Absorption, Excitation and Emission Spectra, Quantum Yield

Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

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

phosphorescencephotoluminescence – absorption of light

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.

The discovery and characterization of luminescence

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.

Sir John Herschel (1845) made the first observation of fluorescence from quinine sulfate

Sir George Stokes (1852) created the term “Fluorescence”.

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!

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

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.

Adolph Von Beyer (1871) a German chemist, synthesized Spiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one, 3',6'-dihydroxy.

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.

FLUORESCEIN!!!

Shimomura, Johnson and Saiga (1962) discovered Green Fluorescent Protein in the Aequorea jellyfish

Most of the basic principles of fluorescence were developed during the 1920's and 1930's.

Fluorescence in the 20Fluorescence in the 20thth Century Century

Fluorescence resonance energy transfer ( T. Förster)

Excited state lifetime (Gaviola)

Quantum yield (Wavilov)

Polarization of fluorescence (Weigert, F. Perrin)

during the 1950's:

Jablonski diagram (A. Jablonski)

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

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

The Jablonski DiagramThe life history of an excited state electron in a luminescent probe

S0

Phosphorescencekph < 106 s-1

T1

Fluorescencekf ~ 107 – 109 s-1

S2

S1

Absorption

Inter-system crossing kx ~ 104 – 1012 s-1

Internal conversion ki ~ 1012 s-1

Radiationless decay knd > 1012 s-1

ki ~ 106 -1012 s-1

kx ~ 10-1 – 105 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.

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.

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

S2

S1

Absorption

Flu

ore

scen

ce

kf ~

10

7 – 1

09 s

-1

The fluorescence excitation spectrumThe 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.

light source

Xenon or arc lamp

excitation monochromator moving

detector

emission monochromator fixed

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).

Iem(ex)

Probability

LOW

HIGH

MEDIUM

Energy

Inter-nuclear distance

S0

S1

v 0

v 1

v 2

v 3

v1 0

v 11

v 12

v1 3

The fluorescence excitation spectrum

anthracene

The fluorescence emission spectrumThe 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).

excitation monochromator fixed

detector

emission monochromator moving

light source

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.

Iem(em)

Probability

LOW

HIGH

MEDIUM

Energy

Inter-nuclear distance

S0

S1

v 0

v 1

v 2

v 3

v1 0

V1 1

V1 2

V1 3

The fluorescence emission spectrum

anthracene

The fluorescence spectra

anthracene

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

emission

excitation

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

The mirror simmetry does not hold exactly when the spectra are plotted in wavelength units.

fluorescein

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

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

• water has a strong O-H stretching band at 3300 cm-1

E1Fluorescence spectra and peptide/protein binding to membranes

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

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

ex = 280 nm

E2Fluorescence 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

Excitation spectra of genetically encoded ratiometric pHluorin, em = 508 nm, from Schulte et al. Plant Methods 2006, 2: 7

Fluorescence spectroscopy can measure pH inside of cells and cellular compartments. Modern pH sensitive dyes can be genetically encoded highly specific location

Quantum Yield

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 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 can be defined:

QY = Number of emitted photons / Number of absorbed photons

r

f

nrf

f

kk

kkk

QY

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