Radiotherapy plays a significant role in cancer treatment, and is thought to be curative by mainly killing tumor cells through damage to their genetic material. However, recent findings indicate that the tumor’s vascular blood supply is also a major determinant of radiation response. The goals of this thesis are to: (1) develop an experimental platform for small animals to deliver ionizing radiation and perform high-resolution optical imaging to treatment targets, and (2) use this toolkit to longitudinally monitor the response of tumors and the associated vasculature. The thesis has achieved: (1) customization of a novel micro-irradiator for mice, (2) technical development of an improved optical coherence tomography imaging system, (3) comprehensive experimental protocol and imaging optimization for optical microscopy in a specialized animal model, and (4) completion of a feasibility study to demonstrate the capabilities of the experimental platform in monitoring the response of tumor and vasculature to radiotherapy.

Radiotherapy plays a significant role in cancer treatment, and is thought to be curative by mainly killing tumor cells through damage to their genetic material. However, recent findings indicate that the tumor’s vascular blood supply is also a major determinant of radiation response. The goals of this thesis are to: (1) develop an experimental platform for small animals to deliver ionizing radiation and perform high-resolution optical imaging to treatment targets, and (2) use this toolkit to longitudinally monitor the response of tumors and the associated vasculature. The thesis has achieved: (1) customization of a novel micro-irradiator for mice, (2) technical development of an improved optical coherence tomography imaging system, (3) comprehensive experimental protocol and imaging optimization for optical microscopy in a specialized animal model, and (4) completion of a feasibility study to demonstrate the capabilities of the experimental platform in monitoring the response of tumor and vasculature to radiotherapy.

AbstractFive bacteria, Escherichia coli, Serratia marcescens, Bacillus subtilis,, Bacillus cereus, and Sarcina flava, were tested for establishment in the yellow mealworm, Tenebrio molitor, using a fluorescent screening technique. Through literature research some of the above bacteria were known to establish in the mealworm.The various test bacteria were dyed with Calcofluor white, a fluorescent dye, and introduced to the mealworm by several techniques. Oral injection, anal injection, drop on head, and petri dish methods were attempted. The best method of bacterial entry into the insect was found to be the drop on head. Since few bacteria were ingested by the insect, a quantitative analysis was impossible.It was found that as the dyed bacteria grew, fluorescence decreased. This was substantiated by allowing dyed Sarcina flava to grow at 37 degrees C. in nutrient broth and examining every hour for ten hours.Although this study did not show a definite screening technique for bacterial establishment in Tenebrio molitor Linnaeus, the results from this project could aid researchers in developing such a technique.Ball State UniversityMuncie, IN 47306

This dissertation describes the synthesis of molecular machines designed to operate on surfaces (nanocars) or in the solution phase (nanosubmarines), and the study of their diffusion using fluorescence techniques. The design of these molecular machines is aimed to facilitate monitoring of their movement and incorporation of a source of energy for propulsion. To complement previous scanning tunneling microscopy studies of the translation of nanocars on surfaces, chapter 1 describes the synthesis of a family of fluorescently tagged nanocars. The nanocars were functionalized with a tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye. Single-molecule fluorescence microscopy (SMFM) studies of one of these nanocars revealed that 25% of the nanocars moved on glass. The SMFM results also suggested that the dye hindered the mobility of the nanocars. Seeking to improve the mobility, chapter 2 presents the synthesis of a new set of fluorescent nanocars, featuring a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye embedded in their axles. The mobility of these inherently fluorescent nanocars on glass was nearly double than that of their TRITC-tagged predecessors. Their diffusion was also studied on reactive-ion-etched glass, and amino-functionalized glass. The results showed that the mobility is affected by the substrate. To equip the nanocars with an energy input for propulsion, two nanocars functionalized with an olefin metathesis catalyst were synthesized, as described in chapter 3. The catalytic activity of these nanocars toward ring-opening metathesis polymerization (ROMP) in solution was similar to that of their parent catalysts. As an alternative approach to investigate if chemical propulsion through a ROMP process can be achieved at the molecular level, chapter 4 presents the synthesis of a fluorescent ROMP catalyst, termed a nanosubmarine, and the study of its diffusion using fluorescence correlation spectroscopy (FCS). FCS results showed an increase of 20 ± 7% in the diffusion constant of this nanosubmarine in presence of its fuel, cis,cis-1,5-cyclooctadiene. Overall, the work accomplished in this dissertation constitutes a step forward toward development of easily tracked and highly mobile nanocars, and paves the way for the synthesis of truly nanosized chemically propelled molecular machines that operate in the solution phase.

