Practical Course Biophysics:
Absorption and Fluorescence Spectroscopy
Katalin Tóth <[email protected]>
Jörg Langowski <[email protected]>
Jan Krieger <[email protected]>
Contents
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
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A Preparatory tasks
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B Tasks during practical course
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C Useful Data
C.1 Constants . . . . . . . . . . . . . . . . . . . . . .
C.2 Unit conversions . . . . . . . . . . . . . . . . . .
C.3 Material properties . . . . . . . . . . . . . . . . .
C.3.1 Water . . . . . . . . . . . . . . . . . . . .
C.3.2 Sucrose Solution . . . . . . . . . . . . . .
C.4 Electromagnetic Spectrum . . . . . . . . . . . . .
C.5 Fluorophore data . . . . . . . . . . . . . . . . . .
C.5.1 Alexa-488 . . . . . . . . . . . . . . . . . .
C.5.2 Alexa-594 . . . . . . . . . . . . . . . . . .
C.5.3 Enhanced green fluorescing protein (EGFP)
C.5.4 Rhodamine 6G . . . . . . . . . . . . . . .
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Chapter 1
Absorption and Fluorescence
Spectroscopy
1.1
Introduction
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: Ephoton = ∆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
1.2
Absorption and the Lambert-Beer law
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:
I0
A := log10
(1.2.1)
I
where I0 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 I0 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, 2c, 3c... 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 I0 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
1.3.1
Fluorescence
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 hνex 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 (S0 ) niveau to an excited state (S1 ) 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 S1 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 S1 without emitting a photon.
3. Fluorescence emission: Finally with a certain probability (see discussion above) a photon
of energy hνfl is emitted, returning the fluorophore to its ground state S0 . 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 hνex . The
energy difference is related to the Stokes shift, which is the wavelength difference between
the absorption and emission maximum:
∆λStokes = λmax. emission − λmax. 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.
For polyaromatic molecules in solution, the discrete electronic transitions represented by hνex
and hνfl 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 φfl (λ ) is defined in terms of the number of
absorbed photons Nabs and the number of emitted photons Nem :
φfl (λ ) =
Nem
.
Nabs (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:
φsample
=
φreference
Nem,sample
Nabs,sample
Nem,reference
Nabs,reference
=
Nem,sample Nabs,reference
Iem,sample Aabs,reference
·
=
·
Nem,reference Nabs,sample
Iem,reference Aabs,sample
(1.3.2)
where the Is denote the measured fluorescence intensity and the As 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.
Appendix A
Preparatory tasks
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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Appendix B
Tasks during practical course
• 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
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Appendix C
Useful Data
C.1
Constants
• Boltzman’s constant: kB = 1.38 · 10−23 J/K
• Avogadro’s number: NA = 6.022 · 1023 mol−1
C.2
Unit conversions
• 1 Pa = 1 kg/(m · s2 ) = 1 N/m2
• 1 J = 1 kg · m2 /s2
• 1 l = 1 dm3 ,
1 fl = 10−15 l = 1 µm3
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C.3
Material properties
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: C12 H22 O11
• 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].
mass %
0.5
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
16.0
20.0
26.0
30.0
40.0
50.0
60.0
70.0
n
1.3337
1.3344
1.3359
1.3373
1.3388
1.3403
1.3418
1.3433
1.3448
1.3463
1.3478
1.3573
1.3639
1.3741
1.3812
1.3999
1.4201
1.4419
1.4654
η(20 ◦ C) [mPa · s]
1.015
1.028
1.055
1.084
1.114
1.146
1.179
1.215
1.254
1.294
1.336
1.653
1.945
2.573
3.187
6.162
15.431
58.487
481.561
η(30 ◦ C)
η(40 ◦ C)
1.49
1.18
2.37
4.37
10.1
33.8
222
1.83
2.34
6.99
21.0
114
Fig. C.2: Viscosity η and refractive index n of sucrose solution at 20 ◦ C
C.4
Electromagnetic Spectrum
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
Fluorophore data
C.5.1
Alexa-488
• max. excitation wavelength: λex,max = 494 nm
• max. emission wavelength: λem,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): τfl = 4.1 ns
• quantum yield (50 mM potassium phosphate, 150 mM NaCl pH=7.2 at 22 ◦ C): φfl = 92%
• diffusion coefficients in water:
D(22.5 ◦ C) = 435 µm2 /s [2], D(25 ◦ C) = 465 µm2 /s, D(37 ◦ C) = 624 µm2 /s
If not state otherwise data was taken from invitrogen.
C.5.2
Alexa-594
relative intensity/absorbtion
[0..1]
1
abs: Alexa594
fl: Alexa594
0.8
0.6
0.4
0.2
0
300
400
500
wavelength λ [nm]
• max. excitation wavelength: λex,max = 590 nm
• max. emission wavelength: λem,max = 617 nm
• molecular weight: 820 Da
• molar extinction: ε(493 nm) = 90000 M−1 cm−1
• quantum yield: φfl = 66%
If not state otherwise data was taken from invitrogen.
600
700
C.5.3
Enhanced green fluorescing protein (EGFP)
• max. excitation wavelength: λex,max = 489 nm
• max. emission wavelength: λem,max = 508 nm
• molecular weight: ≈ 26.9 kDa
• molar extinction: ε(489 nm) = 55000 M−1 cm−1
• quantum yield: φfl = 60%
• diffusion coefficient in 100 mM phosphate-citrate buffer (pH=7.5): D(22.5 ◦ C) = 95 µm2 /s
[2], D(25 ◦ C) = 102 µm2 /s, D(37 ◦ C) = 136 µm2 /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: λex,max = 529.75 nm
• max. emission wavelength: λem,max = 555 nm
• molecular weight: 479.02 Da
• molar extinction: ε(529.75 nm) = 116000 M−1 cm−1
• quantum yield: φfl (300...600 nm, EtOH) = 0.9%
• diffusion coefficient in water: D(22.5 ◦ C) = 426 µm2 /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.
References
[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.
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