Jan Krieger < j.krieger@dkfz.de
>

1 Absorption and Fluorescence Spectroscopy
1.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2
Absorption and the Lambert-Beer law . . . . . . . . . . . . . . . . . . . . . . .
1.3
Fluorescence
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.2
Fluorescence spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.3
Environmental sensitivity of fluorescence . . . . . . . . . . . . . . . . .
1.3.4
Determination of fluorescence quantum yield . . . . . . . . . . . . . . .
A Preparatory tasks
B Tasks during practical course
10
C Useful Data
11
C.1 Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
C.2 Unit conversions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
C.3 Material properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
C.3.1
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
C.3.2
Sucrose Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
C.4 Electromagnetic Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
C.5 Fluorophore data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
C.5.1
Alexa-488 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
C.5.2
Alexa-594 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
C.5.3
Enhanced green fluorescing protein (EGFP) . . . . . . . . . . . . . . . .
17
C.5.4
Rhodamine 6G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
4
5
5
3
3
6
7
8
9
2

The first part of this practical course cover basic absorption and fluorescence spectroscopy, which may be used to quantify different sample properties, such as concentrations and photophysical properties of the dyes. The basic processes that we have to acquaint us with are absorption of photons by dye molecules and the subsequent emission of fluorescence photons.
Fig. 1.1: Absorption of an incident photon and emission of a fluorescence photon in a simplified fluorophore electronic state system.
Figure 1.1 gives an overview of these processes. In a simplified picture, the fluorophore is
described by two electronic states which are separated by an energy gap ∆ E .
• Absorption: When an incident photon hits a dye molecule in its ground state, the dye may be brought into its excited state. The photon is absorbed during this process, as its energy is used to excite the dye. This process only takes place if the photon energy equals the energy gap between the ground and excited state: E photon
=
∆
E .
• Fluorescence: If an excited dye molecule returns to its ground state the energy
∆
E
0 has to be deposited somewhere. One possible process is the emission of a (fluorescence) photon, carrying the energy
∆
E
0
. Although there are other possibilities to deposit
∆
E
0
, there is a class of dyes where fluorescence is the dominant path.
The next sections dig deeper in the theory of these two processes and explain the theoretical and experimental background, needed for this practical course.
3

The Lambert-Beer law describes the effect of the absorption process, when light passes through some material. It connects the expected decrease in transmitted light (absorbance) with the
properties of the material. Figure 1.2 shows a basic setup for absorption measurements using the
Lambert-Beer law.
Fig. 1.2: Setup of an Absorption Measurement
The law may be written in terms of the absorption A which is defined as the logarithmic relative decrease of intensity:
A : = log
10
I
0
I
(1.2.1) where I
0 and I are the intensities before and after the sample. The absorption is also called optical density (OD), so if a solution in a cuvette has OD = 1, this states that only 10% of the light pass
(i.e. 90% are absorbed).
Lambert-Beer’s law states that:
A =
ε
(
λ
) · L · c (1.2.2) where c is the sample concentration and L is the optical path length. The wavelength dependent coefficient
ε
(
λ
) is called molar absorptivity and is given in units of M
− 1 cm
− 1
. Typical values
for the molar absorptivity are (see also appendix C.5):
• Rhodamine 6G: ε
( 529 .
75 nm ) = 116000 M
− 1 cm
− 1
• Alexa 488: ε
( 493 nm ) = 73000 M
− 1 cm
− 1
Using (1.2.1) one can obtain the concentration of a sample solution by measuring
I and I
0 for a given path length L and absorptivity
ε
(
λ
) . If
ε
(
λ
) is not known it can be obtained by plotting the absorption A against a series of concentrations c , 2 c , 3 c ...
the resulting linear graph has a slope of
L ·
ε
(
λ
) .
The absorptivity may also be used to identify different components in the sample, such as
DNA, proteins or dyes. This is usually done in an absorption spectrometer (depicted in Fig. 1.3).
Which measures I
0 in a reference sample to get rid of any influence by the solvent. Thus the measurement of the absorption is absolute, independent of the spectrometer, being the comparison of two measured intensities.

