In recent times, near-field optical microscopy has received increasing attention for its ability to obtain high-resolution images beyond the diffraction limit. Near-field optical microscopy is achieved via the positioning and manipulation of a probe on a scale less than the wavelength of the incident light. Despite many variations in the mechanical design of near-field optical microscopes almost all rely on direct mechanical access of a cantilever or a derivative form to probe the sample. This constricts the study to surface examinations in simple sample environments. Distance regulation between the sample surface and the delicate probe requires its own feedback mechanism. Determination of feedback is achieved through monitoring the shift of resonance of one arm of a 'tuning fork', which is caused by the interaction of the probes tip with the Van der Waals force. Van der Waals force emanates from atom-atom interaction at the top of the sample surface. Environmental contamination of the sample surface with additional molecules such as water makes accurate measurement of these forces particularly challenging. The near-field study of living biological material is extremely difficult as an aqueous environment is required for its extended survival. Probe-sample interactions within an aqueous environment that result in strong detectable signal is a challenging problem that receives considerable attention and is a focus of this thesis. In order to increase the detectible signal a localised field enhancement in the probing region is required. The excitation of an optically resonant probe by morphology dependent resonance (MDR) provides a strong localised field enhancement. Efficient MDR excitation requires important coupling conditions be met, of which the localisation of the incident excitation is a critical factor. Evanescent coupling by frustrated total internal reflection to a MDR microcavity provides an ideal method for localised excitation. However it has severe drawbacks if the probe is to be manipulated in a scanning process. Tightly focusing the incident illumination by a high numerical aperture objective lens provides the degree of freedom to enable both MDR excitation and remote manipulation. Two-photon nonlinear excitation is shown to couple efficiently to MDR modes due to the high spatial localisation of the incident excitation in three-dimensions. The dependence of incident excitation localisation by high numerical aperture objective on MDR efficiency is thoroughly examined in this thesis. The excitation of MDR can be enhanced by up to 10 times with the localisation of the incident illumination from the centre of the microcavity to its perimeter. Illuminating through a high numerical aperture objective enables the remote noninvasive manipulation of a microcavity probe by laser trapping. The transfer of photon momentum from the reflection and refraction of the trapping beam is sufficient enough to exert piconewtons of force on a trapped particle. This allows the particle to be held and scanned in a predictable fashion in all three-dimensions. Optical trapping removes the need for invasive mechanical access to the sample surface and provides a means of remote distance regulation between the trapped probe and the sample. The femtosecond pulsed beam utilised in this thesis allows the simultaneous induction of two-photon excitation and laser trapping. It is found in this thesis that a MDR microcavity can be excited and translated in an efficient manner. The application of this technique to laser trapped near-field microscopy and single molecule detection is of particular interest. Monitoring the response of the MDR signal as it is scanned over a sample object enables a near-field image to be built up. As the enhanced evanescent field from the propagation of MDR modes around a microcavity interacts with different parts of the sample, a measurable difference in energy leakage from the cavity modes occurs. The definitive spectral properties of MDR enables a multidimensional approach to imaging and sensing, a focus of this thesis. Examining the spectral modality of the MDR signal can lead to a contrast enhancement in laser trapped imaging. Observing a single MDR mode during the scanning process can increase the image contrast by up to 1:23 times compared to that of the integrated MDR fluorescence spectrum. The work presented in this thesis leads to the possibility of two-photon fluorescence excitation of MDR in combination with laser trapping becoming a valuable tool in near- field imaging, sensing and single molecule detection in vivo. It has been demonstrated that particle scanned, two-photon fluorescence excitation of MDR, by laser trapping 'tweezers' can provide a contrast enhancement and multiple imaging modalities. The spectral imaging modality has particular benefits for image contrast enhancements.

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

Thesis (M.S.)--University of Nevada, Reno, 2005. / "December, 2005." Includes bibliographical references (leaves 50-53). Online version available on the World Wide Web.

<p>The knowledge of the human genome sequence, as revealed in the HUGO project, has created exciting new possibilities for biomedical research. The Swedish Human Proteome Resource (HPR) program aims to make use of this information to gain further insight into the human proteome. Recombinant proteins are generated from coding sequences identified from the human genome sequence and used to produce specific antibodies to target proteins. Antibodies are subsequently utilized for functional analysis of the corresponding proteins using tissue micro arrays. The aim of my project was to investigate the possibility of distinguishing characteristic distribution patterns of intracellular proteins in the resolution capacity offered by light microscopy. A map of representative distribution patterns was created using immunohistological staining with commercially available antibodies toward well-characterised proteins in the cell. Such a map could then aid in interpreting the results of immunohistological staining of intracellular proteins using antibodies produced within the Human Proteome Resource program. Proteins manifested in nucleus, nuclear membrane and plasma membrane were clearly visible at the expected location. Proteins manifested in different organelles in the cytoplasm however, showed all a similar staining pattern, making determination of exact protein location uncertain. A possible explanation is the resolution of the light microscope not being sufficient to visualize certain proteins specific to organelles in the cytoplasm. Results may also have been influenced by the choice of secondary antibody, where the strenghtened signal generated by an enzyme labelled polymer may have a negative effect on depiction of details in the image generated.</p>

