Antonie J.W.G. Visser1,2 and Olaf J. Rolinski2

1Laboratory of Biochemistry, Microspectroscopy CentreWageningen University, P.O. Box 81286700 ET Wageningen, The Netherlands

antoniejvisser@gmail.com

2Department of Physics, University of StrathclydeScottish Universities Physics Alliance, Photophysics Group

Glasgow G4 0NG, UKo.j.rolinski@strath.ac.uk

Introduction

Basic photophysics in the framework of photobiology is concerned withprocesses that occur when sunlight, filtered through the Earth'satmosphere, interacts with matter (atoms and molecules) present onEarth. The spectrum of solar radiation striking the Earth (Figure 1) spans100 nm to 106 nanometers (1 nm = 10-9 m) and can be divided into theultraviolet (UV) range (100 nm to 400 nm), visible range (400 nm to 700nm) and infrared (IR) range (700 nm to 106 nm). UV radiation has bothdamaging and beneficial effects on living matter. UV radiation is alsoresponsible for the photochemical reaction leading to production of theprotective ozone layer in the atmosphere. As the name suggests thevisible part of the spectrum is the light that human eyes can detect. Animportant part of electromagnetic radiation reaching the Earth is IRradiation.

Figure 1. Solar radiation spectrum above the atmosphere, andat the surface of the earth. [From Wikipedia: Sunlight]

BASIC PHOTOPHYSICS

1 of 34

The spectrum of solar radiation is close to that of a black body with atemperature of 5600 K. A black body is an idealised object that absorbsall electromagnetic radiation falling on it. Because no light is reflected ortransmitted, the object appears black when it is cold. However, a blackbody emits a temperature-dependent spectrum of light, which is termedblack-body radiation. All hot objects radiate a mixture of heat and light,with intensity and color varying with temperature. Think of an iron pokerin a fire that changes color when it becomes hotter, and after taking itout the glow starts to disappear, as does the heat. Historically, studyingthe laws of black-body radiation has led to the development of quantummechanics.

Visible light is only one small part of the electromagnetic spectrum,which is classified by wavelength into radio wave, microwave, infrared,visible, ultraviolet, X-rays and gamma rays (Figure 2).

Figure 2. Electromagnetic spectrum, with the visible lightspectrum enlarged. [From Wikipedia: ElectromagneticRadiation]

The behaviour of electromagnetic radiation depends on its wavelength orenergy (i.e., frequency). Higher frequencies have shorter wavelengthsand, vice versa, lower frequencies have longer wavelengths. Theinteraction of electromagnetic radiation with atoms or molecules dependson the energy. Spectroscopy can be detected at a much wider range thanthe visible range of 400 nm to 700 nm. Depending on the spectral regionof interest, the units used to describe the radiation may be frequency,energy, wavelength or inverse wavelength (wavenumber). Table 1compares different spectral regions.

BASIC PHOTOPHYSICS

2 of 34

Table 1. Characteristics of the Electromagnetic Spectrum.Wavelength ranges of the electromagnetic spectrum have beenclassified by their spectroscopic uses. For example, very highfrequency (VHF) radiation is used in nuclear magneticresonance (NMR), while ultra high frequency (UHF) radiation isused in electron paramagnetic resonance (EPR). The other rowsof Table 1 provide information on frequency, wavelength, wavenumber and energy for a center wavelength of the spectralrange. The last row gives the energy provided by one mole ofphotons of given wavelength, that is the energy per einstein.

Electromagnetic radiation exhibits both wave-like and particle-likeproperties, a concept known as wave-particle duality. Both wave andparticle characteristics have been demonstrated by a large number ofexperiments. Electromagnetic waves are composed of an electric field(E), and a magnetic field (B) perpendicular to it, oscillating in phase inthe propagation direction (k). An arrow on top of a letter gives thedirection of the vector (Figure 3).

Figure 3. Electromagnetic waves as oscillating electric andmagnetic fields. (-q and +q represent electrical charges)[From Wikipedia: Electromagnetic Radiation]

The sinusoidal waves are characterized by frequency ν, which is inverselyproportional to the wavelength λ according to the equation:

where c is the speed of light (in vacuum c = 300,000 km/s; light travelsin one nanosecond 30 cm; 1 ns =10-9 s). The wave model can explainseveral phenomena. Interference is the superposition of two wavesgiving a new wave pattern that can be constructive when two waveshave the same direction or destructive when they have the oppositedirection. Another example is light dispersion into a visible spectrumwhen white light is shone through a prism, because of the wavelength

BASIC PHOTOPHYSICS

3 of 34

dependent refractive index of the prism material. Electromagnetic wavesalso have particle-like properties, as packets of energy or quanta, calledphotons. The energy per photon, E, can be calculated from the Planck-Einstein equation:

where h is Planck's constant and ν is frequency. The particle model canexplain the absorption and emission spectra of light by atoms ormolecules. The absorption of electromagnetic radiation is how photonenergy is taken up. Typically an electron is elevated to a higher energylevel by light absorption. When an excited electron returns to the lowestenergy level, a light photon is emitted.

Molecular Photophysical Processes Relevant for Photobiology

Molecular photophysical processes relevant for photobiology includeabsorption and emission of UV, visible or near-IR light, by aromaticmolecules. In the remainder of this module we will focus on theseprocesses.

Jablonski Diagram: Basic principles of molecular photophysics can beclarified with the help of the Jablonski diagram, named after the Polishphysicist Aleksander Jablonski (Figure 4).

Figure 4. Jablonski diagram representing energy levels andspectra. Solid arrows indicate radiative transitions as occurringby absorption (violet, blue) or emission (green forfluorescence; red for phosphorescence) of a photon. Dashedarrows represent non-radiative transitions (violet, blue, green,red). Internal conversion is a non-radiative transition, whichoccurs when a vibrational state of a higher electronic state iscoupled to a vibrational state of a lower electronic state. In thenotation of, for example, S1,0, the first subscript refers to theelectronic state (first excited) and the second one to thevibrational sublevel (v = 0). In the diagram the followinginternal conversions are indicated: S2,4→S1,0, S2,2→S1,0,

BASIC PHOTOPHYSICS

4 of 34

S2,0→S1,0 and S1,0→S0,0. The dotted arrow from S1,0→T1,0 is anon-radiative transition called intersystem crossing, because itis a transition between states of different spin multiplicity.Below the diagram sketches of absorption-, fluorescence- andphosphorescence spectra are shown.

