This thesis concerns the development and application of time resolved fluorescence which yield new and detailed information on molecular order and motion in both isotropic and ordered systems. Polarised fluorescence studies involve the photoselection of a non-equilibrium distribution of excited state probe orientations whose relaxation is monitored by time resolved fluorescence anisotropy. Chapter 1 introduces the basic concepts of probe photophysics and orientational photoselection. Chapter 2 combines photoselection with single and multiple laser pulses to provide a full description of probe alignment dynamics in the nematic phase of 5-cyanobiphenyl (5CB). In isotropic media the cylindrically symmetric and asymmetric alignment relaxation times are necessarily equal. However, in 5CB, this was seen to correlate strongly with equilibrium order parameters, indicating the need to quantify fully the probe orientational distribution function. In Chapter 3, single and two-photon time resolved fluorescence anisotropy measurements are used to determine the hitherto unmeasured (ground state) K=6 moment of P. The final chapters concern the development of the next generation of photoselection techniques where an excited state population is engineered by stimulated emission depletion (STED). In Chapter 4 STED following two-photon excitation (PUMP) has been demonstrated in a range of well established single photon probes and two recently developed quadrupolar chromophores. Saturation of the DUMP transition due to excited state re-pumping was investigated, and from a two level rate equation model stimulated emission cross-sections and ground state vibrational relaxation times were determined. In Chapter 5 the polarisation properties of STED are investigated; orientation-selective depletion was shown to yield highly polarised excited state arrays. Polarisation resolved STED measurements were shown to circumvent spontaneous emission selection rules (which forbid the measurement of excited state moments of rank K > 2) allowing the first measurements of hexadecapole (K=4) orientational relaxation in two fluorescent probes.

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Materials based upon the aryleneethynylene skeleton are currently of great interest. The fluorescent and electroluminescent properties of both molecular and polymeric systems has prompted speculation about the suitability of these materials as the emitting layer in electroluminescent devices, while the conjugated π-system has led to the development of molecular wire-like architectures, and materials which display negative differential resistance. The degree of conjugation in the π-system is dependent on the relative orientation of the aromatic rings. The barrier to rotation about the aryl-alkynyl single bond for phenyleneethynylenes is very low, leading to a continuum of rotational isomers when in solution. Engineering physical control over the conformation of aryleneethynylenes therefore presents a significant challenge. In this work, a review is presented on the uses, behaviour and synthesis of aryleneethynylenes, followed by an account of the practical work carried out. Initially various series of 1,4-bis(phenylethynyl)benzene (BPEB) analogues containing naphthalene and thiophene moieties were produced. Study of the photophysical behaviour and molecular modelling of these systems suggests that the choice of central ring in a three ring aryleneethynylene system has the greatest effect on the compounds’ characteristics. This can be attributed to the π-electrons of the central aromatic ring giving the largest contribution to the frontier orbitais of these compounds. Conformational control of aryleneethynylenes is then examined. Derivatives of BPEB and 9,10-bis(phenylethynyl)anthracene (BPEA) with bulky alkyl substituents have been synthesised, with the aim of using steric bulk to restrict rotation about the acetylenie bond. Initial attempts at this approach resulted in the additional steric bulk only giving slight conformational restriction in the solid state as well as only having a small effect on the observed photophysics. Subsequently, compounds were successfully synthesised where, in the ground state, the two outer rings were orthogonal to the central ring system, switching off their contribution to the frontier orbitais, and thus leading to significant changes in photophysical behaviour being observed relative to BPEB and BPEA.

