L’avènement de la microscopie STED (Stimulated Emission Depletion) a bouleversé le domaine desneurosciences du au fait que beaucoup de structures neuronale, tels que les épines dendritiques, lesaxones ou les processus astrocytaires, ne peuvent pas être correctement résolu en microscopiephotonique classique. La microscopie 2-photon est une technique d’imagerie photonique très largement utilisée dans le domaine des neurosciences car elle permet d’imager les événements dynamique en profondeur dans le tissu cérébral, offrant un excellent sectionnement optique et une meilleure profondeur de pénétration. Cependant, la résolution spatiale de cette approche est limitée autour de 0.5 μm, la rendant inappropriée pour étudier les détails morphologiques des neurones et synapses. Le but de mon travail de thèse était à A) développer un microscope qui permet d'améliorer l'imagerie 2-photon en la combinant avec la microscopie STED et B) démontrer son potentiel pour l'imagerie à l'échelle nanométrique de processus neuronaux dynamiques dans des tranches de cerveau aigus et in vivo. Le nouveau microscope permet d'obtenir une résolution spatiale latérale de ~ 50 nm à des profondeurs d'imagerie de ~ 50 μm dans du tissu cérébral vivant. Il fonctionne avec des fluorophores verts, y compris les protéines fluorescentes communes telles que la GFP et YFP, offrant le contraste de deux couleurs basé sur la détection spectrale et linéaire ‘unmixing’. S’agissant d’un microscope droit, utilisant un objectif à immersion ayant une grande distance de travail, nous avons pu incorporer des techniques électrophysiologiques comme patch-clamp et ajouter une plateforme pour l'imagerie in vivo. J’ai utilise ce nouveau microscope pour imager des processus neuronaux fins et leur dynamique à l’échelle nanométrique dans différent types de préparations et des régions différentes du cerveau. J’ai pu révéler des nouvelles caractéristiques morphologique des dendrites et épines. En outre, j'ai exploré différentes stratégies de marquage pour pouvoir utiliser la microscopie STED pour imager le trafic des protéines et de leur dynamique à l'échelle nanométrique dans des tranches de cerveau. / The advent of STED microscopy has created a lot of excitement in the field of neuroscience becausemany important neuronal structures, such as dendritic spines, axonal shafts or astroglial processes,cannot be properly resolved by regular light microscopy techniques. Two-photon fluorescence microscopy is a widely used imaging technique in neuroscience because it permits imaging dynamic events deep inside light-scattering brain tissue, providing high optical sectioning and depth penetration. However, the spatial resolution of this approach is limited to around half a micron, and hence is inadequate for revealing many morphological details of neurons and synapses. The aim of my PhD work was to A) develop a microscope that improves on two-photon imaging by combining it with STED microscopy and to B) demonstrate its potential for nanoscale imaging of dynamic neural processes in acute brain slices and in vivo. The new microscope achieves a lateral spatial resolution of ~50 nm at imaging depths of ~50 μm in living brain slices. It works with green fluorophores, including common fluorescent proteins like GFP and YFP, offering two-color contrast based on spectral detection and linear unmixing. Because of its upright design using a long working distance water-immersion objective, it was possible to incorporate electrophysiological techniques like patch-clamping or to add a stage for in vivo imaging. I have used the new microscope to image fine neural processes and their nanoscale dynamics in different experimental preparations and brain regions, revealing new and interesting morphological features of dendrites and spines. In addition, I have explored different labeling strategies to be able to use STED microscopy for visualizing protein trafficking and dynamics at the nanoscale in brain slices.

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"February 2003" / Bibliography: p. 121-135. / xii, 135 p. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis is a theoretical evaluation of the (single) first photon detection (FPD) technique as a limiting case of time-resolved transillumination (TI) for diagnostic purposes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physics and Mathematical Physics, 2003

DNA photocleaving reagents are a unique class of molecules that display the ability to cleave DNA, causing strand breaks, upon exposure to an irradiation source. In terms of biological applications, achieving excitation through a two-photon absorption event provides for unique benefits that can be useful in such applications as photodynamic therapy and cell viability studies. Thus, this thesis pertains to the study of a class of photocleaving reagents that have been shown to become excited through a twophoton process during irradiation with a pulsed femtosecond laser at 775 nm. N-(Alkoxy)pyridinethiones were selected as possible oxygen-based radical generators upon irradiation at two-photon wavelengths. Experiments were carried out with pBR 322 plasmid DNA to determine if these N-(alkoxy)pyridinethiones could cause strand cleavage and if so how efficient they are in doing so. Several compounds were found to be effective DNA strand cleavers when irradiated at two-photon wavelengths, displaying the utility of two-photon excitation in biological studies. Rationale is suggested for the observed variation in cleaving efficiency based on inherent properties of the generated radicals. A second study was done to measure the two-photon cross section of the compound N-(anthracenoyloxy)pyridinethione. The two-photon cross section was found by measuring the fraction of substrate remaining after specific periods of femtosecond laser irradiation at 775 nm, and the two-photon cross section was found to be 0.051 GM.

The attenuation of photons within the body has been recognised as the major limiting factor hindering the ability of single photon emission computed tomography (SPECT) as a quantitative technique. This thesis investigates several aspects of an emission-transmission SPECT system using the Monte Carlo method and experimental techniques. The system was based on a rotating gamma camera fitted with a parallel hole collimator. The simulation of a transmission study was performed using a simple non-uniform mathematical phantom with two different external sources, a collimated line source and a flood source. The results showed that the attenuation maps were highly dependent on the geometry and photon energy of the source. The collimated line source produced improved image quality with lower statistical noise than the flood source. The results showed that, when high atomic number elements are present in the tissue composition, the attenuation coefficients at different energies are related through a second order polynomial transformation. If the object under study is formed of soft tissue equivalent materials, a linear transformation holds. The attenuation maps generated in the transmission study were used to correct for non-uniform attenuation compensation of an emission phantom. The results showed that non-uniform attenuation compensation improved image quality and reduced noise when compared to data without attenuation compensation. The presence of scattered photons in the emission data reduced the quality of the images and precluded accurate quantification. Absolute quantification was performed using the percent air sensitivity criterion. The largest difference between the theoretical and the Monte Carlo simulated images was approximately 8%. An emission-transmission myocardial perfusion study was simulated using an anthropomorphic phantom. Two photon energies of clinical interest were used, 75 keV and 140 keV, corresponding to the main photon emission energies of 201Tl and 99mTC. The results showed that 99mTc provided better image quality than 201Tl. Non-uniform attenuation compensation produced a very good agreement between the theoretical prediction and the simulation when scatter-free data were considered. The results presented in this thesis indicate that it is not possible to accomplish accurate attenuation compensation in general situations if scatter correction is not applied.

Thesis (Ph. D.)--University of Chicago, August 1999. / Includes bibliographical references. Also available on the Internet.