Handbook of Fluorescence Spectroscopy and Imaging: From Single Molecules to Ensembles

Handbook of Fluorescence Spectroscopy and Imaging: From Single Molecules to Ensembles
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Mul tichrornophoric Labels 2. Nanocrystals 3. In Vitro Fluorescence Labeling 3. Fluorescence Labeling in Living Cells 5. Introduction 5. Optical Set-Up 5. Data Acquisition and Evaluation 5. Milliseconds to Seconds: Diffusion and Concentration 5. Single-Focus FCS 5. Antibunching 5. Rotational Diffusion 5. Fluorescence Lifetime Correlation Spectroscopy 5. Conclusion 6. Introduction 6. Single-Laser Excitation 6. Light-Harvesting Systems: Phycobilisomes and Allophycocyanins 6. Hairpin Ribozyme Dynamics and Activity 6. Protein Un folding and Dynamics 7.

Fluorescence Quenching by PET 7. PET Reporter System 7. Biological and Diagnostic Applications 8. Diffraction Barrier of Optical Microscopy 8. Multi-Photon and Structured Illumination Microscopy 8. Stimulated Emission Depletion 8. Single-Molecule Based Photoswitching Microscopy 8. Single-Enzyme Studies and Kinetics 9. Conformational Dynamics 9. Shedding Light on Single-Enzyme Mechanisms 9. Lipase-Catalyzed Hydrolysis of Phospholipid Bilayers 9. Threshold Method 9. Autocorrelation Analysis 9. Conclusions References. Notes Online version of the print title. Formerly CIP.

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Includes bibliographical references and index. Access Conditions Online access restricted to licenced organisations. Mode of access: World Wide Web. View online Borrow Buy Freely available Show 0 more links Set up My libraries How do I set up "My libraries"? Open to the public ; University Library. Open to the public. One of the most widely used applications of absorption spectroscopy is the determination of the concentration of substances in solution.

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Molecular aggregates are macroscopic clusters of molecules with sizes intermediate between crystals and isolated molecules. In the mids Scheibe [4] and, independently Jelley [5] discovered that when increasing the concentration of the dye pseudoisocyanine PIC in water, a narrow absorption band arises, red shifted to the monomer band. The narrow absorption band was ascribed to the optical excitation of the aggregates formed. To form the simplest aggregate, a dimer, the dye—dye interaction must be strong enough to overcome any other forces which would favor solvation of the monomer.

For the majority of possible dimer geometries, two absorption bands arise, one at higher energy relative to the monomer band, termed H-type aggregates absorption band shifted hypsochromic , and at lower energy relative to the monomer j9 j 1 Basic Principles of Fluorescence Spectroscopy 10 H-aggregate S1 S0 J-aggregate S1 Monomer Dimer S0 Monomer Dimer Figure 1. In fact, with the exception of a few examples [13], the non-emissive character of the excited state has become commonly accepted as a general feature of H-aggregates.

According to exciton theory of Kasha et al. After exciting the H-exciton band, a rapid downwards energy relaxation occurs to the lower exciton states that exhibit vanishingly small transition dipole moments. These two different types of aggregates, the J- and Haggregates, are distinguished by the different angle a between the molecular transition dipole moments and the long aggregate axis. The appearance of isosbestic points at and nm indicates the formation of 1 : 1 complexes for tryptophan concentrations less than 20 mM [12].

In multicomponent solutions isosbestic points almost never occur because the probability that three or more compounds have identical molar absorbances at any wavelength is negligibly small. Owing to this low probability, the occurrence of two or more isosbestic points demonstrates the presence of two and only two components absorbing in the observed spectral region.

However, this rule does not apply if two chemically distinct components have identical absorption spectra e. In this case the entire spectrum represents a set of isosbestic points for these two components alone. Isosbestic points are especially useful for the study of equilibrium reactions involving absorbing reactants and products. Both types absorb in the same region. At higher tryptophan concentrations above 20 mM slight deviations can be observed indicating a low probability of the formation of higher aggregated complexes. In addition, collisions and electrostatic interactions with surrounding solvent molecules broaden the lines of vibrational transitions.

This results in a quasicontinuum of states superimposed on every electronic level. The population of the levels in contact with the thermalized solvent molecules is determined by the Boltzmann distribution. In the quantum mechanical picture, vibrational levels and wavefunctions are those of quantum harmonic oscillators and rigid rotors with some corrections for rotation— vibration coupling and centrifugal distortion Figure 1. The more realistic anharmonic potential e. Excitation of a bound electron from the HOMO to the LUMO increases the spatial extent of the electron distribution, making the total electron density more diffuse, and often more polarizable.

