Transcript Fluorescence spectra - UFCH JH

CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Absorption, Excitation and Emission Spectra,
Quantum Yield
Martin Hof, Radek Macháň
Emission of light - Luminescence
Luminescence – the excess of light emitted above thermal
radiation. The emission follows after the molecule has resided
for some time in the excited state.
according to excitation mechanism:
photoluminescence – absorption of light
chemiluminescence – chemical reaction
thermoluminescence – heat
electroluminescence – electric current
…
fluorescence
phosphorescence
The discovery and characterization of luminescence
Nicolás Monardes (1577), a Spanish physician and botanist who wrote
on medicines of the New World, was the first to describe the bluish
opalescence of the water infusion from the wood of a small Mexican tree.
When made into cups and filled with water, a peculiar blue tinge was
observed.
This wood was very popular in XVI - XVII Europe, where it was known as
"Lignum nephriticum" (kidney wood), because of its medicinal virtues for
treating kidney ailments.
In the ensuing centuries the wood was no longer used and the botanic
identity of the LN was lost in a confusion of several species. Safford, in
1915, succeeded in disentangling the botanic problem and identified the
species which produced the Mexican LN as Eynsemhardtia polystachia.
More recently, several highly fluorescent glucosyl-hydroxichalcones were
isolated from this plant.
Robert Boyle (1664) was inspired by Monardes’ report and investigated this
system more fully. He discovered that addition of acid abolished the color and that
addition of alkali brought it back. Hence Boyle was the first to use fluorescence as
a pH indicator!
Sir John Herschel (1845) made the first
observation of fluorescence from quinine
sulfate
Sir George Stokes (1852) created the term “Fluorescence”.
Stokes used a prism to obtain the ultraviolet region of the solar spectrum ( < 400
nm) to illuminate a quinine solution and observed the emission through a stained
glass filter (> 400 nm; blocks the excitation light). This observations led Stokes to
proclaim that fluorescence is of longer wavelength than the exciting light, which led
to this displacement being called the Stokes Shift
Adolph Von Beyer (1871) a German chemist, synthesized
Spiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one, 3',6'-dihydroxy.
FLUORESCEIN!!!
R. Meyer (1897) used the term “fluorophore” to describe chemical groups
which tended to be associated with fluorescence; this word was analogous to
“chromophore” which was first used in 1876 by O.N. Witt to describe groups
associated with color.
Gregorio Weber (1952) synthesized dansyl chloride
for attachment to proteins and used polarization to
study protein hydrodynamics - these studies
initiated the field of quantitative biological
fluorescence.
Shimomura, Johnson and Saiga (1962) discovered Green Fluorescent
Protein in the Aequorea jellyfish
Fluorescence in the 20th Century
Most of the basic principles of fluorescence were developed during
the 1920's and 1930's.
Excited state lifetime (Gaviola)
Quantum yield (Wavilov)
Polarization of fluorescence (Weigert, F. Perrin)
Jablonski diagram (A. Jablonski)
during the 1950's:
Fluorescence resonance energy transfer ( T. Förster)
Virtually all fluorescence data required for any research project will fall
into one of the following categories.
1. The fluorescence emission spectrum
2. The excitation spectrum of the fluorescence
3. The quantum yield
4. The polarization (anisotropy) of the emission
5. The fluorescence lifetime
In this course, we examine each of these categories and briefly discuss
historical developments, underlying concepts and practical
considerations
The Jablonski Diagram
The life history of an excited state electron in a luminescent probe
Internal conversion
ki ~ 1012 s-1
S2
Radiationless decay
knd > 1012 s-1
ki ~ 106 1012 s-1
Inter-system crossing
kx ~ 104 – 1012 s-1
S1
kx ~ 10-1 – 105 s-1
Absorption
Fluorescence
kf ~ 107 – 109 s-1
T1
Phosphorescence
kph < 106 s-1
S0
Key points:
 Most emission/quenching/FRET/chemical reactions occur from the lowest
vibrational level of S1
 Emission has lower energy compared to absorption (Stokes shift)
 Excitation spectra are mirror images of the emission spectra
 Triplet emission is lower in energy compared to singlet emission
 Emission spectra are practically independent of the excitation wavelength
S2
S1
Absorption
The fact that ground state fluorophores, at
room temperature, are predominantly in the
lowest vibrational level of the ground
electronic state (as required from Boltzmann’s
distribution law) accounts for the Stokes shift.
Fluorescence
kf ~ 107 – 109 s-1
Specifically, although the fluorophore may
be excited into different singlet state energy
levels (e.g., S1, S2, etc) rapid thermalization
invariably occurs and emission takes place
from the lowest vibrational level of the first
excited electronic state (S1). This fact
accounts for the independence of the
emission spectrum from the excitation
wavelength.
Finally, the fact that the spacings of the energy levels in the vibrational manifolds
of the ground state and first excited electronic states are usually similar accounts
