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