In general, polyatomic molecules will have 3N — 6 vibrational modes n1, n2, n3,. For each of the 3N — 6 normal vibrations, a potential well exists with a rotational energy ladder. As electronic transitions are very fast compared with nuclear motions, vibrational levels are favored when they correspond to a minimal change in the nuclear coordinates.

In the simplest case of a diatomic molecule, the nuclear coordinate axis refers to the internuclear distance. Figure 1. Upon absorption of a photon of the necessary energy, the molecule makes a so-called vertical transition to the excited electronic state. The occurrence of vertical transitions on the potential energy curve is explained by the Franck—Condon principle and the Born—Oppenheimer approximation. The Born—Oppenheimer approximation is based on the fact that the proton or neutron mass is roughly times that of an electron and that electrons move much faster than nuclei.

Applying the Born—Oppenheimer approximation to transitions between electronic energy levels, led Franck and Condon to formulate the Franck—Condon principle. Classically, the Franck—Condon principle is the approximation that an electronic transition is most likely to occur without changes to the position of the nuclei in the molecular entity and its j13 j 1 Basic Principles of Fluorescence Spectroscopy 14 environment.

The quantum mechanical formulation of this principle is that the intensity of a vibrational transition is proportional to the square of the overlap integral between the vibrational wavefunctions of the two states that are involved in the transitions [21—24]. In other words, electronic transitions are essentially instantaneous compared with the time scale of nuclear motions. Therefore, if the molecule is to move to a new vibrational level during the electronic transition, this new vibrational level must be instantaneously compatible with the nuclear positions and momenta of the vibrational level of the molecule in the originating electronic state.

In the semiclassical picture of vibrations of a simple harmonic oscillator, the necessary conditions can occur at the turning points, where the momentum is zero. This is the reason why electronic transitions cannot occur without vibrational dynamics. Thus, the absorption is practically continuous all across the absorption band. On the other hand, at lower temperatures, the spectral widths are usually reduced and the spectra exhibit enhanced vibrational information Figure 1. Therefore, dye solutions that form a clear organic glass when cooled down to 77 K show spectra comparable to theoretical calculations because of their well-resolved vibrational structure.

Further cooling below the glass point, when the free movement of solvent molecules or parts thereof is inhibited, usually brings about no further sharpening of the spectral features. Generally speaking, the zero-phonon line and the phonon sideband jointly constitute the line shape of individual light absorbing and emitting molecules embedded into a transparent solid matrix.

A phonon is a quantized mode of vibration occurring in a rigid crystal lattice, such as the atomic lattice of a solid. When the host matrix contains many chromophores, each will contribute a zero-phonon line and a phonon sideband to the absorption and emission spectra. The distribution of intensity between the zero-phonon line and the phonon sideband is strongly dependent on temperature. At room temperature the energy is high enough to excite many phonons and the probability of zero-phonon transitions is negligible. At lower temperatures, in particular at liquid helium temperatures, however, being dependent on the strength j15 j 1 Basic Principles of Fluorescence Spectroscopy 16 of coupling between the chromophore and the host lattice, the zero-phonon line of an individual molecule can become extremely narrow.

On the other hand, the centers of frequencies of different molecules are still spread over a broad inhomogeneous band. Inhomogeneous broadening results from defects in the solid matrix that randomly shift the lines of each individual molecule. Therefore, for each particular laser frequency, resonance is achieved for only a small fraction of molecules in the sample [26, 27]. The shape of the zero-phonon line is Lorentzian with a width determined by the excited-state lifetime T10, according to the Heisenberg uncertainty principle. The lattice reduces the lifetime of the excited state by introducing radiationless decay mechanisms.

These perturbations shift the energy of the electronic transition, introducing a temperature dependent broadening of the line width. Upon excitation, the molecule might undergo a number of possible photophysical and photochemical processes, such as intersystem crossing into long-lived triplet states that exhibit different absorption characteristics generally lower at the excitation wavelength. Thus, the sample will absorb less at the frequency of illumination during the lifetime of the triplet state.

If an absorption spectrum of the sample is measured it will show a transient spectral hole at the laser frequency used to excite the sample Figure 1. Some of the product states may have very long lifetimes, in particular if the molecule undergoes a chemical reaction. In that case, the spectral hole is 1. Different environments in a disordered matrix shift the zero-phonon lines of single molecules at random.

The figure shows the inhomogeneous absorption spectrum of an ensemble of molecules before a and after b illumination at a specific laser frequency nL. The sharp spectral hole appears because the narrow lines of the excited molecule are shifted to new frequencies. The resulting antihole is much broader than the hole and can only be seen after very deep and broad holes have been burned. Alternatively, the molecule might be excited into higher singlet states by absorption of a second photon.

