Colorful principles of
absorption and fluorescence
Ctirad Hofr
Advanced methods of biophysics in experimental biology
1
Light is electromagnetic waves
• Light consists of an electric component and a magnetic
component, which oscillate in phase in perpendicular planes
• Light is characterized by frequency f and wavelength λ
• Frequency f determines how many times per second wave
oscilates, unit is Hertz Hz = s-1
• Wavelength determines the spatial period of the wave - the
distance over which the wave's shape repeats, expressed in
nanometers nm = 10-9 m
• Frequence f and wavelength λ is given by
c = λ f
where c is the speed of light (c=299 792 458 m s-1 in vacuum)
• Energy E = h f, where h Planck’s constant (6.626 10-34 J s)
3
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λ
Animation of electromagnetic waves
http://www.edumedia-sciences.com/a185_l2-transverse-electromagnetic-wave.html
Electromagnetic wave
z
xy
c = λ f
c is constant, if wavelength
increases, frequency must be
reduced to get constant
product.
Wavelength λ is inversely
proportional to the
frequency f
E = h f
The greater the frequency, the
greater the energy of the
radiation.
The greater the wavelength
λ, the lower the energy of
the radiation.
B E
5
Visible spectrum
Only a small portion of entire spectrum of
radiation is visible.
The visible spectrum is bordered by
wavelength of 400 nm and 700 nm.
http://science.hq.nasa.gov/kids/imagers/ems/visible.html
400 nm
7.5 1014 Hz
700 nm
4.3 1014 Hz
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Intensity
Intensity – the number of photons passing
through an unit area in a given direction
per unit time
7
Absorpion
• A substance absorbs light
• For absorption of
monochromatic light
• Beer-Lamber Law:
Absorbance is directly proportional to the
concentration and thickness of the
solution layer
lc
II ⋅⋅−
⋅= ε
100
I
I
lcA 0
10log=⋅⋅= ε
ε=molar extinction coefficient, c-concentration, l-length of optical path
8
Absorbance dependence on the
relative intensity of the incident and
transmitted light
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
0 1 2 3 4 5 6 7 8 9 10
I0/I
Abs
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Luminiscence
• Light emission from a substance; occurs
from the electron excited states
Luminiscence is divided to:
1.fluorescence
2.phosphorescence
According to the origin, luminiscence is divided to
1. photoluminescence 2. chemiluminiscence
10
Fluorescence
• Emission from excited singlet states
• Practically: fluorescence is observed during
excitation and disappears quickly after the
shutdown
• Time decay τ (Lifetime) is the average time
that elapses from the excitation to emission the
order of 1 to 10 nanoseconds
• note: light traveles 30 cm in 1 ns
11
Phosphorescence
• Emission from excited (prohibited) triplet
states
• Practically: the lifetime of phosphorescence
is much longer than the lifetime of
fluorescence
Lifetime in order of
milliseconds to seconds
note: light traveles 300 až 300 000 km in that time
12
Frank-Condon principle of laziness
of nuclei during absorption
Absorption of a photon by an electron
(excitation of a molecule) is a very quick
process in the order of femtoseconds (10-
15s). Because the atomic nucleus is much
heavier than the electron, it doesn’t move
during photon absorption. After absorption
of a photon – excitation, the whole
molecule is in an unstable state („is hot")
and vibrates to get rid of energy (to "cool").
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Absorption and emission of energy by
the molecule
Energy
0
1
2
0
1
2
Distance
Ground state
Excited state
14
Radiative and non-radiative transitions
between electronic-vibrational states of a
molecule
absorption fluorescence phosphorescence
λ
τ ≈ 10-15 s τ ≈ 10-8 s τ ≈ 10-3-100 s
absorption fluorescence phosphorescence
T1
S2
inner conversion
intersystem
conversion
S1
Vibration relaxation
EnergieE=hf
Formation of the absorption =
excitation spectrum
15Výukový materiál společnosti Invitrogen
Formation of the emission
spectrum
16
Výukový materiál společnosti Invitrogen
The dependence of the emission
spectrum on the excitation light
17
Výukový materiál společnosti Invitrogen
18
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Stokes shift
The emitted light has always lower energy
(longer wavelength) than the energy of the
absorbed light (smaller λ).
Difference between absorption maximum
and fluorescence emission maximum of
the spectrum is given by specific
characteristic of the fluorophore.