for the fact that the emission and absorption spectra (plotted in energy units such
as wavenumbers) are approximately mirror images
S0
The fluorescence excitation spectrum
The relative efficiencies of different wavelengths of incident light to excite
fluorophores is determined as the excitation spectrum. In this case, the excitation
monochromator is varied while the emission wavelength is kept constant if a
monochromator is utilized - or the emitted light can be observed through a filter.
detector
Iem(lex)
emission monochromator
fixed
light
source
Xenon or arc lamp
excitation monochromator
moving
If the system is “well-behaved”, i.e., if the fluorescence intensity is proportional to
the absorbed energy, excitation spectrum will match the absorption spectrum.
However fluorescence detection is more sensitive (if detected at the wavelength of
the maximum of the emission spectrum).
The fluorescence excitation spectrum
Energy
anthracene
S1
Probability
v1 3
LOW
v 12
HIGH
v 11
MEDIUM
v1 0
S0
v3
v2
v1
v 0 Inter-nuclear distance
The fluorescence emission spectrum
The relative distribution of various wavelengths in the light emitted after excitation
by a single wavelength. In this case, the emission monochromator is varied while the
excitation wavelength is kept constant if a monochromator is utilized - or the sample
can be excited by monochromatic light source (laser).
detector
emission monochromator
moving
Iem(lem)
The shape of the emission
spectrum is within a certain
range of excitation
wavelength practically
independent of the excitation
wavelength, usually the
wavelength of the maximum of
the excitation spectrum is
chosen for the emission
spectrum measurement.
light
source
excitation monochromator
fixed
The fluorescence emission spectrum
Energy
anthracene
S1
V1 3
V1 2
V1 1
v1 0
S0
Probability
v3
LOW
v2
HIGH
v1
MEDIUM
v 0 Inter-nuclear distance
The fluorescence spectra
anthracene
emission
excitation
Because of similarity in spacing of the energy levels in the vibrational
manifolds of the ground state and first excited electronic states, the emission
and absorption spectra (plotted in energy units such as wavenumbers) are
approximately mirror images
The fluorescence spectra
quinin sulphate
emission
excitation
Mirror simmetry can be peturbed by an aditional band in the excitation
spectrum caused by the excitation to S2 state.
The fluorescence spectra
emission
excitation
fluorescein
The mirror simmetry does not hold exactly when the spectra are plotted in
wavelength units.
http://www.fluorophores.tugraz.at/
The fluorescence spectra – possible artifacts
The turbidity of the sample is too high and the excitation
light does not penetrate deep enough and emission light
is reabsorbed or scattered.
• use diluted samples and filtered to
eliminate scattering or measure
close to the surface
Raman scattering from the solvent.
• subtract Raman spectrum of the solvent from
the fluorescence spectrum
• water has a strong O-H stretching band at Dn  3300 cm-1
Optical saturation appears at very high excitation intensities. Most molecules
are at an excited state and cannot be excited any more. Emission intensity is,
therefore, no more proportional to the excitation intensity.
• measure at the range of proportionality between excitation and
emission intensities
Fluorescence spectra and peptide/protein binding to
membranes
E1
Energy levels of molecules (and thus their spectra) are influenced by the
properties of their environment, especially its polarity.
Less polar environment  blue shift
The emission maximum of Trp in
peptide melittin (bee venom) shifts
from ~355 nm (water) to ~325 nm in
hydrophobic environment
lex = 280 nm
The fraction of membrane-associated
melittin and/or its depth of penetration
to the membrane can be deduced from
the spectral shift
a – water
b – DMPC/DHPC bicelles
c – DMPC/DHPC/Chol bicelles
Anderson et al. BBA 2007, 1768: 115
Fluorescence as a pH indicator
Fluorescence spectra of some molecules are sensitive to pH thanks to an
equilibrium between protonated and deprotonated form of the fluorophore
which differ in spectral properties
Fluorescence spectroscopy can measure pH inside of cells and cellular
compartments. Modern pH sensitive dyes can be genetically encoded  highly
specific location
Excitation spectra of genetically encoded ratiometric pHluorin, lem = 508 nm,
from Schulte et al. Plant Methods 2006, 2: 7
E2
Quantum Yield
Quantum yield can be defined:
QY = Number of emitted photons / Number of absorbed photons
The quantum yield of fluorescence (QY) is dependent on the rate of the
emission process divided by the sum of the rates of all other
deactivation processes
kf
kf

QY 


kf  knr
k
r
kf is the rate of fluorescence and knr is the sum of the rates of all
radiationless deexcitation pathways.
If the rates of the ratiationless deactivation processes are slow
compared to kr then the QY is high
However, if the rates of these other processes are fast compared to kr
then QY is low
Quantum Yield is determined by a comparison with a standard
Acknowledgement
The course was inspired by courses of:
Prof. David M. Jameson, Ph.D.
Prof. RNDr. Jaromír Plášek, Csc.
Prof. William Reusch
Financial support from the grant:
FRVŠ 33/119970