The diagram illustrates the electronic states of a molecule and thetransitions between them. The electronic states are arranged verticallyby energy. They are grouped horizontally by spin multiplicity. In the leftpart of the diagram three singlet states with anti-parallel spins areshown: the singlet ground state (S0) and two higher singlet excitedstates (S1 and S2). Singlet states are diamagnetic, as they do notinteract with an external magnetic field. The triplet state (T1) is theelectronic state with parallel spins. A molecule in the triplet stateinteracts with an external magnetic field. Transitions between electronicstates of the same spin multiplicity are allowed. Transitions betweenstates with different spin multiplicity are formally forbidden, but mayoccur owing to a process called spin-orbit coupling. This transition iscalled intersystem crossing. Superimposed on these electronic states arethe vibrational states, which are of much smaller energy. In the followingsections we will address the sequence of processes, which occur when anaromatic molecule absorbs a photon of sufficient energy.

It requires some effort to calculate the energy levels and wavefunctionsof an aromatic molecule of average size. Born and Oppenheimer havemade this task considerably easier by breaking up the quantum-mechanical wavefunction of a molecule into its electronic and nuclear(vibrational and rotational) components:

In the first step of this Born-Oppenheimer approximation the electronicSchrödinger equation is solved yielding the electronic wavefunction,

, depending on electrons only with the nuclei fixed in anequilibrium configuration.

Light Absorption: When a molecule absorbs a photon of appropriateenergy, a valence electron is promoted from the ground state to somevibrational level in the excited singlet manifold (Figure 4). The process oflight absorption is extremely rapid, in the order of one femtosecond (1fs = 10-15 s). It means that the nuclei of the molecule are fixed duringthe transition, because of their much larger mass, and that theBorn-Oppenheimer approximation is valid.

It is to be noted that the point of departure is always the lowestvibrational level of S0 designated S0,0. We can understand this byreferring to the Boltzmann distribution function, which is a measure forthe distribution of (in this case) vibrational states of the molecule:

where N1 is the number of molecules in the first higher vibrational state(S0,1), and N0 that for the lowest vibrational level (S0,0), ∆Evib is theenergy difference between the two vibrational states, kB is the Boltzmann

BASIC PHOTOPHYSICS

5 of 34

constant, and T the absolute temperature. At room temperature, ∆Evib ismuch larger than kBT, implying that only the lowest vibrational level ispopulated.

After light absorption the excited molecule ends up at the lowestvibrational level of S1 (S1,0) via vibrational relaxation and internalconversion. This radiationless process takes place in about onepicosecond (1 ps = 10-12 s).

A sketch of an absorption spectrum consisting of two bands is shown inFigure 4. In the condensed phase, broad absorption (and also emission)bands are observed, not the sharp transitions seen for atoms ormolecules in the gaseous phase. The reason is due to phenomena knownas homogeneous broadening and inhomogeneous broadening.Homogeneous broadening arises from the multitude of vibrational states(many more than shown in Figure 4) and rotational states (superimposedon the vibrational states), which all are superimposed on the electronictransitions preventing the observation of sharp transitions.Inhomogeneous broadening arises from solvent effects, which will bedetailed later for fluorescence.

The strength of the lowest optical transition is very often expressed interms of the (dimensionless) oscillator strength f:

where ε is the molar extinction coefficient connected with the lowestelectronic transition, σ is the wavenumber and the integral is over allwavenumbers of the absorption band. For intense (strongly allowed)transitions f ≈ 1. The oscillator strength has a direct relationship with theelectronic transition dipole μeg, which couples the wavefunctions of theground (ψg) and excited (ψe) electronic states:

with μ=-er (e is charge of electron), and the integration takes place overspatial coordinates r.

The transition dipole is a measure of the dipole moment associated withthe shift of charge that occurs when electrons are redistributed in themolecule upon excitation. The oscillator strength is proportional to themagnitude of the transition dipole:

Fluorescence: In 1852, the British scientist Sir George G. Stokes coinedthe term "fluorescence" after observing blue luminescence in the mineralfluorite. Stokes also discovered the redshift in band maximum of thefluorescence spectrum relative to the band maximum of absorption(Stokes shift).

The lowest vibrational level of S1 is the starting point for fluorescence

BASIC PHOTOPHYSICS

6 of 34

emission to the ground state S0, non-radiative decay to S0 (internalconversion), and transition to the lowest triplet state (intersystemcrossing) (Figure 4). Fluorescence takes place on the nanosecondtimescale (1 ns = 10-9 s) and, depending on the molecular species, itsduration amounts to 1-100 nanoseconds. It is clear from the Jablonskidiagram that fluorescence always originates from the same level,irrespective of which electronic energy level is excited. The emitting stateis the zeroth vibrational level of the first excited state S1,0. It is for thisreason that the fluorescence spectrum is shifted to lower energy than thecorresponding absorption spectrum (Stokes shift). The Stokes shift canbe enhanced by solvent interactions, examples of which will be givenlater. We can also conclude from the sketched spectra in Figure 4 thatvibrational fine structure in a fluorescence spectrum reports aboutvibrations in the ground state, and vibronic bands in an absorptionspectrum provides information on vibrations in higher electronic excitedstates.

Another factor that has to be considered in fluorescence spectroscopy isthe Franck-Condon factor. If we look at the Jablonski scheme in Figure 4,it can be seen that the fluorescence transition S1,0→S0,0 is not the mostintense one. The Franck-Condon principle states that the most intensevibronic transition is from the vibrational state in the ground state to thatvibrational state in the excited state vertically above it (Figure 5, bluearrow).

Figure 5. Energy diagram for explanation of the Franck-Condon principle. The potential wells show favored transitionsbetween vibrational sublevels ν = 0 and v = 2 both forabsorption (blue arrow) and emission (green arrow).[From Wikipedia: Franck-Condon Principle]

The schemes (for absorption and emission) in Figure 5 are simplifiedtwo-dimensional potential energy diagrams, but for the sake ofargument, they are a sufficient representation, since we are dealing with(an)harmonic oscillators. Since the excited state is different from theground state, a displaced minimum nuclear normal coordinate can beexpected. It should be noted further that the time to reach the excited

BASIC PHOTOPHYSICS

7 of 34

state is so short (femtoseconds) that the nuclei positions are virtuallyunchanged during the electronic transition. In the vibronicwavefunctions, the nuclear coordinates can then be uncoupled from theelectronic coordinates (Born-Oppenheimer principle). The transitiondipole can then be factored into an electronic and a nuclear part:

The second integral is the so-called Franck-Condon vibrational overlap,which also determines the strength of the electronic transition (oroscillator strength). In the fluorescent part of the scheme in Figure 4, thesecond and third vibrational transitions (S1,0→S0,1 and S1,0→S0,2) havelarger Franck-Condon factors than the one between fundamentalvibrational wavefunctions (S1,0→S0,0).