Many research questions in biological research lead to numerous microscope images that need to be evaluated. Here digital image cytometry, i.e., quantitative, automated or semi-automated analysis of the images is an important rapidly growing discipline. This thesis presents contributions to that field. The work has been carried out in close cooperation with biomedical research partners, successfully solving real world problems. The world is 3D and modern imaging methods such as confocal microscopy provide 3D images. Hence, a large part of the work has dealt with the development of new and improved methods for quantitative analysis of 3D images, in particular fluorescently labeled skeletal muscle cells. A geometrical model for robust segmentation of skeletal muscle fibers was developed. Images of the multinucleated muscle cells were pre-processed using a novel spatially modulated transform, producing images with reduced complexity and facilitating easy nuclei segmentation. Fibers from several mammalian species were modeled and features were computed based on cell nuclei positions. Features such as myonuclear domain size and nearest neighbor distance, were shown to correlate with body mass, and femur length. Human muscle fibers from young and old males, and females, were related to fiber type and extracted features, where myonuclear domain size variations were shown to increase with age irrespectively of fiber type and gender. A segmentation method for severely clustered point-like signals was developed and applied to images of fluorescent probes, quantifying the amount and location of mitochondrial DNA within cells. A synthetic cell model was developed, to provide a controllable golden standard for performance evaluation of both expert manual and fully automated segmentations. The proposed method matches the correctness achieved by manual quantification. An interactive segmentation procedure was successfully applied to treated testicle sections of boar, showing how a common industrial plastic softener significantly affects testosterone concentrations.

The material properties of fibre networks and fibre reinforced composites are strongly influenced by fibre-fibre interactions. Stress transfer between load bearing elements in such materials is often dictated by the nature of the fibre-fibre interface. Inter-fibre bonding is solely responsible for internal cohesion in paper, because all stresses transferred between fibres operate through fibre-fibre bonds. . The future development of cellulosic fibre materials will require an improved understanding of the fibre-fibre interface. Fluorescence resonance energy transfer (FRET) was proposed as a new tool for the study of fibre interfaces. A protocol for covalent linkage of fluorophores to natural and regenerated cellulosic fibres was developed and the absorptive and emissive properties of these dyes were characterized. The fluorescent response of these dyed fibres in paper sheets was studied using steady-state fluorescence spectroscopy. Fluorescence micrographs of fibre crossings on glass slides were analyzed using the FRETN correction algorithm. Energy transfer from coumarin dyed fibres to fluorescein dyed fibres at the interface was observed. The FRETN surfaces for spruce and viscose rayon fibre crossings were distinctly different. The FRET microscopy method was able to detect statistically significant differences in spruce fibre interface development when fibre fraction and wet pressing were varied. The coalescence of natural cellulosic fibre interfaces during drying was also observed with the technique. Polysaccharide films were employed as model systems for the natural and regenerated cellulose fibre interfaces. It was found that pressing cellulose films did not result in significantly increased FRETN either due to resistance to deformation or the inability to participate in interdiffusion. Conversely, xylan films demonstrated a drastic increase in the FRETN signal with increased wet pressing. These results support the previously observed differences between regenerated cellulose fibres and natural wood fibres. The results of the FRETN analysis of the polysaccharide film model systems suggest that lower molecular weight amorphous carbohydrates are likely to be significant contributors to fibre interface development.