Molecules based on the 1,4-bis(phenylethynyl)benzene (BPEB) architecture are an important class of compounds because of their luminescent properties. The rigid rod-like backbone and extended π-conjugation serves as the base upon which a wide variety of substituted species can be synthesised with a variety of photophysical properties. This thesis represents a detailed study of the photophysical properties of a series of compounds based on BPEB using a combination of steady-state and time- resolved spectroscopic methods. A comparative study of the photophysical properties of BPEB, 1,2,4,5-tetramethyl- 3,6-bis(phenylethynyl)benzene, l,2,4,5-tetramethyl-3,6-bis-(2,4,6-trimethylphenyl- ethynyl)benzene, 1,4-bis(2-(2-tórř-butylphenyl)ethynyl)benzene, 1,4-bis(2-(2-tert- butylphenyl)ethynyl)-2,3,5,6-tetramethylbenzene, 1,4-dibromo-2,5-bis(phenylethy- nyl)benzene and l,2,4,5-tetrafluoro-3,6-bis(phenylethynyl)benzene is reported. These compounds exist as a series of geometric conformers, in solution, in the ground state due the low barrier to rotation about the carbon-carbon triple bond (C≡C). The absorption and emission dipoles of BPEB are found to be almost linear and lie along the long axis of the molecule. The substituents red shift the absorption and emission spectra of BPEB. l,4-Bis(2-(2-tert-butylphenyl)ethynyl)-2,3,5,6- tetramethylbenzene, exhibits fluorescence from both high and low energy conformations at 77 K. Only l,4-dibromo-2,5-bis(phenylethynyl)benzene has a long lived phosphorescence emission detectable at 77 K. Time-resolved Raman spectroscopy reveals that the C≡C stretching vibrations of BPEB, and its substituted derivatives in the excited singlet and triplet states, which shows evidence of only minor structural changes upon excitation. The photophysical properties, and solvatochromism, of a series of compounds having donor and acceptor groups bridged by BPEB are reported. They are methyl- 4-(4-(4-dimethylaminophenylethynyl)phenylethynyl)benzoate, 4-(4-(4-dimethyl- aminophenylethynyl)phenylethynyl)benzonitrile, 4-(4-(4-dimethylammo-3,5-dimeth- ylphenylethynyl)phenylethynyl)benzonitrile, 4-(4-(4-dimethylaminophenylethynyl)- phenylethynyl)-N,N-dimethylbenzenamine, 4-(4-(4-cyanophenyIethynyl)phenylethy֊nyl)benzonitrile, 4-(4-(4-dimethylaminophenylethynyl)phenylethynyl)benzene, 4-(4- (4-cyanophenylethynyl)phenylethynyl)benzene and methyl-4-(4-phenylethynyl)- phenylethynylbenzoate. The donor-acceptor systems exhibit intramolecular charge transfer (ICT) and twisted internal charge transfer in the excited state, when in polar solvents. In non-polar solvents only the locally excited state is observed. The largest change in dipole moment in the excited state occurred when the dimethylamino group was fixed perpendicular to the adjacent phenyl ring. In protic solvents, hydrogen bonds to the amino group in the ICT state to form a hydrogen- bonded ICT state. The photophysical properties of 9,10-bis(phenylethynyl)anthracene (BPEA) and a series of nine of its donor and acceptor substituted derivatives are reported. Compounds with the strongest donor (dimethylamino) and acceptor (nitro) groups exhibit ICT. All substituted BPEA compounds had lower fluorescence quantum yields and lifetimes, and higher non-radiative decay constants than BPEA. The molecular and photophysical properties of 2,5-bis(phenylethynyl)thiophene, 1,4-bis(2-thienylethynyl)benzene and 2,5-bis(2-thienylethynyl)thiophene were investigated with specific emphasis on the bonding character of their triplet excited states. Like the parent, BPEB, these compounds exhibit evidence of triple bond character in the excited singlet and triplet states.

Fluorescence is widely used in DNA sequencing and genetic analysis. Novel fluorescence techniques for studying nucleic acids and other biologically important molecules are constantly being developed. In order to provide efficient probes with desired fluorescent properties, new fluorophores and nucleic acids labelling chemistries are required. The synthesis of various cyanine dyes derivatives is described in this thesis. Dyes were designed for NHS-ester and click chemistry labelling of oligonucleotides. Additionally the synthesis of disperse blue quencher and a 2′-mesyl-modified thymidine monomer are also presented. A simple and convenient method of oligonucleotide labelling was developed. This method utilises the copper-catalysed 1,3-dipolar cycloaddition reaction (known as click chemistry) between alkyne-modified cyanine dyes and azide modified oligonucleotides. The conditions of the labelling were optimised and have proved efficient for solid-phase and solution-phase labelling of oligonucleotides with alkyne-functionalised dyes. Click labelling was applied for the preparation of a series of HyBeacon probes and their fluorescence properties were investigated. The click-labelled HyBeacons were compared with HyBeacons labelled with standard cyanine dye NHS-esters showing improvements in the properties of the probes labelled by click chemistry. The thesis also contains a detailed discussion of the photophysical properties of free cyanine dyes in solution and while attached to oligonucleotide probes.

Förster resonance energy transfer (FRET) has become an important tool for studying biochemical reactions at the single-molecule level, despite its increasing maturity there is an on-going effort to improve and expand the technique. This thesis presents methods for extending conventional two-colour single-molecule FRET measurements; by expanding the range and applicability of single-molecule fluorescence methods a greater variety of biological reactions can be studied, in greater detail than previously possible. To circumvent the complexities of multi-colour FRET measurements and extend the range of observable distances I developed and characterised a new single-molecule fluorescence method termed tethered fluorophore motion (TFM). TFM is based on the existing technique of tethered particle motion (TPM) which relies on Brownian motion of a particle, attached to a surface by DNA, to probe the effective length of the DNA tether. TFM takes this concept and applies it at the single-fluorophore level, allowing simultaneous measurement of other fluorescence observables such as FRET and protein induced fluorescence enhancement (PIFE). Having developed TFM I combined it with FRET to study site-specific recombinase proteins at the single-molecule level, in greater detail than possible by either technique alone. Studying the model tyrosine recombinase Cre, I extend and clarify previous ensemble observations regarding the order of DNA strand exchange, as well as uncovering a previously unobserved complex conformation and molecular heterogeneity. Finally, I used TFM-FRET to study the more complex XerCD recombination system and its interaction with the DNA translocase FtsK. I made observations, for the first time, of synaptic complex formation and of recombination at the single-molecule level, and these suggest intriguing and unexpected intermediates in the recombination reaction. I also combine TFM with PIFE to investigate the mechanism of DNA looping by FtsK. The introduction of TFM, and its combination with other fluorescence techniques, allows observation of complex protein-DNA interactions from a variety of perspectives and will help expand the repertoire and applicability of single-molecule biophysical experiments.