Phosphorescence: In Figure 4, the triplet state is also drawn. Once themolecule has reached this state, it will reside for a very long time there(from microseconds to seconds) before it will decay to the ground state.This is due to the spin-forbidden transitions involved in the (excited)singlet-triplet and triplet-singlet (ground state) transitions. In rigidsolution or in deoxygenated solutions, long-lived phosphorescence(milliseconds to seconds) from this state can be observed. The name"phosphorescence" probably originates from the French physicist EdmondBecquerel, who devised in 1857 an instrument that he called aphosphoroscope. With this instrument he could measure how long ittakes a phosphorescent sample to stop glowing after excitation.

Because of its long lifetime, the triplet state of an aromatic molecule isthe starting point for photochemical reactions. One reaction in particularis very prominent, namely the production of very reactive singlet oxygen.The oxygen molecule, O2, possesses a triplet ground state. In solution,frequent collisions between an aromatic molecule in the triplet state andoxygen result in energy transfer, and generation of singlet oxygen, whichcan oxidise (and destroy) the aromatic molecule.

Practical Aspects of Fluorescence

Absorption Spectrum: The measurement of an absorption spectrum(Figure 6) is based on the Lambert-Beer law, and shows the ability of theinvestigated sample to absorb light at different wavelengths. As lightabsorption occurs almost always from the lowest vibrational level of theelectronic ground state, the absorption spectrum characterizes theenergetic structures of the electronic excited states of an aromaticmolecule.

BASIC PHOTOPHYSICS

8 of 34

Figure 6. Measurement principle of light absorption spectra.The values measured directly in the spectrophotometer are theintensities of light transmitted through sample and referencecuvettes.The light source S generates a broad spectrum of lightand the dispersion device (grating or prism) M selects amonochromatic light of specific wavelength λ. The light is thendivided into two identical beams directed to two cuvettes, asample cuvette, SC, containing the solution of the aromaticcompound and a reference cuvette, RC, with solvent only.Changing the wavelength over the required range enablesmeasuring the intensities of the light transmitted through bothcuvettes, I(λ) and I0(λ).

The absorbance Aλ at wavelength λ is then defined as:

and is equal according to the Lambert-Beer law to:

where ε(λ) is the extinction coefficient at wavelength λ for the particularmolecule, c its concentration, and d the path length of the cuvette, forinstance 1 cm.

Examples of absorption (absorbance, left side) and fluorescence (rightside) spectra of three widely investigated fluorophores are presented inFigure 7.

BASIC PHOTOPHYSICS

9 of 34

Figure 7. Normalised absorption (left), and fluorescence(right) spectra of three fluorophores. Shown are the spectra of2,5-diphenyl-oxazole (PPO, a molecule used in scintillationcounting) in an organic solvent, N-acetyl-tryptophan amide(NATA), and the protein human serum albumin (HSA) inaqueous buffer.

Fluorescence Spectrum: A fluorescence spectrum Iflu(λ) represents theintensity of the fluorescence light emitted by the sample as a function ofemission wavelength (Figure 8). As fluorescence transitions start in mostcases from the lowest vibrational level of the first electronic excitedstate, Iflu(λ) characterises the energetic structure of the electronic groundstate.

Figure 8. Measurement of fluorescence spectra.Fluorescence is detected at right angle to the excitation beam.A monochromatic light beam of selected wavelength λexc

excites the molecules in the sample. Fluorescence light isemitted from the excited state of these molecules in alldirections. Fluorescence is usually collected at right angles tothe excitation direction in order to minimize the presence ofscattered excitation light in the fluorescence channel. Themonochromator M2 allows measurements of fluorescenceintensity as a function of the emission wavelength. Thefluorescence spectra of PPO, NATA and HSA are shown in

BASIC PHOTOPHYSICS

10 of 34

Figure 7.

Sometimes it is useful to measure how fluorescence intensity depends onthe excitation wavelength. In this case we will detect fluorescenceintensity at a fixed wavelength (for instance at the wavelength ofmaximal fluorescence) as a function of the excitation wavelength (M1),thus obtaining an excitation spectrum. An excitation spectrum isessentially an absorption spectrum, because the fluorescence intensityIflu(λ) is proportional to:

where ε(λ) is the extinction coefficient at excitation wavelength λ, I0 isthe monochromatic light intensity, and Φ is the quantum yield offluorescence (see below). This equation is valid only for dilutefluorophore solutions, in which the absorbance of the aromatic compoundnever exceeds 0.05. Excitation spectra turn out to be useful in obtainingabsorption spectra of very dilute samples.

Quantum Yield of Fluorescence: The quantum yield, Φ, is defined asthe ratio of the number of fluorescence photons emitted by the sample nE

to the number of photons absorbed nA. It can be shown also that Φ is theratio of the rate of the radiative transition (kr) to the rates of alltransitions (kr+knr), in which the excited state is involved. Therefore, anymolecular mechanism leading to a non-radiative depopulation of theexcited state reduces the quantum yield:

The quantum yields of some popular fluorophores, naturally occurringmono- and dinucleotides, wild type green-fluorescent protein (GFP), andother visible fluorescent protein variants, are collected in Table 2.

Table 2. Quantum yields of selected fluorophores.

Fluorescence Lifetime: The other important characteristic feature offluorescence is its time response, namely the decay of fluorescenceintensity following infinitesimally short or δ-type excitation. The schemepresented in Figure 9 is a simplified Jablonski diagram, which can be

BASIC PHOTOPHYSICS

11 of 34

used to explain the basic kinetics of fluorescence.

Figure 9. Kinetic scheme to explain fluorescence lifetime. δ(0)is excitation with a δ-pulse, kr is rate constant of radiativetransition, and knr is rate constant of non-radiative transition.

The population of the excited molecules [M*] generated at the momentof excitation, t=0, starts to decrease exponentially through the radiative(kr) and non-radiative (knr) transitions to the ground state.

The characteristic time of this process, 0, is called the fluorescencelifetime:

The intensity of fluorescence, F(t), emitted at any moment of thisprocess is proportional to [M*](t), thus:

Note that F(t) is a shorthand notation for Iflu(t). The fluorescence lifetime0 has the physical meaning of the time needed for the fluorescence

intensity to decrease to 1/e (= 1/2.71) of its initial value F0 (Figure 10).

Figure 10. Fluorescence decay F(t) = F0 exp(-t/5) with t in ns.F(t) is the fluorescence intensity that decays exponentially witha fluorescence lifetime, 0, equal to 5 ns. The δ-pulserepresents an excitation light pulse of infinitesimally smallduration, for instance, a duration of 1 femtosecond (10-15

BASIC PHOTOPHYSICS

12 of 34

second).

In practice the generation of a δ-pulse for excitation is not possible,because the instrumental response time needed to detect fluorescencedecays is often comparable to the fluorescence lifetimes that we want tomeasure. The experimentally detected fluorescence decays are not puredecays like F(t) in Figure 10, but convolutions of the ideal decay functionwith the so called prompt function (or instrumental response function).Measuring the prompt function, and then extracting the undistortedfluorescence decay function will be described later in this module.