Fig. 1.3: absorption spectrometer
1.3.1
Introduction
Fluorescence is the result of a three-stage process in the electron shell of certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. This process
is illustrated in the simplified electronic state diagram (Jablonski diagram) in Fig. 1.4.
Fig. 1.4: Jablonski diagram and spectra, illustrating the processes involved on the creation of an excited state by optical absorption and subsequent emission of fluorescence.
The three processes involved in fluorescence are:
1.
Excitation: A photon of energy h
ν ex is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore. The energy is used to push an electron from a ground state (S
0
) niveau to an excited state (S
1
) niveau. The absorption occurs in about 1 fs = 10
− 15 s.

2.
Non-radiating transitions: The electron spends a finite time (typically 1 − 10 ns) in the excited state. During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment (collisions
...). These processes have two important consequences. First the energy of the initial S
1 sub state is partially dissipated, yielding a relaxed singlet excited state from which fluorescence emission originates. Second, not all the molecules initially excited by absorption return to the ground state by fluorescence. Other processes such as collisional quenching, fluorescence energy transfer and intersystem crossing may also depopulate S
1 without emitting a photon.
3.
Fluorescence emission: Finally with a certain probability (see discussion above) a photon of energy h
ν fl is emitted, returning the fluorophore to its ground state S
0
. Due to energy dissipation during the excited state lifetime (non-radiative relaxation), the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon h ν ex
. The energy difference is related to the Stokes shift, which is the wavelength difference between the absorption and emission maximum:
∆ λ Stokes
=
λ max. emission
−
λ max. absorbtion
.
1.3.2
Fluorescence spectra
The entire fluorescence process is cyclic. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon called photobleaching), the same fluorophore can be repeatedly excited and detected.
and
For polyaromatic molecules in solution, the discrete electronic transitions represented by h
ν ex h ν fl are replaced by rather broad energy spectra called fluorescence excitation spectrum and
fluorescence emission spectrum, respectively (see Fig. 1.5). The widths of these spectra are of
particular importance for applications in which two or more different fluorophores are detected simultaneously.
Fig. 1.5: Excitation and emission cpectra of a fluorophore. The emission spectra are plotted for excitation at three different wavelengths (EX1, EX2, EX3).
With few exceptions, the fluorescence excitation spectrum of a single fluorescent species in dilute solution is identical to its absorption spectrum. Under the same conditions, the form of the fluorescence emission spectrum is independent of the excitation wavelength. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength
(see Fig. 1.5).

Fig. 1.6: Principle of a fluorescence spectrometer
Fluorescence spectra can be measured in a fluorescence spectrometer (see Fig. 1.6) which
consists of a monochromized excitation light source (like the absorption spectrometer in Fig.
1.3) and a detection channel, also with a monochromator. The detection channel is arranged
perpendicularly to the excitation to suppress as much detection of the emission light as possible.
Such a spectrometer may be used in two modes:
1.
detect emission spectra: Here the excitation wavelength is fixed and the detection wavelength is scanned over a given range. The results is the emission spectrum of the sample.
2.
detect excitation spectra: Here the detection wavelength is fixed to the emission maximum and the excitation wavelength is scanned. The result is an excitation spectrum of the sample.
When using fluorimeters, the characteristics of the lamp, monochromators and detectors are all wavelength dependent, so the measured spectra must be corrected for these parameters. For correction of the different incoming light intensity, we use a solution of rhodamin dye, which due to its wavelength independent quantum yield (300-600nm) transforms incoming photons of different wavelength with the same probability into an emitted photon. To correct for the other instrument dependent factors, a spectrum has been registered placing a calibrated emitter into the sample box. Fluorescence spectra obtained on two different fluorimeters may be compared only after such corrections.
1.3.3
Environmental sensitivity of fluorescence
Fluorescence spectra and quantum yields are generally more dependent on the environment than absorption spectra and extinction coefficient. The most important factors that influence fluorescence properties are:
• solvent polarity (solvent in this context includes interior regions of cells, proteins, membranes and other biomolecular structures)
• proximity and concentration of quenching species
• pH of the aqueous medium
• temperature
Due to the possibility of reabsorption of an emitted fluorescence photon by neighboring dyes, the linear dependence of the fluorescence intensity on the dye concentration is limited to dilute solutions.

