Practical Course Biophysics:

Absorption and Fluorescence Spectroscopy

Katalin Tóth <kt@dkfz.de>Jörg Langowski <jl@dkfz.de>

Jan Krieger <j.krieger@dkfz.de>

Contents

1 Absorption and Fluorescence Spectroscopy 31.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 Absorption and the Lambert-Beer law . . . . . . . . . . . . . . . . . . . . . . . 41.3 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.3.2 Fluorescence spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.3.3 Environmental sensitivity of fluorescence . . . . . . . . . . . . . . . . . 71.3.4 Determination of fluorescence quantum yield . . . . . . . . . . . . . . . 8

A Preparatory tasks 9

B Tasks during practical course 10

C Useful Data 11C.1 Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11C.2 Unit conversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11C.3 Material properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

C.3.1 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12C.3.2 Sucrose Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

C.4 Electromagnetic Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14C.5 Fluorophore data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

C.5.1 Alexa-488 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15C.5.2 Alexa-594 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16C.5.3 Enhanced green fluorescing protein (EGFP) . . . . . . . . . . . . . . . . 17C.5.4 Rhodamine 6G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Chapter 1

Absorption and FluorescenceSpectroscopy

1.1 Introduction

The first part of this practical course cover basic absorption and fluorescence spectroscopy, whichmay be used to quantify different sample properties, such as concentrations and photophysicalproperties of the dyes. The basic processes that we have to acquaint us with are absorption ofphotons 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 simplifiedfluorophore electronic state system.

Figure 1.1 gives an overview of these processes. In a simplified picture, the fluorophore isdescribed 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 maybe brought into its excited state. The photon is absorbed during this process, as its energy isused to excite the dye. This process only takes place if the photon energy equals the energygap between the ground and excited state: Ephoton = ∆E.

• Fluorescence: If an excited dye molecule returns to its ground state the energy ∆E ′ has tobe deposited somewhere. One possible process is the emission of a (fluorescence) photon,carrying the energy ∆E ′. Although there are other possibilities to deposit ∆E ′, there is a classof dyes where fluorescence is the dominant path.

The next sections dig deeper in the theory of these two processes and explain the theoreticaland experimental background, needed for this practical course.

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1.2 Absorption and the Lambert-Beer law

The Lambert-Beer law describes the effect of the absorption process, when light passes throughsome material. It connects the expected decrease in transmitted light (absorbance) with theproperties of the material. Figure 1.2 shows a basic setup for absorption measurements using theLambert-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 logarithmicrelative decrease of intensity:

A := log10

(I0

I

)(1.2.1)

where I0 and I are the intensities before and after the sample. The absorption is also called opticaldensity (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 dependentcoefficient ε(λ ) is called molar absorptivity and is given in units of M−1cm−1. Typical valuesfor the molar absorptivity are (see also appendix C.5):

• Rhodamine 6G: ε(529.75nm) = 116000M−1cm−1

• Alexa 488: ε(493nm) = 73000M−1cm−1

Using (1.2.1) one can obtain the concentration of a sample solution by measuring I and I0 for agiven path length L and absorptivity ε(λ ). If ε(λ ) is not known it can be obtained by plotting theabsorption A against a series of concentrations c,2c,3c... the resulting linear graph has a slope ofL · ε(λ ).

The absorptivity may also be used to identify different components in the sample, such asDNA, 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 themeasurement of the absorption is absolute, independent of the spectrometer, being the comparisonof two measured intensities.

Fig. 1.3: absorption spectrometer

1.3 Fluorescence

1.3.1 Introduction

Fluorescence is the result of a three-stage process in the electron shell of certain molecules (generallypolyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. This processis 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 anexcited 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 incandes-cent lamp or a laser and absorbed by the fluorophore. The energy is used to push an electronfrom a ground state (S0) niveau to an excited state (S1) niveau. The absorption occurs inabout 1fs = 10−15 s.

2. Non-radiating transitions: The electron spends a finite time (typically 1− 10ns) in theexcited state. During this time, the fluorophore undergoes conformational changes and isalso 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 substate is partially dissipated, yielding a relaxed singlet excited state from which fluorescenceemission originates. Second, not all the molecules initially excited by absorption return tothe ground state by fluorescence. Other processes such as collisional quenching, fluorescenceenergy transfer and intersystem crossing may also depopulate S1 without emitting a photon.

3. Fluorescence emission: Finally with a certain probability (see discussion above) a photonof energy hνfl is emitted, returning the fluorophore to its ground state S0. Due to energydissipation during the excited state lifetime (non-radiative relaxation), the energy of thisphoton is lower, and therefore of longer wavelength, than the excitation photon hνex. Theenergy difference is related to the Stokes shift, which is the wavelength difference betweenthe 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 theexcited state (an important phenomenon called photobleaching), the same fluorophore can berepeatedly excited and detected.

For polyaromatic molecules in solution, the discrete electronic transitions represented by hνexand hνfl are replaced by rather broad energy spectra called fluorescence excitation spectrum andfluorescence emission spectrum, respectively (see Fig. 1.5). The widths of these spectra are ofparticular importance for applications in which two or more different fluorophores are detectedsimultaneously.

Fig. 1.5: Excitation and emission cpectra of a fluorophore. The emission spectra are plotted forexcitation at three different wavelengths (EX1, EX2, EX3).