Fluorescence Anisotropy: Photons can be absorbed only when theirenergy fits to the energy gap between the ground and excited energylevels of a particular molecule. Another condition for light absorption isthat the electric component or vector of the electromagnetic wave mustbe parallel or close to parallel, to the transition moment of the molecule.

In solution, the orientations of the transition moments are completelyrandom. Therefore, if we excite such a system with linearly polarizedlight, the excitation will be efficient only for those molecules whosetransition moments are, at the moment of excitation, oriented similarlyto the direction of polarization. The initial distribution of orientations ofthe excited molecules will then be highly anisotropic. This ordering effectis called 'photoselection'.

After excitation, the molecules start to fluoresce with their characteristicfluorescence lifetime 0, and, simultaneously, Brownian rotational motioncauses the initial orientational order of the excited molecules to vanish.

Polarization of fluorescence is determined by the orientation of thefluorophores' transition moment at the instant of fluorescence emission.This gives an opportunity to determine rotational diffusion offluorophores by detecting the anisotropy of their fluorescence (Figure11).

Figure 11. Geometry of fluorescence anisotropy experiment.Fluorophores in the cuvette are excited with short pulses of

BASIC PHOTOPHYSICS

13 of 34

vertically polarized light (z) causing photoselection. A polarizerin the fluorescence channel (x) can be rotated from the verticalto the horizontal position. In the first part of the experiment,the decay of intensity of vertically polarized fluorescence Ivv(t)is measured. Then the polarizer is moved to horizontalorientation, and Ivh(t) is detected.

Both decays, Ivv(t) and Ivh(t), are combined to form the fluorescenceanisotropy function r(t), defined as:

For spherical and freely rotating fluorophores the anisotropy decaysexponentially:

with the characteristic decay constant r, called rotational correlationtime, and r0 = 2/5 for randomly distributed molecular systems. Accordingto the theory of rotational diffusion, r is related to the radius r of arotating sphere by the equation:

Here, kBT is the Boltzmann factor, and η is the viscosity of the solvent.The relation between fluorescence anisotropy decays, and the dimensionsof rotating molecules is one of the fundamentals of the newly emergingresearch discipline of 'nanometrology'.

Solvent Effects on Fluorescence: The concept of solvation can beunderstood from interactions between a fluorophore (the solute), and thesurrounding solvent molecules. The dominating solute-solventinteractions arise from electrostatic dipole-dipole interactions, which leadto lowering the potential energies of all energy levels involved inabsorption and fluorescence processes. This effect can be explained byOnsager's model of solvation (Figure 12).

BASIC PHOTOPHYSICS

14 of 34

Figure 12. Changes in solute-solvent interactions lead tosolvatochromic shifts in absorption and fluorescence spectra ofthe same fluorophore. [see text for definitions of symbols]

According to this model, the dipole moment of the fluorophore in theground state, μg, interacts with the dipole moments of the surroundingsolvent molecules, rearranging them in a way that minimises thepotential energy of the whole system. If we would "freeze" the moleculesfor a while and remove the fluorophore, the special arrangement of thesolvent dipole moments would result in a non-balanced electric field Rg,called the "reaction field".

In Onsager's model, the solute-solvent interaction is identified as aninteraction of the fluorophore dipole moment, μg, with the reaction fieldRg, namely:

The energy level of the ground state is therefore lowered by this value.The symbol 'rel' indicates that the solvent is in a state of thermodynamicequilibrium (relaxed). Electronic excitation of the fluorophore (Figure 12)causes a rapid (~10-15 s) change of its dipole moment to μe. This time ismuch too short for the solvent molecules to rearrange their orientations.Thus, immediately after excitation the interaction energy will be:

indicating that the reaction field will be still the same as it was beforeexcitation. The symbol 'FC' indicates a non-equilibrated, Franck-Condonstate. The solvent molecules need usually picoseconds (10-12-10-10 s) toperform solvent relaxation achieving finally the solute-solvent interactionenergy:

BASIC PHOTOPHYSICS

15 of 34

The process of fluorescence brings the fluorophore dipole moment backto its ground-state value μg, so just after fluorescence:

which finally evolves during ground-state solvent relaxation to .

Direct consequences of the different solute-solvent interaction energiesat different stages of absorption and fluorescence events are the spectralshifts in absorption (∆Uabs) and fluorescence (∆Uflu) spectra:

The fluorescence spectra of the solvent-sensitive fluorophore "badan" ina number of solvents are shown in Figure 13. For "badan", μe is muchlarger than μg. The larger the dipole moment of the solvent molecule thestronger is the "red-shift" of the spectrum.

Figure 13. Fluorescence spectra of "badan" (i.e., the2-mercapto-ethanol adduct of 6-bromoacetyl-2-dimethylaminonaphtalene) in: (1) toluene, (2) chloroform,(3) acetonitrile, (4) ethanol, (5) methanol and (6) water.Figure from Handbook of Molecular Probes.

Quenching of Fluorescence by External Molecules: Any moleculethat interacts with the fluorophore and reduces its quantum yield, iscalled a quencher. There are a number of different molecularmechanisms of quenching.

In case of static quenching, external molecules Q, simply formground-state complexes with the fluorophore M (Figure 14). Thesecomplexes can be excited, but are not fluorescent.

BASIC PHOTOPHYSICS

16 of 34

Figure 14. Schematic illustration of static quenching. The MQcomplex is not fluorescent yielding reduction in fluorescenceintensity, but no change in fluorescence lifetime (see text).

The equilibrium between free and complexed fluorophores is controlledby an association constant Ks:

where [M], [Q] and [MQ] are the concentrations of fluorophore, quencherand fluorophore-quencher complexes, respectively. [M]0 is the totalconcentration of the fluorophore. The above equation can be easilyconverted into the well-known Stern-Volmer equation, which describesthe decrease of fluorescence intensity with increasing quencherconcentration:

Here, F0 and F are the fluorescence intensities measured without, and inthe presence of the quencher. Due to the nature of static quenching,there is no change in fluorescence lifetime, since only the non-complexedfluorophore is fluorescent. However, the quencher-fluorophore complexcan still be fluorescent, but displays an ultra-short fluorescence lifetimein the picosecond time-range yielding no significant steady-statefluorescence intensity. The static quenching arises from competingprocesses that induce non-radiative pathways to the ground state. Thenon-radiative rate constant (k'nr) is much larger than the radiative rateconstant (kr), and emission is almost completely eliminated.

In the case of dynamic quenching the quenching molecule Q collides withthe excited fluorophore M* (Figure 15). The excited state kinetics areaffected by forming an additional way of depopulating the excited state.Consequently, the fluorescence decay is modified. Both steady-state andtime-resolved fluorescence yield the famous Stern-Volmer constantKSV = 0kq, in which o is the fluorescence lifetime without quencher, andkq is the diffusion controlled rate constant of quenching. KSV has thedimension M-1.