Fluorescence spectra may be strongly dependent on the solvent. Representative fluorophores include the aminonaphtalenes such as prodan and dansyl, which are effective probes of environmental polarity in, for example, protein’s interior. Also, one has to consider that binding of a fluorophore to a target can dramatically affect its quantum yield. Newly developed fluorophores (e.g. Alexa dyes, Atto dyes) are designed to be independent of the solvent pH over a wider range.
Extrinsic quenchers, the most ubiquitous of which are paramagnetic species such as oxygen and heavy ions such as iodide, reduce fluorescence quantum yields in a concentration dependent manner. If quenching is caused be collisional interactions, as it usually is the case, information on the proximity of the fluorophore and quencher and their mutual diffusion rate can be derived.
1.3.4
Determination of fluorescence quantum yield
The wavelength-dependent fluorescence quantum yield
φ fl
(
λ
) is defined in terms of the number of absorbed photons N abs and the number of emitted photons N em
:
φ fl
(
λ
) =
N em
N abs
(
λ
)
.
(1.3.1)
As mentioned above it quantifies the relation between radiating and non-radiating decays of the excited state in fluorescence.
While the oldest and most fundamental methods for calculating the quantum yield are based on measuring the absolute luminescence, these methods are difficult and require great precision.
Certain fluorophores have been established as standards with well accepted quantum yields, fluorescein is the most common among them. Quantum yields of new compounds are calculated by comparing emission rates to those of the known standards following the equation:
φ sample
φ reference
=
N em,sample
N abs,sample
N em,reference
N abs,reference
N em,sample
=
N em,reference
·
N abs,reference
N abs,sample
I em,sample
=
I em,reference
·
A abs,reference
A abs,sample
(1.3.2) where the I s denote the measured fluorescence intensity and the A s are the measured absorbances.
Note that the last equality is only valid, if all the intensities and absorbances were measured at the same excitation/detection wavelengths.
You may also want to take a look at this document: http://www.nanoco.biz/download.
aspx?ID=77 .

1. In Fig. A.1 below you see an eppendorf tube containing a rather concentrated solution
of EGFP. You find the absorption and emission spectrum in section C.5.3. Explain the
yellow-green color of the solution and the yellow color of the transmitted daylight.
Fig. A.1: EGFP solution illuminated by day light
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• concentration determination
• comparison of absorption and excitation spectra
• demonstration of the inner filter effect
• demonstration of the dependence of quantum yield on environmental factors
• FRET measurements of DNA and nucleosome samples (in bulk) in a spectrofluorimeter and single-particle measurements in a confocal microscope
10

• Boltzman’s constant: k
B
= 1 .
38 · 10
− 23
J / K
• Avogadro’s number: N
A
= 6 .
022 · 10
23 mol
− 1
• 1 Pa = 1 kg / ( m · s
2 ) = 1 N / m
2
• 1 J = 1 kg · m
2
/ s
2
• 1 l = 1 dm
3
, 1 fl = 10
− 15 l = 1
µ m
3
11

C.3.1
Water
• refractive index (
ϑ
= 20
◦
C ,
λ
= 589 .
29 nm): n = 1 .
3330
Fig. C.1: Viscosity of water

C.3.2
Sucrose Solution
• Sucrose, molecular formula: C
12
H
22
O
11
• Sucrose, molar mass: 342 .
30 g / mol
This table shows the refractive index n and the dynamic viscosity η for a sucrose solution. All data
was taken from [3, 4].
9.0
10.0
16.0
20.0
26.0
30.0
40.0
50.0
60.0
70.0
3.0
4.0
5.0
6.0
7.0
8.0
mass %
0.5
1.0
2.0
n
η
( 20
◦
C ) [ mPa · s ]
η
( 30
◦
C )
η
( 40
◦
C )
1.3337
1.015
1.3344
1.028
1.3359
1.055
1.3373
1.3388
1.3403
1.084
1.114
1.146
1.3418
1.179
1.3433
1.215
1.3448
1.254
1.3463
1.294
1.3478
1.336
1.3573
1.653
1.3639
1.945
1.3741
2.573
1.3812
3.187
1.3999
1.4201
1.4419
1.4654
6.162
15.431
58.487
481.561
1.49
2.37
4.37
10.1
33.8
222
1.18
1.83
2.34
6.99
21.0
114
Fig. C.2: Viscosity η and refractive index n of sucrose solution at 20
◦
C