Formation of Stokes shift
20
Educational material of Invitrogen
21
Stokes law
The wavelength of the emitted light is greater
than or equal to the wavelength of the
excitation light
λem ≥ λex
This is due to the fact that after
the light absorption, a partial loss
of energy (heat) often occurs
during transition from higher
excited electron states to the
lowest excited metastable state.
λEx
520 nm
λEm
560 nm
22
Stokes shift
Emission has always smaller energy
(longer wavelength) than is absorbed
energy (smaller λ).
Difference between absorption maximum
and fluorescence emission maximum of
the spectrum is given by specific
characteristic of the fluorophore.
23
Experiment G. G. Stokese
Sun
Blue glass
windows in the church
Transmits light with
λ < 400 nm
Excitation filtr
Quinine
solution
Glass of wine
Transmits light with
λ > 400 nm
Emission filtr
G.G.
Stokes
1852, Cambridge
24
After filtr exchange – fluorescence
disappears
After filter exchange, ie. if we put a glass of wine in the path of the sun's rays,
transmitted light can no longer excite the solution of quinine.
25
Colorful animated introduction
to the principle of fluorescence
http://probes.invitrogen.com/resources/educ
ation/tutorials/1Intro/player.html
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Typical fluorophores
Fluorophores or fluoreoscent dyes are molecules, that
emit fluorescence. Fluorescence is exhibited especially by
aromatic compounds (polyaromatic hydrocarbons or
heterocycles)..
Typical flourophores are for example:
•quinine (tonic)
•fluorescein, rhodamine B (antifreeze, fluorescent labeling)
•POPOP (scintillators)
•Acridine orange, ethidium bromide (DNA)
•umbelliferone (ELISA)
•anthracene, perylene (environmental pollution by oils)
27
The use of fluorescence
in geography
28J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,Springer, 2006
29
Quantum yield
Quantum yield Q is the ratio of emitted and
absorbed photons.
Indicates the efficiency with which photons excite
fluorescence.
Quantum yield can be up 1.
In fact, it is lower thanks to the non-radiative
transitions of molecules from the excited state.
Rhodamine flourophores (~1) and fluorescein (0.95)
has the highest quantum yields
http://www.iss.com/resources/reference/data_tables/FL_QuantumYieldStandards.html
Reduction of the quantum yield with temperature-thermal
quenching of luminescence – is characteristic
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Excitation spectrum
The dependence of fluorescence intensity
on the excitation wavelength at the
constant wavelength of the emitted light
λEm= const.λEx scan
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Emission spectrum
The dependence of fluorescence intensity on
the wavelength at the constant excitation
wavelength
λEx= const. λEm scan
Spectraviewer
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Unchanged shape of the emission
spectrum
The shape of the emission spectrum is
independent of the excitation
wavelength.
This phenomenon is due to the fact that
the duration of the excited state and the
quantum yield of the complex molecules in
solution does not depend on the
wavelength of the excitation light
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The shape of the emission spectrum is
unchanged at different excitation light
http://probes.invitrogen.com/resources/educ
ation/tutorials/2Spectra/player.html
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Mirror symmetry of absorption and
excitation spectrum
Energy
0 2
0 2
0
1
2
0
1
2
Distance
Abs Emis.
0 2
0 10 1
0 2
0 0
Wavelength λ
Ground
state
Excit.
state
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The law of mirror symmetry between
absorption and emission spectrum
Structure of vibration levels is the same in
ground and excited state, therefore the
absorption and emission of corresponding
vibration levels may occur with equal probability.
This results in a mirror symmetry of absorption
spectrum and fluorescence emission spectrum.
Practically: at very low concentrations of the sample, we can
determine the shape of absorption spectrum from flourescent
emission spectrum without using the amount of the samples higher
in several orders of magnitude.
250 300 350 400 450 500 550
-200
0
200
400
600
800
1000
1200
1400
1600
1800 Excitation
Emission
FluorescenceIntensity
λ
Quinine Solution
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Fluorescent excitation and emission
spectrum of the real solution
Mirror symmetry distorts during measurements of real samples due
to fluorophore ionization at different pH, fluorophore complexation
with other molecules in solution, or by simple contribution of other
non-fluorescent molecules to the absorption (excitation) spectrum.
37
Next:
• What is needed to be able to measure the
spectrum of the fluorophore?
• How can we detect fluorescent molecules
in the gel?