Several compounds behave as dynamic quenching agents when presentat sufficiently high concentration, including oxygen, heavy-atom halidessuch as bromide and iodide, mercury and caesium ions, tetrachloro-methane (CCl4), amines and acrylamide.

BASIC PHOTOPHYSICS

17 of 34

Figure 15. Schematic illustration of dynamic quenching andrelevant rate equations. The steady-state fluorescence intensityratio (F0/F) (F is the fluorescence intensity in presence ofquencher Q) is the same as the ratio of fluorescencelifetimes 0/ q ( q is the fluorescence lifetime in the presenceof quencher Q). The Stern-Volmer constant KSV is equal to

0kq.

Förster Resonance Energy Transfer (FRET): Theodor Försterdeveloped the quantitative theory for resonance energy transfer in thelate 1940s. Therefore, we call this process Förster resonance energytransfer or FRET. FRET is a photophysical process where theexcited-state energy from a donor molecule is transferred non-radiativelyto an acceptor molecule at close distance via weak dipole-dipole coupling(Figure 16). FRET is sometimes referred to as "fluorescence" resonanceenergy transfer, but this is a misconception, since no fluorescence takespart in the coupled transition.

Figure 16. Kinetic scheme of FRET.

Förster derived the following expression for the rate constant of transferkT:

in which R is the distance between donor and acceptor molecules and D

is the fluorescence lifetime of the donor (without acceptor). Since thetransfer rate is proportional to the inverse 6th power of the distance R,the transfer rate is an extremely sensitive parameter for obtainingdistances in the range of 1-10 nm (or 10-100 Å). The distance at whichthe excitation energy of the donor is transferred to the acceptor withprobability 0.5, is called the Förster or critical distance R0 (in units of

BASIC PHOTOPHYSICS

18 of 34

cm), and can be calculated using the relevant spectroscopic properties ofthe participating molecules:

in which κ2 is the orientation factor, Φ0 the quantum yield of donorfluorescence (without acceptor), NA is Avogadro's number, and n is therefractive index of the intervening medium. The integral (J, in units of M-1cm3) is the degree of spectral overlap between donor fluorescencespectrum (FD, its spectrum normalized so that the integral is equal toone), and acceptor absorption spectrum (scaled to its maximum molarextinction coefficient, εA, in units of M-1cm-1), given by eitherwavenumber (σ) or wavelength (λ) scale (Figure 17):

Figure 17. Illustration of spectral overlap integral between thefluorescence spectrum of cyan-fluorescent protein (CFP), andthe absorption spectrum of yellow-fluorescent protein (YFP).

The orientation factor κ2 is given by:

For a definition of the angles we refer to Figure 18, in which somelimiting situations have been depicted illustrating that 0 < κ2 < 4. Forsystems without any 3-dimensional, spatial information the orientationfactor is the indeterminate parameter in R0. All other parameters can bemeasured or evaluated.

BASIC PHOTOPHYSICS

19 of 34

Figure 18. The orientation factor in FRET. μD and μA are thetransition moments of donor and acceptor molecules and R isthe separation vector.

When the critical transfer distance, R0 (see equation above), is expressed

in units of Å, the scaling constant is equal to 0.2108, themolar extinction coefficient, εA, in units of M-1cm-1, the wavelength, λ, inunits of nm, and the overlap integral, J, in units ofM-1cm-1nm4, and R0 is given by:

Förster also introduced the transfer efficiency E, which is only a functionof actual (R) and critical (R0) distances:

There are several methods available for quantification of FRET, of whichthe one based on donor fluorescence lifetimes is the moststraightforward, because the fluorescence lifetime is a concentration-independent property, while fluorescence intensity is not. Donorfluorescence lifetimes in the absence ( D) and presence ( DA) of acceptormolecules are often measured for the observation of FRET and adecreased fluorescence lifetime of the donor is then an indication ofmolecular interactions (Figure 19). From this reduction in lifetimes weimmediately obtain the experimental FRET efficiency:

BASIC PHOTOPHYSICS

20 of 34

Figure 19. Time-resolved fluorescence detection of FRET.Shown are a donor molecule D and an acceptor molecule Aseparated at distance R in a macromolecule. A very short lightpulse (δ(0)) excites donor D. Analysis of the generated donorfluorescence decay (F(t)) gives information on the rateconstant of transfer κT from which the distance R can beobtained.

In Figure 20 we have summarised the main concepts of FRET, which areillustrated with the widely used FRET couple CFP (cyan-fluorescentprotein, donor) and YFP (yellow-fluorescent protein, acceptor). FRET isused extensively for monitoring interactions and conformational changesbetween or within biological macromolecules conjugated with suitabledonor-acceptor pairs. Because of its sensitivity FRET also forms the basisfor "sensing" important biological molecules in many applications.

Figure 20. Main concepts of FRET. The occurrence of FRETdepends on distance (upper left panel) and the spectral overlap(upper right panel). The reduction of a lifetime from 2.5 to 2.0ns corresponds to a transfer efficiency of 0.2 (20%) and to adistance between CFP and YFP of 6.3 nm.

BASIC PHOTOPHYSICS

21 of 34

Time-Resolved Fluorescence Instrumentation: Measurement of thefluorescence lifetime, or generally of the parameters that govern thecourse of fluorescence intensity with time can be performed with twodifferent techniques that are both widespread: the pulse method and thephase-modulation method.

The pulse method is frequently used in combination with time-correlatedsingle photon counting (TCSPC). The sample is excited with a short pulseof light, and the time between the pulse and detection of the first emittedphoton is measured. The course of fluorescence intensity with time(Figure 21) is recorded by multiple repetition of this procedure.

Figure 21. Schematics of time-correlated single photoncounting. The arrival time of the first photon after an excitationpulse is measured and stored in memory. The histogram ofmany arrival times of photons represents the "fluorescenceintensity versus time" curve.

The resulting signal is a convolution of the real decay over time (i.e.,following the δ-pulse of excitation) of fluorescence intensity with theinstrument response function (Figure 22).

Figure 22. Illustration of the convolution principle. L(t) is theinstrumental response function and F(t) is the experimentaldecay function. The channel number gives the time axis, as itcorresponds to a certain time increment. F(t) is a convolutionproduct of L(t) with the real decay function Fc(t):

BASIC PHOTOPHYSICS

22 of 34

Analysis of the measured fluorescence intensity curves and determinationof fluorescence lifetimes are often performed by a nonlinear least-squares fitting method. Let us assume that a model function of a sum oftwo exponential terms adequately describes the course of fluorescenceintensity with time. Such situation may occur, when the fluorophoreexists in two distinct environments each characterised by its ownfluorescence lifetime.

in which n=2, and 1 and 1 are the amplitude and lifetime ofcomponent 1, and 2 and 2 are the amplitude and lifetime ofcomponent 2. The result of the convolution of the model function withthe instrument response function is fitted to the measured fluorescenceintensity curves by optimising the parameters 1, 1 and 2, 2.