With few exceptions, the fluorescence excitation spectrum of a single fluorescent species indilute solution is identical to its absorption spectrum. Under the same conditions, the form of thefluorescence emission spectrum is independent of the excitation wavelength. The emission intensityis 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) whichconsists 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 arrangedperpendicularly 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 wavelengthis 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 maximumand 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 areall wavelength dependent, so the measured spectra must be corrected for these parameters. Forcorrection of the different incoming light intensity, we use a solution of rhodamin dye, which due toits wavelength independent quantum yield (300-600nm) transforms incoming photons of differentwavelength with the same probability into an emitted photon. To correct for the other instrumentdependent factors, a spectrum has been registered placing a calibrated emitter into the samplebox. Fluorescence spectra obtained on two different fluorimeters may be compared only after suchcorrections.

1.3.3 Environmental sensitivity of fluorescence

Fluorescence spectra and quantum yields are generally more dependent on the environment thanabsorption spectra and extinction coefficient. The most important factors that influence fluorescenceproperties are:

• solvent polarity (solvent in this context includes interior regions of cells, proteins, membranesand 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 dilutesolutions.

Fluorescence spectra may be strongly dependent on the solvent. Representative fluorophores in-clude the aminonaphtalenes such as prodan and dansyl, which are effective probes of environmentalpolarity in, for example, protein’s interior. Also, one has to consider that binding of a fluorophoreto a target can dramatically affect its quantum yield. Newly developed fluorophores (e.g. Alexadyes, 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 oxygenand heavy ions such as iodide, reduce fluorescence quantum yields in a concentration dependentmanner. If quenching is caused be collisional interactions, as it usually is the case, information onthe 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 ofabsorbed 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 theexcited state in fluorescence.

While the oldest and most fundamental methods for calculating the quantum yield are basedon measuring the absolute luminescence, these methods are difficult and require great precision.Certain fluorophores have been established as standards with well accepted quantum yields, fluo-rescein is the most common among them. Quantum yields of new compounds are calculated bycomparing emission rates to those of the known standards following the equation:

φsample

φreference=

Nem,sampleNabs,sample

Nem,referenceNabs,reference

=Nem,sample

Nem,reference· Nabs,reference

Nabs,sample=

Iem,sample

Iem,reference· Aabs,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 thesame 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 solutionof EGFP. You find the absorption and emission spectrum in section C.5.3. Explain theyellow-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 andsingle-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

• 1Pa = 1kg/(m · s2) = 1N/m2

• 1J = 1kg ·m2/s2

• 1l = 1dm3, 1fl = 10−15 l = 1 µm3

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C.3 Material properties

C.3.1 Water

• refractive index (ϑ = 20 ◦C,λ = 589.29nm): n = 1.3330

Fig. C.1: Viscosity of water

C.3.2 Sucrose Solution

• Sucrose, molecular formula: C12H22O11

• Sucrose, molar mass: 342.30g/mol

This table shows the refractive index n and the dynamic viscosity η for a sucrose solution. All datawas taken from [3, 4].

mass % n η(20 ◦C) [mPa · s] η(30 ◦C) η(40 ◦C)0.5 1.3337 1.0151.0 1.3344 1.0282.0 1.3359 1.0553.0 1.3373 1.0844.0 1.3388 1.1145.0 1.3403 1.1466.0 1.3418 1.1797.0 1.3433 1.2158.0 1.3448 1.2549.0 1.3463 1.29410.0 1.3478 1.33616.0 1.3573 1.65320.0 1.3639 1.945 1.49 1.1826.0 1.3741 2.57330.0 1.3812 3.187 2.37 1.8340.0 1.3999 6.162 4.37 2.3450.0 1.4201 15.431 10.1 6.9960.0 1.4419 58.487 33.8 21.070.0 1.4654 481.561 222 114

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 ofsome important fluorescence dyes, as well as often used laser lines.

C.5 Fluorophore data

C.5.1 Alexa-488

• max. excitation wavelength: λex,max = 494nm

• max. emission wavelength: λem,max = 517nm

• molecular weight: 643.41Da

• molar extinction: ε(493nm) = 73000M−1cm−1

• fluorescence lifetime (20 ◦C,pH = 7.4): τfl = 4.1ns

• quantum yield (50mM potassium phosphate, 150mM 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

300 400 500 600 700

wavelength λ [nm]

0

0.2

0.4

0.6

0.8

1

rela

tive

inte

nsi

ty/a

bso

rbtio

n

[0..1]

abs: Alexa594fl: Alexa594

• max. excitation wavelength: λex,max = 590nm

• max. emission wavelength: λem,max = 617nm

• molecular weight: 820Da

• molar extinction: ε(493nm) = 90000M−1cm−1

• quantum yield: φfl = 66%

If not state otherwise data was taken from invitrogen.

C.5.3 Enhanced green fluorescing protein (EGFP)

• max. excitation wavelength: λex,max = 489nm

• max. emission wavelength: λem,max = 508nm

• molecular weight: ≈ 26.9kDa

• molar extinction: ε(489nm) = 55000M−1cm−1

• quantum yield: φfl = 60%

• diffusion coefficient in 100mM 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 = 529.75nm

• max. emission wavelength: λem,max = 555nm

• molecular weight: 479.02Da

• molar extinction: ε(529.75nm) = 116000M−1cm−1

• quantum yield: φfl(300...600nm,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 us-ing 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. CRCpress Boca Raton, FL, 1988.

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