This thesis is dedicated to the study of fluorescent conjugated polymers made of fluorene labelled with rylene moieties. Those polymers are important candidates for use in Organic Light Emitting Devices (OLEDs). The dyes present in the polymers were studied at the single-molecule level. The first part of the work is devoted to the construction and validation of an epi-fluorescent confocal/widefield/Total Internal Reflection microscope. The ensemble properties of the samples are first measured in solution. The combination of steady-state and time-resolved spectroscopies allows us to unravel the photophysics of the conjugated polyfluorene polymer containing perylenediimides in its backbone. Energy transfer is found to occur between the polyfluorene and the perylenediimide units. Beside energy transfer, a photoinduced electron transfer is also supposed to take place. Widefield microscopy is used to measure the end-to-end distance in single polymer chains. From those measurements the polymer is shown to present a quasi linear shape inside its host matrix. From the simulation of the end-to-end distance distribution, a conjugation length of 4-6 fluorene units is found. The introduction of a new subtraction method associated with defocused imaging allows us to study a more complicated polymer containing more perylenediimide units. The location and the 3D orientation of the incorporated dyes were measured at the same time by this new technique named SPIDER. Finally, the sequential two-color measurements allow us to get useful informations concerning the energy transfer occurring between polyfluorene backbone and perylenediimide units at the single molecule level.

Fluorescence microscopy offers the opportunity to image noninvasively biological samples in real time. However, the phenomenon of diffraction limits the resolution of conventional fluorescence microscopes to submicrometer dimensions in both the horizontal and vertical directions. This limitation can be overcome by photoswitchable fluorescent probes able to undergo reversible saturable optically linear fluorescence transitions (RESOLFT). In this study, firstly, a photoswitchable fluorescent probe based on BODIPY fluorophore and Spiropyran photochrome were designed and its photophysical and photochemical properties were investigated in organic and aqueous environments. Also, its imaging with patterned illumination was showed by trapping them in PMMA matrix. Secondly, photochromic [1,3]oxazines with different substituents as well as polymers incorporating them were synthesized and their photochemical and photophysical properties were investigated. Thirdly, to improve the switching speeds and fatigue resistance of the BODIPY-Spiropyran conjugate, the photochromic part was replaced by [1,3]oxazines and dyads incorporating BODIPY fluorophore and [1,3]oxazine photochromes were synthesized. Lastly, a new strategy was designed to switch the fluorescence of fluorophores by a modular approach. It is based on photoinduced elongation of the absorption wavelength of a fluorescent chromophore with the aid of an appended photochromic auxochrome.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xii, 146 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 134-146).

M.S. / Applied Physics / A noble gas ion laser with strong transitions in the 196-225 nm wavelength region has been developed for use as an illuminator in a photoelectron microscope. The laser is pulsed, and it can be operated at repetition rates up to 200 Hz to produce average output powers up to 5.0 mW at 219 nm. This is comparable to the output of the brightest available incoherent source, a Hg-Xe-Cd arc lamp that produces 2.6 mW of usable light in the 221-226 nm range. The laser has the advantage that it can be focused to produce much higher intensities than the arc lamp, and less total power is necessary. But the pulsed laser has a low duty cycle (~ 10[superscript minus 5]), and the corresponding peak powers (~ 300 watts) result in a space-charge-limited resolution of approximately 500 Å when the laser illuminates a phthalocyanine target. The magnitude of this aberration is proportional to beam current. Consequently, the resolution o can be improved to about 50 Å by decreasing the input power, or increasing the duty cycle, by a factor of 100-1000. Techniques for achieving such an improvement are suggested.

No description available.

The way in which the myocardium responds to its mechanical environment must be understood in order to develop reasonable treatments for congestive heart failure. The first step toward this understanding is to characterize and quantify the cardiac microstructure in healthy and diseased hearts. Myocardium has a laminar architecture made up of myolaminae, which are sheets of myocytes surrounded by a collagen weave. By enhancing the contrast between the myocytes and the surrounding collagen, the myocardium can be investigated and its laminar structure can be quantified. Many of the techniques that have been used to view the microstructure of the heart require the use of toxic or caustic chemicals for fixation or staining. An efficient imaging method that uses polarization microscopy and enhances the contrast between the collagen and myocytes while minimizing the use of harmful chemicals was developed in this research. Collagen is birefringent; therefore its visibility should be enhanced through polarization microscopy and image processing. The sheet angles were viewed directly by cutting slices of a rat septum perpendicular to the fiber angle. Images of different polarization combinations were taken and a region of interest was selected on the sample. Image processing techniques were used to reduce the intensity variation on the images and account for the variable gain of the camera. The contrast between the collagen and myocytes was enhanced by comparing adjusted images to the background and looking at a single image this comparison produced. Although the contrast was enhanced, the embedding media reduced the collagen signal and the enhancement was not as striking as expected.