Singlet—singlet absorption S1! Sn and subsequent ionization of the molecule represents a possible photobleaching pathway. According to the Franck—Condon principle, the vertical transition to higher excited vibrational levels of S0 is followed by vibrational relaxation until thermal equilibrium, according to the Boltzmann distribution, is reached. The probability 1. For example, the lowest singlet vibrational level can overlap one of the higher vibrational levels of the triplet state. However, it is known [31] that the presence of heavy atoms can substantially increase the intersystem crossing rate constant.

A molecule in a high vibrational level of the triplet state can lose energy through collisions with solvent molecules vibrational relaxation , leaving it at the lowest vibrational level of the triplet state. It can then again undergo intersystem crossing to a high vibrational level of the electronic singlet ground state, where it returns to the lowest vibrational level through vibrational relaxation. As in the case of singlet states, triplet states can be excited into higher excited triplet states, Tn, by absorption of a second photon. Because internal conversion and other radiationless transfers of energy compete so successfully with radiative deactivation, phosphorescence is usually seen only at low temperatures or in highly viscous media.

Owing to the long lifetime of the triplet state and distinct overlap of the T1! Tn and the S0! S1 absorption spectra of most organic dye molecules, triplet states are most probably involved in photobleaching pathways [32, 33]. Furthermore, as higher excited states are in general more reactive than their underlying ground states, they are the most likely to be involved in photobleaching pathways.

Generally, rhodamine, oxazine, or carbocyanine derivatives are used in applications requiring high sensitivity. To minimize triplet—triplet absorption, the triplet state has to be depopulated by addition of triplet quenchers such as cyclooctatetraene COT or molecular oxygen.

Thus, the dye will be transferred into the singlet ground state upon contact formation with triplet quenchers. On the other hand, triplet quenchers such as anthracence, stilbene, or naphthalene derivatives all exhibiting low lying triplet states can be j19 j 1 Basic Principles of Fluorescence Spectroscopy 20 Table 1. Internal conversion Internal conversion Vibrational relaxation Singlet—singlet absorption Fluorescence Intersystem crossing Phosphorescence Triplet—triplet absorption Sn!

S1, Tn! T1 S1! Sn S1! S0 S1! T1, Sn! Tn, Tn! Sn T1! S0 T1! Table 1. Because the electron distribution changes upon excitation, different bonding forces and dipole moments arise. In contrast, the orientational polarization of the solvent molecules does not change 1. Therefore, the orientational polarization is not in equilibrium with the excited molecule.

Typically, dielectric relaxation is completed in 10 ps. A thermal blooming measurement is, in essence, a calorimetric determination of the very small temperature gradients induced by the absorption of light energy. The technique can be extremely sensitive and allows one to measure exceptionally weak absorption [52—56]. The basic idea involving power conservation is very simple. The laser power that is incident on any sample must be 1.

The thermal lens develops over a period of a few tenths of seconds. The pulse method, that is, time-correlated single-photon counting TCSPC [59—61], features high sensitivity and the ability to deal with low photon count rates with a time resolution down to the ps region, that is, typical parameters to be handled in single-molecule experiments.

As it is a counting process it is inherently digital in nature. Usually, the excitation light pulse is split such that a photodiode is triggered at the same time that the sample is excited. It is as if a stopwatch is started at this point. The TCSPC measurement relies on the concept that the probability distribution for emission of a single photon after an excitation yields the actual intensity versus time distribution of all photons emitted as a result of the excitation.

Pile-up results from the fact that a TCSPC experiment can record only one photon per excitation pulse. To prevent pile-up effects in TCSPC measurements, the power of the excitation light should be adequately reduced. The charging linear voltage ramp of the TAC is stopped by the regular electronic output of the photodiode, which represents the highly stable and exact repetition rate of the optical excitation. Subsequently, a pulse is output from the TAC, the amplitude of which is proportional to the charge on the ramp, and, hence, the time between start and stop.

It has to be pointed out here that the TAC is run in the inverted mode, so that each photon that is detected is counted. The pulse height is digitized by an analog-to-digital converter and a count is stored in a multi-channel analyzer MCA in an address corresponding to that number. These components are contained on a PC card. Depending on the number of channels used, at least several thousand counts should be accumulated at the peak Figure 1. The resolution of the TCSPC measurements is limited by the spread of the transit times in the detector, by the timing accuracy of the discriminator that receives the detector pulses, and by the accuracy of the time measurements.

The width of the time channels of the histogram can be made to be less than 1 ps. For excitation, a pulsed diode laser with a repetition rate of 10 MHz and a pulse length of ps FWHM at nm is used. The IRF is measured at the excitation wavelength using a scattering solution. One limitation to the analysis of TCSPC measurements is the fact that, in general, the length of the laser pulse can not be neglected Figure 1. Therefore, alternative strategies appear to be more suitable.