The resolution that can be achieved with TCSPC depends much on thelight source and detector. Using mode-locked lasers in combination withmicrochannel-plate photomultipliers, instrument response function widthsof thirty picoseconds are possible. This allows for the measurement ofdecay times in the range of ten to twenty picoseconds. Much cheaperdiode lasers and light-emitting diodes (LEDs) give longer excitationpulses (width in the range 50-200 ps), but decay times of severalhundred picoseconds can still be resolved.

In the phase-modulation method, the sample is excited by an intensitymodulated light source, which can either be a xenon arc lamp, LED (seebelow) or a laser (pulsed or CW). The excitation is sinusoidal-modulatedat angular frequency (rad/s). The fluorescence is delayed in phase andpartially demodulated. The experimental quantities to be determined arethe phase difference ( ), and the modulation ratio ( ), as shown inFigure 23.

BASIC PHOTOPHYSICS

23 of 34

Figure 23. Determination of the phase difference ( ) andmodulation ratio ( ) in the phase-modulation method.

For a single fluorescence lifetime, phase difference and modulation ratioare related to the lifetime ( ) by:

For the precise determination of fluorescence lifetimes, the phasedifference ( ) and the modulation ratio ( ) are measured as functionof different frequencies . The curves can be analysed by the method ofnonlinear least squares using theoretical expressions of the sine andcosine Fourier transforms of the δ-pulse response. The resolution of thephase-modulation method depends on the modulation frequency.Fluorescence lifetimes in the picosecond range can be measured usingmodulation frequencies between 2 and 10 GHz. For lifetimes in the 1-10nanosecond range, modulation frequencies between 2 and 200 MHz areappropriate.

Luminescence From Semiconductor (Nano-)Crystals

Semiconductors: Until now we have treated light emission fromaromatic molecules such as fluorescence and, in less detail,phosphorescence. A general term of light emission is luminescence, aterm derived from the Latin "lumen", light, and introduced by theGerman scientist Eilhard Wiedemann in 1880 ("Lumineszenz"). Becauseluminescence from crystalline semiconductor materials has a completelydifferent origin than that of fluorescence, it is worthwhile to pay attentionto it, as we are surrounded nowadays by many applications of them.

To understand how semiconductors work we must have backgroundknowledge in solid-state physics. That is beyond the scope of thismodule, and we prefer to give a qualitative picture of the physics behindsemiconductors. A semiconductor is a material that has an electricalconductivity between that of a conductor and an insulator, that is,generally in the range 103 Siemens/cm (conducting) to 10-8 S/cm(non-conducting). Devices made from semiconductor materials are usedin modern electronics, including computers, telephones, LCD displays,etc. Semiconductors have also found applications in opto-electronics andphotophysics. Below we will briefly describe the working principles ofquantum dots, light emitting diodes (LEDs), diode lasers and solar cells.

There is another motivation to know more about semiconductors,especially solar cells. The working principle of solar cells shows aremarkable analogy with that of the photosynthetic apparatus. Plants useradiation in the wavelength range of 400-700 nm to reduce CO2 toglucose, with the concomitant oxidation of water to O2. The radiationenergy is in the range of the band gap of many semiconductor materials(Table 3).

BASIC PHOTOPHYSICS

24 of 34

Table 3. Some semiconductor materials and their band gaps.[adapted from Wikipedia: Band Gap]

This photosystem consists of protein complexes, which combine lightharvesting proteins and reaction centers. The light harvesting complexhaving bound a large number of chlorophylls and carotenoids, absorbssolar energy and rapidly transfers this energy to the reaction center,which is a special pair of two chlorophyll molecules. Efficient lightharvesting requires that energy must be transferred via weak(Förster-type) and strong (exciton-type) coupling mechanisms. This isanalogous to the creation of electrons, holes and excitons insemiconductor materials after illumination, which are transportedefficiently in the crystal lattice to a p-n junction (see below). The functionof a p-n junction may be compared to that of the reaction center of thephotosystem, since charge carriers are separated in both systems.Negative charge carriers are electrons in both systems. Positive carriersare holes in semiconductors, and protons in reaction centres. Bothphotosystems and solar cells must operate reversibly. They share,therefore, the same process of recombination of charge carriers.

Impregnating impurities into their crystal lattice may easily modify theconductivity of semiconductors. The process of the controlled adding ofimpurities to a semiconductor is known as 'doping'. The amount ofimpurity, or dopant, added to a pure semiconductor varies its level ofconductivity, and also its band gap. By incorporating impurity atoms intothe crystal lattice of pure semiconductors, the electrical conductivity maybe varied not only by the number of impurity atoms but also, by the typeof impurity atom.

BASIC PHOTOPHYSICS

25 of 34

In general, dopants that produce the desired controlled changes areclassified as either electron acceptors or donors. A donor atom donatesweakly bound valence electrons to the material creating excess negativecharge carriers. These weakly bound electrons can move in the crystallattice relatively freely, and can facilitate conduction in the presence ofan electric field. Conversely, an activated acceptor produces an electronhole (usually referred to simply as a hole), which carries a positivecharge. The holes do not actually move, but a neighboring electron canmove to fill the hole, leaving a hole at the place that the electronoccupied previously. In this way, holes can also move freely in the crystallattice. Both conduction-band electrons and valence-band holescontribute to electrical conductivity. Semiconductors doped with donorimpurities are called n-type (n meaning negative), while those dopedwith acceptor impurities are known as p-type (p meaning positive).

Quantum mechanical theory predicts that electrons in semiconductorscan have energies only within certain bands corresponding to electronstightly bound to the atomic nuclei of the material, and the free electronenergy, which is the energy required for an electron to escape entirelyfrom the material. Each energy band corresponds to a large number ofdiscrete quantum states of the electrons. Most of the states with lowenergy (closer to the nucleus) are filled with electrons, up to a particularband, which is called the valence band, and which is also filled withelectrons. Semiconductors and insulators are distinguished from metalsbecause the valence band in the semiconductor materials is nearly filledunder usual operating conditions, thus causing more electrons to beavailable in the conduction band, which is the band immediately abovethe valence band (and empty at absolute zero temperature, 0 K). Theease with which electrons in a semiconductor can be excited from thevalence band to the conduction band depends on the band gap betweenthe bands, and it is the size of this energy band gap that serves as anarbitrary dividing line (roughly 4 eV) between semiconductors andinsulators (Figure 24).

