET,

exciting D, the energy is transferred to A, fluorescence of A is detectable

The FRET effect is proportional to 1/r⁶

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Example for application of FRET: study of DNA – phospholipid interaction

C. Madeira, Biophys. J. 85, 3106 (2003)

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Acceptor

Donor: EtBr

(ethidium bromide)

N

C₂H₅

NH₂ H₂N

+

Br-

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EtBr absorption

BODIPY fluorescence

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Conformational changes of proteins can be monitored