In particular, for identifying molecules based on 1. For ideal single-molecule data, it was found that the neural networks and the MLE perform almost equally well. However, the maximum anisotropy, r0, which corresponds to the limit of a transparently frozen solvent, can only be measured infrequently because of reabsorption and the different energy transfer processes that promote depolarization.

Complete loss of anisotropy occurs when detection is performed below an angle of Multiexponential anisotropy decays imply that the molecule under investigation exhibits unsymmetrical geometry. With the aid of the rotational correlation time, measured References from time-resolved anisotropy measurements, the rotational volume of the molecule V in the solvent of viscosity g at temperature T can be determined using the gas constant, R. Kuhn, H. Scheibe, G. Jelley, E. Herz, A. Colloid Interface Sci. Kasha, M.

Czikklely, V. Rabinowitch, E. Doose, S. Heinlein, T. B, , — Seidel, C. Mataga, N. Ketelaar, J. Pays-Bas, 71, Franck, J. Faraday Soc. Condon, E. Shpolski, E. Moerner, W. Tamarat, Ph. A, , 1— Orrit, M. Wild, U. Labhart, H.

Single-molecule FRET of protein structure and dynamics - a primer

Liphardt, B. Tsien, R. Pawley Plenum Press, New York, p. Widengren, J. A, , Asimov, M. Menzel, R. Tinnefeld, P. A, , — Maroncelli, M. Jarzeba, W. Akesson, E. Jiang, Y. Reichardt, C. Lippert, E. Suppan, P. A: Chem. Drexhage, K. Strickler, S. Demas, J. Flu, C. Long, M. Twarowskl, A. Brannon, J. Lakowicz, J. Bollinger, T. Becker, W. Enderlein, J. A, , 48— Maus, M. Herten, D. B, 71, Sauer, M. Bowen, B. Tellinghuisen, J. Kullback, S. Most proteins and all nucleic acids are colorless in the visible region of the spectrum.

However, they exhibit absorption and emission in the ultraviolet UV region. Upon binding to proteins, the quantum yield of NADH generally increases drastically, thus enabling the relatively sensitive detection of native proteins carrying an NADH residue. They are found in light harvesting structures phycobilisomes and are used as accessory or antenna pigments for photosynthetic light collection. The phycobilisomes allow the various pigments to be arranged geometrically in a manner that helps to optimize the capture of light and transfer of energy. Phycobiliproteins are composed of a number of subunits, each having a protein backbone to which linear tetrapyrrole chromophores are covalently bound.

All phycobiliproteins contain several phycocyanobilin or phycoerythrobilin chromophores [12—14, 16]. Each bilin has unique j33 j 2 Fluorophores and Fluorescent Labels 34 Table 2. B-phycoerythrin is compromised of three polypeptide subunits forming an aggregate containing a total of 34 bilin chromophores. Phytochromes are large proteins with covalently bound linear tetrapyrrole, that is, bilin, chromophores that transduce light signals by reversibly photointerconverting between red-light-absorbing and far-red-light-absorbing species, a process that typically initiates a transcriptional signaling cascade [26].

The N-terminal region of the protein contains two domains of about amino acids LOV1 and LOV2 , which are regulated by environmental factors that affect their redox status: light, oxygen, or voltage LOV [32—34]. As the transition moment for this process is typically very large, the corresponding absorption bands exhibit oscillator strengths of the order of unity. The reverse process S1! Furthermore, time- and position-resolved detection without contact with the analyte is possible. Furthermore, the dye is fairly hydrophilic.

Subsequently, the isocyanate has been replaced as the active intermediate for covalent coupling by isothiocyanate and N-succinimidyl esters NHS , being more convenient and safe derivates. The process involves the conversion of the lowest vibronic level of the excited state to a higher vibronic level of the ground state. The mechanism is of minor 2.

Firstly, owing to the relatively long lifetimes of triplet states, the chromophore might be excited into higher excited triplet states and undergo irreversible chemical reactions, that is, it might photobleach. Furthermore, the intrinsic intersystem crossing rate can be enhanced if the dye is substituted with heavier elements, which increase the spin—orbit coupling [46].

Coumarins can essentially be described by two mesomeric forms, one nonpolar form with a low dipole moment and a more polar form with a higher dipole moment where a positive charge is located on the nitrogen atom and a negative charge is on the oxygen atom Figure 2. In the electronic ground state S0 of coumarins, the nonpolar mesomeric structure is predominant and the polar form makes only a minor contribution to the actual p-electron distribution. The more polar mesomeric form is stabilized if the dye molecule is surrounded by polar solvent molecules.

Therefore, the absorption maximum of coumarin dyes is generally shifted to longer wavelengths with increasing solvent polarity. In the electronic excited state S1, the more polar mesomeric form is predominant. That is, the electric dipole moment in coumarin dyes increases upon optical excitation.