Polymers are added to asphalt binders primarily to stiffen the binder at higher temperatures and thus to protect the pavement against rutting at summertime temperatures early in the pavement's life. Also, it has been noted that polymers typically increase the ductility of a binder and that some polymer-asphalt combinations are especially effective. Furthermore, it is hypothesized that enhancing a binder's ductility, and maintaining this enhancement with binder oxidative aging, contributes to enhanced binder durability in pavements. However, polymer-asphalt interactions and how they might contribute to improved binder performance is not well understood. The goal of this work was to probe the relationship of polymer morphology on asphalt binder rheology and mixture durability. Experiments were conducted on asphalt mixtures and binders, and as a function of oxidative aging. PFC mixtures, which are an open mixture designed to allow enhanced water drainage, were of specific interest. These mixtures were tested for Cantabro Loss, an indicator of a mixture's likelihood of failure by raveling. Asphalt binders were tested using dynamic shear rheometry (DSR), which provided the DSR function, (G' /η'/G'), a measure of binder stiffness that includes both the elastic modulus and the flow viscosity), ductility (used to measure the elongation a binder could withstand before failure), gel permeation chromatography (GPC), used to estimate the relative amount of polymer) and fluorescence microscopy (used to image the polymer morphology in the asphalt binder). From these data, relationships were assessed between binder morphology and binder rheology and between binder rheology and mixture durability, all as a function of binder oxidative aging. Polymer morphology related to ductility enhancement. Polymer morphology related to a change in the DSR function, relative to the amount of polymer, as measured by the polymer GPC peak height. Cantabro loss correlated to the DSR function (R2=0.963). The overall conclusion is that polymer morphology, as indicated by fluorescence microscopy, relates to both the rheological properties of the binder and the Cantabro loss of the mixture. These relationships should yield a better understanding of polymer modification, increased mixture durability (decreased raveling) and improved rheological properties (DSR function and ductility).

Photophysical characterization of several species of fluorescent silver nanoclusters, encapsulated in oligonucleotide scaffolds, was achieved at the bulk and single molecule level. These studies reveal the presence of a short-lived microsecond blinking component which leads to higher emission rates than exhibited by common organic dyes. This dark state was found to be photo-accessible with a very efficient depopulation transition leading to repopulation of the emissive state. Secondary excitation on resonance with this transition significantly shortens the residence time in the dark state giving rise to as much as 5-fold fluorescence enhancement. Manipulation of the secondary laser can be used to impose a regularly modulated waveform onto the fluorescent signal. Signal processing techniques can be employed to extract the modulated signal from large backgrounds, leading to drastically improved sensitivity. This new imaging concept can be extended, beyond Ag nanoclusters, to common organic fluorophores that demonstrate large dark state quantum yields. These fluorophores simultaneously illustrate the utility of this technique and help to define a general set of parameters for engineering ideal dyes for modulated signal extraction. Ideally suited for fluorescence enhancement, FRET pairs can be used to engineer a wide range of modulatable systems, based on detecting donor emission in the presence of a laser directly exciting the acceptor. The utility of Ag nanoclusters, organic dyes, and FRET systems for improved sensitivity are investigated in this work.

Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio. Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence. The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio. By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular biosynthesis of membranes and steroids. Using multi-color single particle tracking fluorescence microscopy, the intracellular degradation of LDL was probed in live, intact cells. Unique to these experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles were labeled with multiple fluorophores resulting in a quenched fluorescent signal. The characteristics of the vesicle responsible for degradation were determined and the vesicle dynamics involved in LDL degradation were quantified. Visualization of early endosomes, late endosomes and lysosomes was accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle were measured simultaneously. These studies, which are the first to directly observe the degradation of LDL within a cell, strive to completely describe the endo-lysosomal pathway and quantify the dynamics of LDL degradation in cells.