Figure 24. Energy bands of electrons in a metal,semiconductor and insulator. The Fermi level refers to theenergy of the highest occupied quantum state in a system offermions at absolute zero temperature. A fermion is anyparticle that has an odd half-integer (like 1/2, 3/2, and soforth) spin. [From Wikipedia: Electronic Band Structiure]

p-n Junctions: A p-n junction is a junction formed by joining p-type and

BASIC PHOTOPHYSICS

26 of 34

n-type semiconductors together in very close contact (Figure 25). Theterm junction refers to the boundary interface where the two regions ofthe semiconductor meet. Although they can be constructed of twoseparate pieces, p-n junctions are more often created in a single crystalof semiconductor by growing a layer of crystal doped with one type ofdopant on top of a layer of crystal doped with another type of dopant.

Figure 25. p-n Junctions in silicon in an open circuit (top),forward-bias (bottom, left), and reverse-bias circuits (bottom,right). [From Wikipedia: p-n Junction]

The p-n junction possesses some interesting properties, which haveuseful applications in modern electronics. A p-doped semiconductor isrelatively conductive. The same is true of an n-doped semiconductor, butthe junction between them is a non-conductor. This non-conductinglayer, called the depletion (or space charge) region, arises because theelectrical charge carriers in doped n-type and p-type semiconductors(electrons and holes, respectively) attract and eliminate each other in aprocess called recombination. Transport of charge carriers can occur intwo ways. One mode of transport is by 'drift', which is caused by anelectrostatic field across the device. The other mode of transport is bydiffusion of carriers from zones of high carrier concentration to zones oflow carrier concentration. Drift and diffusion have opposite directions.Figure 26 gives a schematic picture of a p-n junction. P-n junctions areessentially electronic diodes and are elementary building blocks of almostall opto-electronic devices such as LEDs, diode lasers, solar cells, etc.

Figure 26. A p-n junction in thermal equilibrium with zero bias

BASIC PHOTOPHYSICS

27 of 34

voltage applied. Blue and red lines indicate the concentration ofelectrons and holes, respectively. Gray and white regions haveneutral charges. The light red zone is positively charged(holes). The light blue zone is negatively charged (electrons).The direction of the electric field is shown on the bottom. Alsoshown are the electrostatic force on electrons and holes (drift),and the direction in which the diffusion tends to move electronsand holes. [From Wikipedia: p-n Junction]

Luminescence of Semiconductors: By the absorption of a photon, anelectron can be promoted from the valence band into the conductionband. Electrons excited to the conduction band also leave behind electronholes in the valence band.

Luminescence can originate from direct light absorption, sincerecombination of the excited electron with the hole in the valence band isaccompanied by light emission. Luminescence can also be created byexcitons. An exciton is an electron-hole pair, bound by Coulomb forces,with the electron in the conduction band, and the hole in the valenceband. Since an exciton is a bound state of an electron and a hole, theoverall charge for this quasi-particle is zero. Hence it carries no electriccurrent. The whole exciton can move through the molecular crystal, andimpurities (so-called carrier traps) are sometimes added to stabilize theexciton at one position. With this additional kinetic energy the excitonicenergy may lie above the band-gap. This extra energy can be releasedby radiation. This light emission in semiconductors is taking place atlower temperature, when the Boltzmann factor (κBT) is less than theexciton binding energy.

In Figure 27, we have redrawn the quantum-mechanical band scheme tounderstand the luminescence of semiconductors.

Figure 27. Band scheme of semiconductors, and the two waysof light emission.

Quantum Dots: Nanometer-sized crystals of certain semiconductors,such as cadmium selenide (CdSe), have interesting luminescentproperties. After photon absorption, the wavelength of luminescencedepends on the nature of the semiconductor material, and on the size ofthe crystal. The light emission is due to a phenomenon known asquantum confinement. Therefore these nanocrystals are called 'quantum

BASIC PHOTOPHYSICS

28 of 34

dots'. In quantum dots, electrons, holes and excitons are confined in allthree spatial dimensions. The smaller the quantum dot is, the shorter isthe emission wavelength. The color of luminescence of CdSe quantumdots varies from violet to red, when the size increases from 2 nm to 7nm. The emission color of quantum dots can be tuned from the visiblethroughout the infrared by the careful choice of materials and sizes(Figure 28). The quantum yield of luminescence can be increased bycoating the quantum dot with a layer of zinc sulfide (ZnS), which has amuch larger band gap (see Table 3), and finally with a polymer. Thisprotective layer will reduce non-radiative decay processes responsible forthe low quantum yield.

Figure 28. Luminescence of various quantum dots illuminatedwith UV light. Picture from Evident Technologies.

Over the past decade, quantum dots have been used in the confocalfluorescence microscopy of cells, thereby replacing organic fluorescentdyes. The improved photo-stability of quantum dots allows acquisition ofmany consecutive two-dimensional confocal images that can bereconstructed into a high-resolution three-dimensional confocal image.Another application taking advantage of their photostability is thetracking of certain biological molecules attached to quantum dots("biosensors") in living cells and animals (mice) over extended periods oftime (even months). First attempts have been made to use quantumdots for tumor targeting. One of the remaining issues with quantum dotprobes is their in vivo toxicity, as they have to be excited with UV light.Quantum dots with a stable polymer coating seem to be essentiallynontoxic.

Light Emitting Diode (LED): An LED consists of a chip ofsemiconducting material doped with impurities to create a p-n junction.When the p-n junction is forward biased (switched on) (Figure 25),current flows easily from the p-side (anode) to the n-side (cathode), butnot in the reverse direction. Charge carriers - electrons and holes - flowinto the junction from electrodes with different voltages. When anelectron meets a hole, it falls into a lower energy level, and releasesenergy in the form of a photon (Figure 29). This effect is calledelectroluminescence and the color of the light is determined by the bandgap energy of the materials forming the p-n junction. An LED is usuallysmall in area (less than 1 mm2), and integrated optical components areused to shape its radiation pattern and assist in reflection to improve theoutput (Figure 29).

BASIC PHOTOPHYSICS

29 of 34

Figure 29.Working principle (left), and schematics of an LED(right). [From Wikipedia: LED]

LEDs present many advantages over other light sources, including lowerenergy consumption, longer lifetime, smaller size, faster switching andgreater reliability. LED development began with infrared and red devicesmade with gallium arsenide (see Table 3). Advances in material sciencehave made possible the production of LEDs having band gaps withenergies corresponding to near-infrared, visible or ultraviolet light. LEDsmade from silicon or germanium are not effective, since electrons andholes recombine by a non-radiative transition, producing no lightemission.

