Photophysics Basics Avisser Orolinski Strath Ac

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24/04/2016 BASIC PHOTOPHYSICS

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BASIC PHOTOPHYSICS

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

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

[email protected]

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

Glasgow G4 0NG, [email protected]

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 both

damaging 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]

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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 poker

in 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 or

energy (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.

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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 rows

of 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 of photons 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 of experiments. Electromagnetic waves are composed of an electric field(E), and a magnetic field (B) perpendicular to it, oscillating in phase in

the 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 waves givinga new wave pattern that can be constructive when two waves have thesame direction or destructive when they have the opposite direction.

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Another example is light dispersion into a visible spectrum when whitelight is shone through a prism, because of the wavelength dependentrefractive index of the prism material. Electromagnetic waves also haveparticle-like properties, as packets of energy or quanta, called photons.The energy per photon, E , can be calculated from the Planck-Einsteinequation:

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 for

fluorescence; 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 the


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electronic 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,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 vertically byenergy. They are grouped horizontally by spin multiplicity. In the left partof the diagram three singlet states with anti-parallel spins are shown: thesinglet ground state (S0) and two higher singlet excited states (S1 andS2). Singlet states are diamagnetic, as they do not interact with anexternal magnetic field. The triplet state (T1) is the electronic state withparallel spins. A molecule in the triplet state interacts with an externalmagnetic field. Transitions between electronic states of the same spinmultiplicity are allowed. Transitions between states with different spinmultiplicity are formally forbidden, but may occur owing to a processcalled spin-orbit coupling. This transition is called intersystem crossing.Superimposed on these electronic states are the vibrational states, whichare of much smaller energy. In the following sections we will address thesequence of processes, which occur when an aromatic molecule absorbsa 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 of light 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 the Born-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 N 1 is the number of molecules in the first higher vibrational state


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(S0,1), and N 0 that for the lowest vibrational level (S0,0), ΔE vib is theenergy difference between the two vibrational states, k B is the Boltzmannconstant, and T the absolute temperature. At room temperature, ΔE vib ismuch larger than k BT , 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 internal

conversion. 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 lowest

electronic 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 (ψ g) and excited (ψe) electronic states:

with μ=-er (e is charge of electron), and the integration takes place over

spatial 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).


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The lowest vibrational level of S1 is the starting point for fluorescenceemission 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 state

is 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 of argument, they are a sufficient representation, since we are dealing with(an)harmonic oscillators. Since the excited state is different from the

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ground state, a displaced minimum nuclear normal coordinate can beexpected. It should be noted further that the time to reach the excitedstate is so short (femtoseconds) that the nuclei positions are virtuallyunchanged during the electronic transition. In the vibronic wavefunctions,the nuclear coordinates can then be uncoupled from the electroniccoordinates (Born-Oppenheimer principle). The transition dipole can thenbe 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

[see Module on Basic Spectroscopy]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.

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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 light

and 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 I 0(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.


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Figure 7. Normalised absorption (left), and fluorescence(right) spectra of three fluorophores. Shown are the spectra of 2,5-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 of emission 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 λexcexcites the molecules in the sample. Fluorescence light is

emitted 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 of scattered excitation light in the fluorescence channel. Themonochromator M2 allows measurements of fluorescence


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intensity as a function of the emission wavelength. Thefluorescence spectra of PPO, NATA and HSA are shown in Figure7.

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 of maximal 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, I 0 isthe monochromatic light intensity, and Φ is the quantum yield of fluorescence (see below). This equation is valid only for dilutefluorophore solutions, in which the absorbance of the aromatic compound

never 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 n A. It can be shown also that Φ is theratio of the rate of the radiative transition (k r ) to the rates of alltransitions (k r +k nr ), 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 of


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fluorescence 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 beused to explain the basic kinetics of fluorescence.

Figure 9. Kinetic scheme to explain fluorescence lifetime. δ(0)is excitation with a δ-pulse, k r is rate constant of radiativetransition, and k nr 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(k r ) and non-radiative (k nr ) 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 F 0 (Figure 10).


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Figure 10. Fluorescence decay F (t ) = F 0 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

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 is

that 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 of the 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 of fluorophores by detecting the anisotropy of their fluorescence (Figure11).


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Figure 11. Geometry of fluorescence anisotropy experiment.Fluorophores in the cuvette are excited with short pulses of 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 I vv (t )is measured. Then the polarizer is moved to horizontalorientation, and I vh(t ) is detected.

Both decays, I vv (t ) and I vh(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 r 0 = 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, k BT 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'.


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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).

Figure 12. Changes in solute-solvent interactions lead tosolvatochromic shifts in absorption and fluorescence spectra of the 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 R g,

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 fieldR g, 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 thermodynamic

equilibrium (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:


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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:

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 (ΔU abs) and fluorescence (ΔU flu) 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 the

stronger is the "red-shift" of the spectrum.

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

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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 molecular mechanismsof quenching.

In case of static quenching, external molecules Q, simply form ground-state complexes with the fluorophore M (Figure 14). These complexes

can be excited, but are not fluorescent.

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 K s:

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, F 0 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 (k r ), 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 constantK SV = 0k q, in which o is the fluorescence lifetime without quencher, and


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k q is the diffusion controlled rate constant of quenching. K SV 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.

Figure 15. Schematic illustration of dynamic quenching andrelevant rate equations. The steady-state fluorescence intensityratio (F 0 /F ) (F is the fluorescence intensity in presence of quencher 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 K SV is equal to

0k q.

