fluorescence - linköping university the emission wavelength •depends on the electronic states...

Fluorescence

Fluorescence and phosphorescence

The emission wavelength

•Depends on the electronic states within the system and how they are related

•is independent of the excitation wavelength as long as it is within the absorbtion range

• is always longer than the excitation wavelength

Etidiumbromide fluorescence in the visible range when in complex with DNA

• Absorbtion spectrum• Emission spectrum

Time scales

How much of the absorbed light is emitted as fluorescence?

The quantum yield

(ibland ) is defined as

= number of emitted photons / number of absorbed photons total

The quantum yield is affected by how quickly (s-1) energy leaves the system through alternative processes:

fluor = kfluor / (kfluor + kNR + kICS )NR: nonradiative processes

ICS: intersystem crossing

Normally, alternative processes occur at significantly higher rates compared to fluorescence; the quantum yield will then be low (<< 1).

If we have an average rate of decay (s-1), we could deinfe an average lifetime (s) for the fluorescence:

Does this work: F = 1/ kfluor ???

NO, because F is also affected by how rapid the alternative processes are! In fact,

= R = 1/ kfluor will be

a maximum value for the life time (if all other decay rates are 0)

A more real value for the fluorescence lifetime is

F = 1 / (kfluor + kNR + kICS )

Then we get

= kfluor / (kfluor + kNR + kICS ) = F / R

How do we estimate the sensitivity in a fluorescence probe?

…gives a handy magnitude between 0.01 and 10

Remember the orders of magnitude: Maximal absorbtion for a molecule the size of an aromatic ring is :

= 105 M-1

cm-1 = 100 000 M-1 cm-1

For a strong absorbent:

= 104 M-1 cm-1 = 10 000 M-1 cm-1 , i e 10% of all absorbed light

The sensitivity is dependent on how much light that can be absorbed by the system, as well as on the quantum yield.

Sensitivity = max *

* 10-2

Internal fluorofores in biomolecules

Normally

the quantum

yield, F

, is small

F = F / R << 1

The larger

the quantum

yield, the larger

the fluorescence

intensity.

Which

factors

affect

the quantum

yield, expect

the prerequisites

of the system? Let

us

look at the

formula

again:

= kfluor / (kfluor + kNR + kICS ) = F / R

How

do

non-ratiative

(NR) and intersystem crossing

(ICS) mechanisms

affect

relaxation, and thereby

intensity?

The Quantum yield, and thereby the intensity, is affected by:

Internal

rearrangements

•excitation energy

is lost

by internal

vibrations

•Increase

with temperature

–

difficult

to measure

temperature-

related

biological

phenomena

(folding

for example)

Quenching

Collision

with so called

quenchers

: molecules

that take

energy

and thereby

compete

with spontaneous

emission : solvent, acryl

amide, O2

, I-

Intersystem crossing

transition

to other

states

-

phosphorescence

The amount of quenching depends on: * the rate constant for quenching * the concentration of the quencher.

= kfluor / (kfluor + kNR + kICS + kQ * [Q]) = F / R

Altered quantum yield can have various reasons:

• Quenching by solvent or buffer components• Change of temperature• Quenching by nearby changes in charge

(protonation/deprotonation) • Very small local changes can still give large

effects in spectra

Stern-Volmer-plots can be used to judge accessibility

Altered quantum yield often occurs when the system is altered

no Ca2+ With

four

Ca2+

External fluorophores

Fluorescence is strongly quenched in water solution, but is drastically increased in non-polar or restraining environments.

ANS-binding to proteins is used to detect folding

Ehrhard et al., Biochemistry 1996

which protein is most/least well-folded?

The emission wavelength

•Depends on the electronic states within the system and how they are related

•is independent of the excitation wavelength as long as it is within the absorbtion range

• is always longer than the excitation wavelength

Calmodulin,

a calcium-binding

protein, regulates

a multitude

of calcium-ion

mediated

events, such

as muscle

contraction, and is a central protein in signal transduction

by activating

protein kinases.

without

Ca2+

with four

Ca2+With peptide

ligand

and calcium

The emission wavelength can change if the system changes.

Altered wavelength can be used in binding studies

Above: the effect on dansyl fluorescence at the bindning of biosensor peptides to human carbonic anhydrase II (HCAII)

Folded and unfolded proteins

N: native, U: unfolded

A fluorophore in a less polar environment usually shows fluorescence at shorter wavelengths – ’red shift’ – together wtih increased intensity.

How is fluorescence measured?

The fluorescence

of a fluorophore

in the membrane surface

depends

largely

on charge properties

–very

sensitive to pH, environmental

changes

etc

Fluorescence polarizations assays

Fluorescence polarization assays

The membrane

bound

fluorophore

reports

on changes

in surface

potential at binding

of proteins

and/or peptides.

FRAP: Fluorescence recovery after photobleaching

FRAP can be performed in-vivo

A fluorescent molecule can quench itself at high concentration

Application: the effect of a toxic pepide penetrating cell membranes, detected by fluorescence

Sal-Man et al., Biochemistry 2002

Vad ser vi med fluorescens? Jfr med CD!