This induces the rearrangement of the surrounding solvent molecules and stabilizes the excited state, which lowers the energy of the excited state considerably. Therefore, coumarin derivatives exhibit a large Stokes shift as compared with, for example, rhodamine or oxazine dyes. Figure 2. Usually, coumarin derivatives are coupled covalently to biomolecules using activated carboxyl functions, as for example in the case of 7-diethylaminocoumarinacetic acid Figure 2.

In methanol Coumarin exhibits an absorption and an emission maximum of and nm, respectively. A dye that is appropriate for this purpose is Coumarin Depending on the redox properties of the DNA base, the dye is reduced or oxidized in its excited state. Hence, this additional photobleaching pathway limits the applicable irradiance for OPE. Furthermore, TPE is deteriorated by other competing nonlinear processes e. They concluded that the single-molecule detection sensitivity of Coumarin molecules is enhanced substantially by using TPE, primarily due to the higher background with OPE at UV wavelengths.

The emission maxima of the three coumarins are located around , , , and nm, respectively. Today, most bio analytical applications requiring high sensitivity use xanthene dyes that absorb and emit in the wavelength region from to nm. The p-electron distribution in the chromophore of the xanthene dyes can be described approximately by two identical mesomeric structures, in which the positive charge is located on either of the two nitrogen atoms Figure 2. Unlike the coumarin dyes, the two forms have the same weight, and thus in xanthene dyes there is no static dipole moment parallel to the long axis of the molecule in either the ground or excited states.

The transition moment of the main long-wavelength absorption band is oriented parallel to the long axis of the molecule. The absorption spectrum of xanthene dyes is determined by the symmetrical p-electron system extending across the diaminoxanthene frame. Because the dipole moment does not change upon excitation, the absorption maximum shows only little dependence on the polarity of the solvent.

On deprotonation of the carboxyl group, for example, in Rhodamine B or Rhodamine Figure 2. These effects can be attributed to some type of mobility of the diethylamino groups in the excited state, which is enhanced by increasing temperature and reduced by increasing viscosity.

Usually, xanthene dyes are coupled to analytes via an additional reactive j41 j 2 Fluorophores and Fluorescent Labels 42 Table 2. Fluorescence quantum yields are given only for ethanolic dye solutions. To ensure protonation of the o-carboxyl group in Rhodamine B and Rhodamine , measurements were performed in aqueous buffer at pH 3. As the carboxyphenyl substituent is held in a position almost perpendicular to the xanthene moiety, it is not part of the chromophore system.

In Rosamine 1 Figure 2. Rhodamine dyes that carry a free o-carboxyl group can exist in several forms. The deprotonation is enhanced by dilution or by adding a small amount of a base. The deprotonated zwitterionic form exhibits an absorption maximum shifted to shorter wavelengths 3—10 nm. In nonpolar solvents such as acetone, the zwitterionic form is not stable and forms an intramolcular lactone in a reversible fashion. The lactone is colorless because the p-electron system of the dye is interrupted. A further interesting characteristic of xanthene derivatives consists in the fact that they are selectively quenched upon contact formation with the DNA base guanine and the amino acid tryptophan via photoinduced electron transfer [66—74].

Both guanine and 2. The molecular structures of the Alexa derivatives Alexa , Alexa , Alexa , Alexa , Alexa , and Alexa are also based on the xanthene chromophores shown in Figure 2. This fact has prompted current efforts towards the use of NIR dyes for bioanalytical applications. As already mentioned, hydrogen vibrations in particular lead to a decreased quantum yield in the red-near-IR region.

The quanta of hydrogen vibrations have the highest energies for organic compounds. Thus, hydrogen vibrations are most likely to contribute to internal conversion between S1 and S0. The most important of these advantages is the reduction of the background signal, which ultimately improves the sensitivity achievable. Cyanine dyes are widely used in ultrasensitive imaging and spectroscopy, especially for biological applications. The absorption can be tuned through the visible and near-infrared region by variation of the length of the polymethine chain joining the two heads of the cyanine dye.

As can be seen in Table 2. In addition, cyanine dyes are less photostable than xanthene dyes, especially under single-molecule conditions [84] and some derivatives Cy5 are destroyed by environmental ozone [85]. Cy5, for example, exhibits a fluorescence quantum yield of 0. Cyanine derivative labs nm lem nm Cy3 Cy3B Cy3. The rate constant for cis—trans isomerization in water was measured to be 2.

To reduce cis—trans isomerization in Cy3B, the polymethine structure has been made more rigid by the incorporation of three 6-membered rings Figure 2. However, a method of stabilizing normal cyanine dyes in solution has been developed and is used extensively in single-molecule experiments, by applying an oxygen scavenging system and thus retracting the main reason for the photobleaching of the cyanine dyes [90].