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 the excited-stateenergy from a donor molecule is transferred non-radiatively to anacceptor 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 transferk T :


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in which R is the distance between donor and acceptor molecules and Dis 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 obtaining

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

in which κ 2 is the orientation factor, Φ0 the quantum yield of donor

fluorescence (without acceptor), N A is Avogadro's number, and n is therefractive index of the intervening medium. The integral ( J , in units ofM-1cm3) is the degree of spectral overlap between donor fluorescencespectrum (F D, its spectrum normalized so that the integral is equal toone), and acceptor absorption spectrum (scaled to its maximum molarextinction coefficient, ε A, 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).CFP and YFP are mutants of Green Fluorescent Protein.

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The orientation factor κ 2 is given by:

For a definition of the angles we refer to Figure 18, in which somelimiting situations have been depicted illustrating that 0 < κ 2 < 4. Forsystems without any 3-dimensional, spatial information the orientation

factor is the indeterminate parameter in R0. All other parameters can bemeasured or evaluated.

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, ε A, 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:

For an exercise how to calculate the critical transfer distance fromspectroscopic data see Module in Experiments for Students: (A.J.W.G.Visser, E.S. Vysotski and J. Lee)

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 most

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straightforward, 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 of molecular interactions (Figure 19). From this reduction in lifetimes weimmediately obtain the experimental FRET efficiency:

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 light

pulse (δ(0)) excites donor D. Analysis of the generated donorfluorescence decay (F (t )) gives information on the rateconstant of transfer κ T 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 suitable

donor-acceptor pairs. Because of its sensitivity FRET also forms the basisfor "sensing" important biological molecules in many applications.


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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.

Time-Resolved Fluorescence Instrumentation: Measurement of thefluorescence lifetime, or generally of the parameters that govern the

course 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 of


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many 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 F c (t):

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 of two 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 of component 1, and 2 and 2 are the amplitude and lifetime of component 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 with

microchannel-plate photomultipliers, instrument response function widthsof thirty picoseconds are possible. This allows for the measurement of decay 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 several


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hundred 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 in

Figure 23.

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 of nonlinear 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 are

appropriate.

Luminescence From Semiconductor (Nano-)Crystals


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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 used inmodern electronics, including computers, telephones, LCD displays, etc.Semiconductors have also found applications in opto-electronics andphotophysics. Below we will briefly describe the working principles of quantum 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).


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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 is analogousto the creation of electrons, holes and excitons in semiconductormaterials after illumination, which are transported efficiently in thecrystal lattice to a p-n junction (see below). The function of 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 of impurities to a semiconductor is known as 'doping'. The amount of impurity, or dopant, added to a pure semiconductor varies its level of conductivity, and also its band gap. By incorporating impurity atoms intothe crystal lattice of pure semiconductors, the electrical conductivity may

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p-n Junctions: A p-n junction is a junction formed by joining p-type andn-type semiconductors together in very close contact (Figure 25). Theterm junction refers to the boundary interface where the two regions of the 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 of dopant 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, but

the junction between them is a non-conductor. This non-conducting layer,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 of low 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 almost

all opto-electronic devices such as LEDs, diode lasers, solar cells, etc.

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Figure 26. A p-n junction in thermal equilibrium with zero biasvoltage applied. Blue and red lines indicate the concentration of

electrons 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 conduction

band. 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 (κ BT ) is less than theexciton binding energy.

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

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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 of the crystal. The light emission is due to a phenomenon known asquantum confinement. Therefore these nanocrystals are called 'quantumdots'. 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 visible

throughout 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.

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 of many consecutive two-dimensional confocal images that can be


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reconstructed 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 of time (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 essentially

nontoxic.Light Emitting Diode (LED): An LED consists of a chip of semiconducting 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).

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 light

emission.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 injection

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distinguishes diode lasers from all other, optically pumped lasers.Therefore diode lasers are called "injection lasers". The initial mode of action 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 a

nanosecond 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 polarization andphase as the first photon. This means that stimulated emission causesgain in an optical wave in the injection region. The gain increases as thenumber of electrons and holes injected across the junction increases.

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 theyare 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 is

confined to the region where the amplification takes place.

Figure 30. Diagram of front view of a double heterostructure


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laser 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 as anti-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 cellshas 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 the

junction 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).

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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 and p-typesides of the solar cell (Figure 32).

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 until

they reach the p-type semiconductor-metal contact (aluminum). Herethey recombine with a hole. The hole was either created as an electron-hole pair on the p-type side of the solar cell or swept across the junctionafter being created at the n-side. The voltage measured is equal to thedifference in energy levels of the minority carriers, i.e., electrons in the

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n-type portion and holes in the p-type portion (V0 in panel 4 of Figure31).

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.

Engelborghs, Y. and Visser, A.J.W.G. Visser (eds.), FluorescenceSpectroscopy and Microscopy: Methods and Protocols, Methods inMolecular Biology, vol. 1076, Springer Science + Business Media, LLC,2014.

Jameson, D.M., Introduction to Fluorescence, CRC Press, 2014.

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. and Berberan-Santos, M.N., Molecular Fluorescence, 2nd ed.,Wiley-VCH, Weinheim, 2012.

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

A very useful review on cellular applications of quantum dots can befound in: Michalet, X., Pinaud, F.F., Bentolila, L.A., Tsay, J.M., Doose, S.,Li, J.J., Sundaresan, G., Wu, A.M., Gambhir, S.S. and Weiss, S., QuantumDots for Live Cells, in Vivo Imaging, and Diagnostics, Science 307, 538-544, 2005.

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

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

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