Radiationless excitation energy transfer r equires interaction between donor and acceptor

IPC Friedrich-Schiller-Universität Jena1

Radiationless excitation energy transfer requires interaction between donor and acceptor

Emission spectrum of donor must overlap with absorption spectrum of acceptor.

Several vibronic transitions within donor have the same energy than in the acceptor

Resonant coupling of the transitions

RET = resonance energy transfer

Resonant transitions

7.3 FRET microscopy

7. Fluorescence microscopy


Assumption: 2 electrons one at the donor D and one at the acceptor A are involved in the transition:

Antisymmetric wavefunction (Fermions) for initially excited state i (D excited, but not A) and final state f (A excited, but not D):

Overall Hamiltonian:Interaction energy:

Coulomb term UC Exchange term Uex

Radiationless excitation energy transfer


7.3 FRET microscopy


Coulomb Interaction (CI)

Exchange Interaction


7.3 FRET microscopyRadiationless excitation energy transfer


Different interaction mechanism lead to excitation energy transfer:

Coulombinteraction

Inter molecular orbital overlap

Dipolar(Förster)

Multipolar

Electron exchange(Dexter)

Charge resonance interaction

Singlet energy transfer

TripletEnergy transfer

„LongRange“

„ShortRange“




Coulomb interaction dominates for allowed i.e. singlet-singlet-transitions.

For forbidden transitions i.e. singlet-triplet-transitions exchange-interaction (only acting for short distances < 10 Å because overlap of orbitals is necessary) dominates.

Coulomb interactions appears also for larger distances up to 80 – 100 Å.

Interaction strength depends on interaction energy (U), energy distance between D* and A* (E), absorption bandwidth (w) and vibronic bandwidth ().

Strong coupling: U>>E U>>w,Weak coupling: U>>E w>>U>>Very weak coupling: U<<<<w




Förster Resonance Energy Transfer (very weak coupling):

D + h1 D* Absorption

D* + A A* + D Energy transfer

A* A + h2 Emission

The following conditions must hold:

D must be a fluorophore with sufficiently long life-time

Partial spectral overlap between emission spectrum of D and absorption spectrum of A

Transition dipole moments D and A must

be oriented properly to each other;

Distance between D and A shouldn‘t be too large


7.3 FRET microscopy


Coulomb interaction can be developed in a multipole series in which the dipole

term exhibits the term with the longest range Energy transfer via dipole-dipole transfer has been first calculated by Förster and

is therefore called Förster process Energy transfer rate from molecule D to molecule A at a distance r:

kD = radiative decay rate of donor

tD0 = donor life-time in absence of energy transfer

r-6-dependency as a result of dipole-dipole interaction

R0 = critical distance or Förster-radius (distance at which intensity

decrease caused by energy transfer and spontaneous decay are

equal ( = kD)).



7.3 FRET microscopy


R0 can be determined via spectroscopic values:

For R0 in Å, in nm, A() in M-1 cm-1 (overlap integral in M-1 cm-1 nm4)

Typical values for Förster-radii R0, i.e. for distances, over which energy transfer is

important lie in the range of 15 -60 Å

2 = orientational factor0

D = quantum yield of donor in absence of energy transfer n = average refractive index for wavelength area of spectral overlapID() = normalized fluorescence spectrum of donor ( )A() = molar absorption coefficient of acceptor.

Overlap between fluorescence of donor and absorption of acceptor



7.3 FRET microscopy


Transfer efficiency can be expressed by:

In combination with changed lifetime:

It follows:

D und D0 are excited state life-times of

donor in absence and presence of acceptor, respectively



7.3 FRET microscopy

distance dependency:ddDA

DD

k 0

11


Besides the distance between the two chromophores also the relative orientation

of the transition dipole moments of the donor D and acceptor A plays a crucial

role for the energy transfer efficiency The orientation factor 2 is given by:

D

A

A: angle between D-A connecting line and

acceptor transition dipole moment

D: angle between D-A connecting line and

donor transition dipole moment

T: angle between donor and acceptor

transition dipole moment



7.3 FRET microscopy


For systems where the orientation stays constant during the energy transfer (e.g. usage of highly viscose solvents or rigid coupling of chromophores to large and stiff molecules) can reach values between 0 (transition dipole moments are orthogonal) and 4 (collinear arrangement); = 1, for a parallel arrangement

If both acceptor and donor can rotate the orientational factor 2 must be replaced by an average value:

In case both chromophores undergo a fast isotropic rotation i.e. the rotation is considerably faster than the energy transfer rate the average orientation factor is given by = 2/3

In case donor and acceptor are freely movable but the rotation is significantly slower than the energy transfer the orientation factor results in: 2 = 0.476



7.3 FRET microscopy


RET is utilized as „optical nano ruler“ (10 – 100 Å) in biochemistry and cell biology

Distance between donor and acceptor should be in the range of:

because R0 is a benchmark for donor-acceptor distances which can be determined

by FRET.



7.3 FRET microscopy


RET as „optical nano ruler“ in biochemistry and cell biology



7.3 FRET microscopy



RET as „optical nano ruler“ in biochemistry and cell biologyFörster Resonance Energy Transfer (very weak coupling):

7.3 FRET microscopy


RET as „optical nano ruler“ in biochemistry and cell biologyOne requires appropriate method to label specific intracellular proteins with suitable fluorophores (fluorescent proteins genetics):

Green Fluorescent Protein (GFP) first isolated from the jellyfish Aequorea victoria GFP can be combined with just about any other protein by attaching its gene to the gene of a target protein, thereby introducing it into a cell. Thus by recording the GFP fluorescence the spatial and temporal distribution of this target protein can be directly monitored in living cells, tissue and organism.

Several GFP mutants with altered fluorescence spectra exist. These mutants are named according to their color e.g. CFP (cyan) or YFP (yellow)



7.3 FRET microscopy

Excitation maxima at 395 und 475 nmEmission wavelength at 509 nm


Agar plate of fluorescent bacteria

colonies


7.3 FRET microscopy


RET as „optical nano ruler“ in biochemistry and cell biology :GFP-mutants

no FRET

FRET

protein folding protein-protein interaction

R0 = 4.7 – 4.9 nm



7.3 FRET microscopy

Date post:	24-Jan-2016
Category:	Documents
Upload:	majed
View:	41 times
Download:	0 times

Radiationless excitation energy transfer r equires interaction between donor and acceptor

Documents