The oxygen scavenging system is composed of phosphate-buffered saline PBS , pH 7. To quench the lifetime of the triplet states in the absence of oxygen, mM b-mercaptoethylamine MEA is added as a triplet quencher. Consequently, cyanine derivatives have emerged as a set of standard dyes in many multicolor singlemolecule assays.

In addition to cyanine derivatives, xanthene derivatives are also available with absorption and emission wavelengths above nm. For example, it has been known for a long time that if the central carbon group of a pyronin or rhodamine dye is replaced by a nitrogen atom, a compound is obtained whose absorption and emission are shifted by about nm to longer wavelengths [46]. Such planar oxazine derivatives are rigid and exhibit suitable spectroscopic characteristics in the wavelength region — nm. The exchange of the carboxyphenyl substituent by an electron-accepting group at the central carbon has an effect similar to the introduction of a nitrogen atom see Figure 2.

In the case of a cyano group, the resulting rhodamine derivative exhibits absorption and emission spectra shifted by about nm. By introduction of an electron acceptor at this position, as in case of Rhodamine , or exchange of the methane group by a more electronegative atom, as in case of oxazine 1, the energy of the LUMO decreases, resulting in a decreased excitation energy. Owing to the two additional double bonds, the absorption and emission maxima shifts to longer wavelengths are of about 30 nm Table 2. Alternatively, the oxygen atom in rhodamine derivatives can be exchanged by a tetrahedral carbon atom.

The absorption maxima of the resulting carbopyronin or carborhodamine dyes e. Exceptions are dyes derived from JA 22 Figure 2. Finally, it has to be noted that all long-wavelength absorbing rhodamine, oxazine, and carborhodamine or carbopyronin derivatives show a much higher photostability than their related cyanine derivatives. These so-called Bodipy dyes are unusual in that they are relatively nonpolar, they exhibit only a small Stokes shift, and the chromophore is electrically neutral.

Unfortunately, all triphenylmethane and related dyes show a tendency to react at the central carbon with nucleophiles, for example, hydroxide ions, if this carbon is sterically available. Therefore, some of the above mentioned derivatives become colorless on addition of a base to their aqueous solutions, due to the formation of a so-called pseudobase. In the pseudobase the p-electron system is interrupted and therefore the long-wavelength absorption is lost.

Although this process is reversible, subsequent reactions, for example, with oxygen, may lead to an irreversible destruction of the dye. It has to be pointed out that the tendency for the formation of the pseudobase is strongly controlled by the structure of the dye. Furthermore, other destructive, that is, irreversible, reactions also tend to increase with increasing absorption maximum.

Therefore, rhodamine derivatives, such as JA 22 or Alexa , are completely stable even in strong basic solutions. The comparably high photostability of rylene dyes immobilized in different polymeric matrices enables the observation of photophysical processes, for example, photon antibunching and electron or energy transfer, at the single-molecule level over extended periods of time [93—98].

Perylenetetracarboxdiimide PDI represents the key structure from 2. Depending on the substitution pattern, PDI exhibits an absorption maximum between and nm with a typical Stokes shift of 30—40 nm. One limitation of perylene derivatives is represented by their low water solubility. Therefore, strategies based on the incorporation of sulfonated phenoxy groups have been developed to make new red-absorbing water-soluble TDI derivatives available for biological labeling applications [93]. Classical semiconductor materials are based on the electronic structure of the elements of the fourth row IV of the Periodic Table.

Although most of the outer shell electrons are located in the valence band, even at room temperature, there is a certain fraction that is excited into the conduction band and is responsible for the conductive properties of semiconductors. Generally, electrons and holes can move freely within the bands. For such a particle within a boundary, that is, a particle in the box, the allowed energy states can be calculated according to Section 1.

Thus, the exciton energy can be controlled by size reduction. The crucial step for fabrication of such small particles is the controlled growing procedure, which ideally leads to crystals of similar composition, similar structure and nearly equal size monodisperse particles.

Growing of particles starts from an atomic precursor of both materials involved. As soon as clusters have been formed, there are two competing reactions in the supersaturated solution: growth of initially formed clusters and formation of new nuclei. As the latter will lead to increasing heterodispersity, the nucleation process has to be interrupted instantaneously, for example, by a sudden change in temperature. The emission maximum is only slightly red-shifted to the longestwavelength absorption shoulder corresponding to the ground state of the exciton.

The absorption in NCs stems from the photoinduced creation of discrete e—h pair combinations.

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New citations to this author. However, true understanding of protein folding requires deciphering the sequence of structural events along the folding pathways between the folded and unfolded states. Both CW or pulsed lasers can be used as excitation source. Probing the stoichiometry of membrane proteins by single-molecule localization microscopy. Zheng, Q. INS Plasmonics and its applications [Cr:4, Lc:3, Tt:1, Lb:0] Course Outline Plasmonics has been an exponentially growing field for the past century and is a key concept and research tool in biology, chemistry and physics.