Fig. C.3: The electromagnetic spectrum, together with the emission and absorption maximum of some important fluorescence dyes, as well as often used laser lines.

C.5.1
Alexa-488
• max. excitation wavelength:
λ ex,max
= 494 nm
• max. emission wavelength:
λ em,max
= 517 nm
• molecular weight: 643 .
41 Da
• molar extinction:
ε
( 493 nm ) = 73000 M
− 1 cm
− 1
• fluorescence lifetime (20
◦
C , pH = 7 .
4):
τ fl
= 4 .
1 ns
• quantum yield (50 mM potassium phosphate, 150 mM NaCl pH=7 .
2 at 22
◦
C):
φ fl
= 92%
• diffusion coefficients in water:
D ( 22 .
5
◦
C ) = 435
µ m
2
/
s [2],
D ( 25
◦
C ) = 465
µ m
2
/ s, D ( 37
◦
C ) = 624
µ m
2
/ s
If not state otherwise data was taken from invitrogen.

C.5.2
Alexa-594
1
0.8
0.6
0.4
0.2
0
300 400 500 wavelength λ [nm]
600
• max. excitation wavelength:
λ ex,max
= 590 nm
• max. emission wavelength:
λ em,max
= 617 nm
• molecular weight: 820 Da
• molar extinction:
ε
( 493 nm ) = 90000 M
− 1 cm
− 1
• quantum yield:
φ fl
= 66%
If not state otherwise data was taken from invitrogen.
abs: Alexa594 fl: Alexa594
700

C.5.3
Enhanced green fluorescing protein (EGFP)
• max. excitation wavelength:
λ ex,max
= 489 nm
• max. emission wavelength:
λ em,max
= 508 nm
• molecular weight: ≈ 26 .
9 kDa
• molar extinction:
ε
( 489 nm ) = 55000 M
− 1 cm
− 1
• quantum yield:
φ fl
= 60%
• diffusion coefficient in 100 mM phosphate-citrate buffer (pH=7 .
5): D ( 22 .
5
[2],
D ( 25
◦
C ) = 102
µ m
2
/ s, D ( 37
◦
C ) = 136
µ m
2
/ s
◦
C ) = 95
µ m
2
/ s
If not state otherwise data was taken from [1].

C.5.4
Rhodamine 6G
Note, the given spectrum was taken in ethanol.
• max. excitation wavelength:
λ ex,max
= 529 .
75 nm
• max. emission wavelength:
λ em,max
= 555 nm
• molecular weight: 479 .
02 Da
• molar extinction: ε
( 529 .
75 nm ) = 116000 M
− 1 cm
− 1
• quantum yield:
φ fl
( 300 ...
600 nm , EtOH ) = 0 .
9%
• diffusion coefficient in water: D ( 22 .
5
◦
C ) = 426
µ m
2
/
s [2]
If not state otherwise data was taken from http://omlc.ogi.edu/spectra/PhotochemCAD/ html/rhodamine6G.html
or http://omlc.ogi.edu/spectra/PhotochemCAD/html/ rhodamine6G.html
.

[1] G. Patterson, R. Day, and D. Piston. Fluorescent protein spectra.
J Cell Sci , 114(5):837–838,
2001.
[2] Z. Petrásek and P. Schwille.
Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy.
Biophysical Journal , 94:1437–
1448, 2008.
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=
2212689&blobtype=pdf .
[3] P. Reiser.
Sucrose: properties and applications . Springer Netherlands, 1995.
[4] R. Weast, M. Astle, and W. Beyer.
CRC handbook of chemistry and physics , volume 69. CRC press Boca Raton, FL, 1988.
19