L'objet de ce travail de thèse concerne la synthèse par réaction cascade pallado-catalysé d'une nouvelle famille de chromophores pentacycliques azotés dont les propriétés de fluorescence peuvent être modulées en fonction des groupements fonctionnels présents dans la molécule. Ces chromophores sont obtenus par réaction one-pot entre des acides 2-formyl boroniques et des 2,5-dihalopyridines. Le processus cascade est initié par un couplage de Suzuki suivi par deux cyclisations successives. La première se fait sur l'azote de la pyridine et la seconde se produit de façon régio-sélective sur le carbone adjacent à l'azote. Les structures cristallines et le calcul théorique DFT ont montré la régio et la stéréo-sélectivité de la réaction. De plus, des études préliminaires ont montré que ces composés polycycliques possèdent d'excellentes propriétés de fluorescence ainsi qu'une activité biologique qui devraient nous permettre d'étendre le champ d'application de ces nouveaux chromophores vers le domaine médical et l'électronique moléculaire / The aim of this work concerns the synthesis of a new family of aza-pentacyclic chromophore whose fluorescence properties can be modulated according to the functional groups present in the molecule. These chromophores were obtained through a cascade process between 2-formylbenzene boronic acid and 2,5-dihalopyridines. The cascade process is initiated by a palladium-catalyzed cross-coupling reaction and is followed by two successive nucleophilic cyclizations; the first cyclization performed on the pyridine nitrogen and the second occurred regioselectively on the adjacent carbon atom. This new cascade reaction allowed the formation of a pentacycle as a single regioisomer with four new bonds and two contiguous stereocenters with trans relationships. In addition, preliminary studies have shown that these polycyclic compounds have excellent fluorescence properties as well as biological properties that should enable us to extend the scope of these new chromophores to the medical field and molecular electronics

La pulvérisation cathodique magnétron est un procédé plasma très répandu dans l'industrie pour le dépôt de couches minces. Néanmoins, les exigences des nouvelles applications nécessitent de mieux comprendre, contrôler et maîtriser les processus fondamentaux gouvernant le transport de la matière pour optimiser le procédé. Ce travail de thèse porte sur l'étude du transport des atomes pulvérisés de tungstène (W) en décharge magnétron continu (DC direct current) et pulsée haute puissance (HiPIMS_high power impulse magnétron sputtering). La fluorescence induite par diode laser (TD-LIF) a été mise au point afin de mesurer les fonctions de distribution en vitesse des atomes W pulvérisés. Les mesures ont été calibrées par absorption laser et validées en corrélant avec les vitesses de dépôt. En procédé DC, l'étude de l’influence des paramètres de la décharge (puissance, tension, mélange gazeux Ar/He, distance par rapport à la cible, etc.) a mis en évidence l'évolution spatiale des régimes de transport balistique (atomes énergétiques), diffusif (atomes thermalisés), et mixte (balistique+diffusif). Pour l'étude du procédé HiPIMS, le plasma pulsé a nécessité de développer la TD-LIF résolue en temps (TR-TDLIF). Le degré de liberté supplémentaire qu'offre la dimension temporelle du plasma HiPIMS a permis de mieux comprendre le transport mixte qui représente le cas le plus compliqué. En effet, cela a permis de mesurer la cinétique du transport des atomes pulvérisés en ayant la possibilité de séparer les temps caractéristiques des différents processus / Magnetron sputter deposition is an established and widely used method for the growth of thin films. Nevertheless, the high level of expectations regarding new applications require a better understanding, controlling, mastering of basic processes governing atoms transport in the view of process optimization. This work consist in the study of transport of sputtered W atoms in direct current and high power impulse magnetron discharges (DC and HiPIMS). A tunable diode laser induced fluorescence technique (TD-LIF) has been developed, in order to measure W sputtered atom velocity distribution function. Measurements were calibrated using laser absorption and were corroborated by deposition rate. In DC, the study of the influence of discharge parameters (power, voltage, Ar/He gas mixture, and distance from target, etc.) highlighted spatial evolution of different regimes of transport: ballistic (energetic atoms), diffusive (thermalized atoms), and mixed (ballistic + diffusive). In HiPIMS, pulsed plasma required to develop a time resolved TD-LIF technique (TR-TDLIF). The additional degree of freedom, given by time dimension allowed for a better understanding of mixed transport which represents the most complicated situation. This technique allowed to measure the kinetic of sputtered W atoms while at the same time providing the possibility to separate characteristic time scales of different processes