Hence, the absorption would probably be expected to consist of narrow bands instead of a broad band distribution. However, owing to the slight heterodispersity of the sample, inhomogeneous line width broadening results. The overlap of the broadened bands with different absorption cross-sections results in the observed absorption spectra. Cooling of the reaction mixture to temperatures 0. Other materials used to increase the water solubility, and through this enabling biological imaging applications to be made, include coating with silanes [, ], peptides [—], and ambiphilic polymers [—].

Although reports showing NC biocompatibility actually appeared a few years ago [, ], their breakthrough for biological targeting was only very recent [—]. At present, NCs can be easily functionalized, for example with streptavidin, to facilitate mild coupling to biotinylated biomolecules Figure 2. However, NCs also posses serious limitations. Instead, during the labeling step, NCs tend to bind to several molecules simultaneously. Another problem is blinking, which is strongly controlled by the excitation intensity Figure 2.

In addition, not all semiconductor particles are generally active, that is, luminescent, but they can be photoactivated []. To explain the strong blinking phenomenon it is assumed that trapping of an electron occurs, induced by photon absorption. Trapping means that the electron is emitted into the surrounding of the NC, thus leaving a charged NC behind.

After a certain time, the electron can be released back into the NC, neutralizing the charge and resulting in an emissive NC. Furthermore, the technique that was applied enabled the exact determination of the photoluminescence quantum yield of single NCs. In this study, average quantum yields of 0. Very recently [], it was shown that single NCs show multiexciton emission under high power excitation. This means that it is possible to create more than one e—h pair per NC. In contrast to organic dyes e—h pairs can coexist in NCs because they are Bosons.

Another issue is whether such particles, which are composed of seemingly toxic material, are well suited for in vivo studies and whether they retain biological functionality. For example, besides their core—shell structure, commercially available NCs have a third layer — an organic surface coating — to provide chemical and photophysical stability, inertness in different environments, buffer solubility, and to introduce reactive groups for linking to biomolecules. Ultimately, this results in particle sizes of 15—25 nm in diameter Figure 2. Nontoxic noble metal nanoclusters composed of only a few atoms also show very strong, robust, discrete, size-dependent emission but with much smaller sizes than those of semiconductor nanocrystals [—].

References 1 Teale, F. USA, 99, — Acta, , — Acta, 83, — References 8 Wellner, D. Weber, G. Lu, H. MacColl, R. Glazer, A. Acta, , 29— Krause, G. Plant Physiol. Plant Mol. Kronick, M. Methods, 92, 1— Mathies, R. Wehrmeyer, W. Wu, M. Nguyen, D. Peck, K. USA, 86, Oi, V. Cell Biol. Ying, L. Butler, W. USA, 45, — Briggs, W. Rockwell, N. Plant Biol.

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Handbook of Fluorescence Spectroscopy and Imaging | Wiley Online Books

Fleury, L. Soep, B. Corrie, J. Perkin Trans. Reynolds, G. Jones, G. Han, K. Eggeling, C. Brand, L. Davidson, R. Jakobi, H. Knemeyer, J. Neuweiler, H. Panchuk-Voloshina, N. Soper, S. Boyer, A. Southwick, P. Mujumdar, R. Terpetschnig, E. Mujumdar, S. Buschmann, V.

Chem A, , — References 85 Fare, T. Ha, T. Heilemann, M. Bates, M. Herrmann, A. Hofkens, J. Vosch, T. Int Ed. Vogt, R. Methods, , 57— Zhao, X. Schaertl, S. Diamanidis, E. Kessler, M. Juris, A. Liebsch, G. Lacoste, T. USA, 97, — Roederer, M. Gerstner, A.

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Molecular Probes Tutorial Series— Anatomy of Fluorescence Spectra

Hohng, S. Cordero, S. Brokmann, X. Biebricher, A. Fisher, B. Zheng, J. Wilcoxon, J. Rabin, I. Fluorescence labeling is used to investigate localization, interactions, and movement of interesting biological molecules. Reactive groups able to couple with amine-containing molecules are by far the most common functional groups used. Most of these reactions are rapid and occur in high yield to give stable amide or secondary amine bonds. For labeling experiments to the amine groups of bio molecules it has to be considered that buffers containing free amines such as tris hydroxymethyl aminomethan Tris , ammonium sulfate, and glycine must be avoided or removed before the reaction.

Handbook of Fluorescence Spectroscopy and Imaging. Aliphatic amines such as the e-amino group of lysine are moderately basic and reactive towards most acylating reagents. However, the concentration of the free base form of aliphatic amines below pH 8. Therefore, pH values of 8. Furthermore, it has to be considered that acylation reagents tend to degrade in the presence of water with increasing pH value.