Diode Laser: Like many other semiconductor devices, a diode laser isformed by doping a very thin layer on the surface of a crystalline wafer.The crystal is doped to produce an n-type region and a p-type region,one above the other, resulting in a p-n junction. Charge injectiondistinguishes diode lasers from all other, optically pumped lasers.Therefore diode lasers are called "injection lasers". The initial mode ofaction is not different from that of an LED. Electron-hole recombinationbelow a laser threshold may result in spontaneous emission, as in anLED. Spontaneous emission is necessary to initiate laser oscillation. Inthe absence of stimulated emission, electrons and holes may coexist inproximity to one another for a certain time before they recombine. Thischaracteristic time is termed the "recombination time" (about ananosecond for typical diode laser materials). Then a nearby photon withenergy equal to the recombination energy can cause recombination bystimulated emission. This generates another photon of the samefrequency, traveling in the same direction, with the same polarizationand phase as the first photon. This means that stimulated emissioncauses gain in an optical wave in the injection region. The gain increasesas the number of electrons and holes injected across the junctionincreases.

As in other lasers, the gain region is surrounded with an optical cavity toform a laser. In the simplest form of a diode laser, an optical waveguideis made on the crystalline wafer, such that the light is confined to arelatively narrow line. The two ends of the crystal are cleaved to formperfectly smooth, parallel edges, forming an optical resonator. Photonsemitted into a mode of the waveguide will travel along the waveguide,and be reflected several times from each end face (mirror) before they

BASIC PHOTOPHYSICS

30 of 34

are emitted. As a light wave passes through the cavity, it is amplified bystimulated emission, but light is also lost due to absorption, and byincomplete reflection from the end mirrors. Finally, if there is moreamplification than loss, the diode begins to perform laser action. Due todiffraction, the beam diverges rapidly in vertical and lateral directionsafter leaving the chip. A lens must be used in order to form a collimatedbeam like that produced by a laser pointer.

In Figure 30, a diagram of a double hetero-structure diode laser isshown. In this device, a layer of low band-gap material is sandwichedbetween two high band-gap layers. One commonly used pair of materialsis gallium arsenide (GaAs) with aluminum-doped gallium arsenide(AlGaAs). Each of the junctions between different band gap materials iscalled a hetero-structure, hence the name "double hetero-structure"laser or DH laser. The advantage of a DH laser is that the active regionwhere free electrons and holes exist simultaneously, is confined to thethin middle layer with lower band gap energy. This means that manymore of the electron-hole pairs can contribute to amplification. Inaddition, light is reflected from the hetero-junction; hence, the light isconfined to the region where the amplification takes place.

Figure 30. Diagram of front view of a double heterostructurelaser diode (not to scale). Material A is, for instance, AlGaAs(band gap ~2.8 eV). Material B is, for instance, GaAs (bandgap 1.43 eV). [From Wikipedia: Diode Laser]

The relatively low cost of mass-produced diode lasers makes themattractive for a wide range of applications in diverse areas. The small sizeof diode lasers is also advantageous. Diode lasers are used in fiber opticscommunication, optical data recording and barcode readers. Red andgreen lasers are common as laser pointers. Both low and high-powerdiode lasers are used extensively in the printing industry, both forscanning of images and for very high-resolution printing platemanufacturing. Infrared and red diode lasers are common in CD and DVDplayers. Diode lasers are also indispensable light sources in confocalfluorescence microscopy and time-resolved fluorescence spectroscopy.High-power diode lasers are used for pumping other, solid-state laserslike Ti-Sapphire. Medical applications include, among others,photodynamic therapy to photo-activate porphyrin derivatives asanti-cancer agents.

Solar Cells: A solar cell is a device that converts the energy of sunlightdirectly into electricity by the photovoltaic effect. In contrast to LEDs anddiode lasers that produce light, solar cells use light to produce electricity.An assembly of solar cells makes a solar panel. Construction of solar cells

BASIC PHOTOPHYSICS

31 of 34

has a long history, starting in the 1880s, and resulting in semiconductor(silicon) devices with a sunlight energy conversion efficiency of around6% in the 1950s. Highly effective hetero-structure solar cells werecreated from 1970 on, until conversion efficiencies have been reachedclose to 40%. A current challenge to bring down the cost of solar energyis to increase the photovoltaic efficiency.

Solar cells are essentially p-n junctions under illumination. Lightgenerates electrons and holes on both sides of the junction, in the n-typeemitter and in the p-type base. Charges build up on either side of thejunction and create an electric field. The generated electrons (from thebase) and holes (from the emitter) then diffuse to the junction and areswept away by the electric field, thus producing electric current acrossthe device. The electric currents of the electrons and holes reinforce eachother, since these particles carry opposite charges. The p-n junction,therefore, separates the carriers with opposite charge, and transformsthe generation current between the bands into an electric current acrossthe p-n junction. (Figure 31).

Figure 31. Photovoltaic effect on p-n junctions. Panels 1 and 2are explained in Figure 26. In panel 3 the electric fields areplotted. EF is the Fermi level. In panel 4 the actual current isgenerated by light. Note that electrons and holes move inopposite directions. [From Wikipedia: Solar Cells]

Ohmic-metal semiconductor contacts are made to both n-type andp-type sides of the solar cell (Figure 32).

BASIC PHOTOPHYSICS

32 of 34

Figure 32. Basic structure of a silicon based solar cell, and itsworking mechanism. The electrodes are connected to anexternal resistance load. [From Wikipedia: Solar Cells]

Electrons that are created on the n-type side of the junction may travelthrough the wire, power the load, and continue through the wire untilthey reach the p-type semiconductor-metal contact (aluminum). Herethey recombine with a hole. The hole was either created as anelectron-hole pair on the p-type side of the solar cell or swept across thejunction after being created at the n-side. The voltage measured is equalto the difference in energy levels of the minority carriers, i.e., electronsin the n-type portion and holes in the p-type portion (V0 in panel 4 ofFigure 31).

Suggested Reading

Birks, J.B., Photophysics of Aromatic Molecules, Wiley-Interscience,London, 1970.

Cantor, C.R. and Schimmel, P.R., Biophysical Chemistry, Part II:Techniques for the Study of Biological Structure and Function, W.H.Freeman, San Francisco, 1980.

Lakowicz, J.R., Principles of Fluorescence Spectroscopy, 3rd ed.,Springer, New York, 2006.

Saleh, B.E.A. and Teich, M.C., Fundamentals of Photonics, 2nd ed., JohnWiley & Sons, Hoboken, New Jersey, 2007.

Steinfeld, J.I., Molecules and Radiation. An Introduction to ModernMolecular Spectroscopy, Harper & Row Publishers, New York, 1974.

Valeur, B., Molecular Fluorescence: Principles and Applications,Wiley-VCH, Weinheim, 2002.

Many topics treated in this module can be found in: Wikipedia.

A very useful introduction to quantum dots can be found in:Quantum Dots.

Fluorescence microscopy, and history can be found in: Microscopy.

BASIC PHOTOPHYSICS

33 of 34

A wide selection of fluorescent probes can be found in: Molecular Probes.

03/15/1012/03/10

[ TOP ]

BASIC PHOTOPHYSICS

34 of 34