Therefore, a compromise between the reactivity of the amine group and the degradation of the acylation reagent in aqueous buffers has to be found for each coupling reaction. In other words, reaction time and pH value have to be carefully optimized. Aromatic amines are very weak bases and thus they are unprotonated at pH 4.

Handbook Of Fluorescence Spectroscopy And Imaging. From Ensemble To Single Molecules

In aqueous solution, acylating reagents are virtually unreactive with the amino group of peptide bonds and with the side-chain amides of glutamine and asparagine residues, the guanidinium group of arginine, the imidazole group of histidine and the amines found in natural nucleotides.

Today N-hydroxysuccinimide NHS esters are most commonly used for coupling to amino groups. To prepare stable NHS ester derivatives, the activation has to be performed in nonaqueous solvents. Thus, in protein molecules, NHS esters can be used to couple principally with the a-amines at the N-terminals and the e-amines of lysine side chains, depending on the pH value, that is, on the degree of deprotonation of the amines.

The reaction of NHS esters with thiol or hydroxyl groups does not yield stable conjugates. NHS esters can also be prepared in situ to react immediately with amines of the target molecules in aqueous solvents. Furthermore, sulfo-NHS esters hydrolyze more slowly in water. Usually, NHS esters have a half-life of the order of hours under physiological pH conditions, but both hydrolysis and amine reactivity increase with increasing pH. Fluorophores can be activated as NHS esters or derivatives, isothiocyanates or sulfonyl chlorides to form carboxamides, thioureas or sulfonamides upon reaction with aliphatic amines.

As an example, fluorescein-NHS and its reaction with the amino group of the aromatic amino acid tryptophan is shown in the first line. TFP esters are stable for several hours even under basic pH 8. Isothiocyanates react with nucleophiles such as amines, thiols, and the phenolate ion of tyrosine side chains [1, 4]. The only stable product of these reactions, however, is with primary amine groups. On the other hand, the isothiocyanate group is relatively unstable in aqueous solution.

Alternatively, isocyanates exchanging the sulfur in an isothiocyanate by an oxygen atom can be used to react with amines. However, the reactivity of isocyanates is even greater than that of the isothiocyanates, which renders their application more complicated due to stability and storage problems. Reaction of a sulfonyl chloride with an amine is best performed at pH 9. In addition, sulfonyl chlorides can be used to couple to target molecules in organic solvents.

On the other hand, sulfonyl chlorides should be stored under nitrogen or in a desiccator to prevent degradation by moisture. Furthermore, thiol-reactive groups are frequently present on one of the two ends in heterobifunctional cross-linkers. The other end of such cross-linkers is often an amine-reactive functional group that is coupled to a target molecule before the thiol-reactive end, due to the comparable labile nature of the amine alkylation chemistries. However, such approaches are cumbersome and, relatively, not very promising.

Furthermore, many proteins are either devoid of cysteine or intrinsic cysteine residues can be removed by site-directed mutagenesis. The common thiol-reactive functional groups are primarily alkylating reagents, including maleimides, iodoacetamides, and aziridines. Reaction of these functional groups with thiols proceeds rapidly at or below room temperature in the pH range 6.

The high reactivity of most thiols even at pH values below 7. Maleic acid imides maleimides are derivatives of the reaction of maleic anhydride with amines. The double bond of the maleimide undergoes an alkylation reaction with the thiol groups to form stable thioether bonds. At pH 7. At higher pH values some cross-reactivity with amino groups takes place. Maleimides do not react with methionine, histidine or tyrosine. Fluorophoremaleimides are usually synthesized in a two-step reaction. Firstly, one amino group of a diamine, for example, ethylenediamine, is converted into a maleimide by reaction with maleic anhydride.

Iodoacetamides readily react with thiols, including those found in peptides and proteins, to form stable thioethers Figure 3. As an example, Alexa Fluor C5maleimide and its reaction with cysteine is shown in the first line. Besides iodoacetamides, aziridines can be used to modify thiol groups in proteins. Thiols react with aziridines in a ring-opening process, forming thioether bonds Figure 3. However, in aqueous solution considerable hydrolysis occurs as an undesired side reaction.

This intramolecular cross-linking strategy, which has only been used for proteins with an existing high-resolution structure, can be used advantageously to monitor the orientation and dynamics of protein domains or other protein structural elements [14, 15]. Nevertheless, the determination of the number of actual double labeled products, that is the exact fraction of cross-linked proteins, is challenging and seriously complicates the data interpretation.

Further detailed description of bioconjugation chemistry would go beyond the scope of this book. The interested reader is referred to the literature where excellent books about bioconjugation chemistry can be found, for example, [1]. This is where the full length protein is assembled from differently labeled synthetic or biosynthetic peptide fragments.