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.

This thesis is intended as an introductory lab manual on the experimental methods using fluorescence microscopy in modern radiobiology research. It is written for those who are unfamiliar with biology research. It first covers the proper use of laboratory equipment and growth of cell cultures in the lab. Subsequent chapters provide overviews of relevant modern experimental techniques for the quantification of radiation induced DNA damage in cells, and detailed protocols for performing these procedures. Techniques covered include immunostaining with fluorescent antibodies, the comet assay, and plasmid DNA transfections. Results of some straightforward experiments using these techniques are presented.

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.

Fluorescence microscopy is a powerful analytical tool for visualizing biological processes at the subcellular level. In this regard, 1,3,5-triarylpyrazoline based fluorescent probes which act as "turn-on" probes, have been extensively researched. These probes achieve their fluorescence "turn-on" response by inhibition of fluorescence quenching by acceptor-excited photoinduced electron transfer upon binding of an analyte. It has been recently shown that some fluorescent probes used in biological research form colloids composed of nanoparticles, due to their hydrophobic character. This hydrophobic character can also lead to partitioning of the probe into cellular membranes. Colloid formation and membrane partitioning may affect the probes' photophysical properties such as absorption and emission wavelength and quantum yields. Recently, a series of 1,3,5-triarylpyrazolines synthesized in our group by M. T. Morgan, showed no formation of aggregates in aqueous buffer. Surprisingly, these probes increased their fluorescence intensity in the presence of liposomes. The photoinduced electron transfer process is greatly affected by the polarity of the medium in which the probe is used. In this study, the effect of membrane proximity on the photoinduced electron transfer process for pyrazoline based "turn-on" probes has been investigated. A series of water soluble 1,3,5-triarylpyrazolines have been synthesized in which a N,N-dialkylaniline moiety acts as an electron donor and a proton acceptor and an alkylated sulfonamide moiety acts as a molecular anchor for interaction with neutral and anionic liposomes.

Nanoparticles are promising platforms for biomedical applications ranging from diagnostic tools to therapeutic delivery agents. During the course of these applications, nanoparticles are exposed to a complex mixture of extracellular serum proteins that nonspecifically adsorb onto the surface. The resulting protein layer, or protein "corona," creates an interface between nanoparticles and the biological environment. Protecting the nanoparticle surface can reduce protein adsorption, but complete inhibition remains a challenge. As a result, the corona, rather than the nanoparticle itself, mediates the cellular response to the nanoparticle. The following dissertation describes the fundamental characterization of the cellular binding of charged nanoparticles, interactions of protein-nanoparticle complexes with cellular receptors, and the structural and thermodynamic properties of adsorbed corona proteins.

Different optical and calorimetric techniques were utilized to gain a better understanding of aggregated macromolecules. This research looked at two different macromolecules: poly(9,9'-dioctylfluorene), a conjugated polymer that forms aggregates in organic solvents; and bovine insulin, which forms amyloid fibrils. Conjugated polymers are of increasing interest due to their thermal stability and ease of solution processing for use in devices. A member of the polyfluorene family, poly(9,9'-dioctylfluorene) (PFO), has been studied due to its blue-emitting spectral properties. However, PFO has been found to form aggregates in solution, which is detected by the presence of a red-shifted absorption peak. This peak is caused when a section of the backbone planarizes forming the [beta]-phase. The [beta]-phase can be removed from the solution upon heating and will not return until the solution is cooled, making it a non-equilibrium process. The dissolution and reformation of the -phase were monitored using absorption spectroscopy and differential scanning calorimetry. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were able to probe the aggregates in films. It is important to understand polymer properties in solution in order to understand film morphology. Amyloid fibrils contribute to over 20 different neurodegenerative diseases, in which cures have yet to be found. The fibrils form when a soluble protein misfolds and self-assembles to form insoluble protein aggregates, and the cause of the fibril formation in vivo has still yet to be determined. Spectroscopy studies have been made possible with the use of fluorescent dyes: thioflavin T (ThT), BTA-2, and Congo red (CR). These dyes bind to amyloid fibrils and exhibit changes in their spectral properties. However, the exact mechanism for the binding of these dyes has only recently been studied. Through the use of calorimetry, the forces involved with binding of ThT and CR to amyloid fibrils can be determined. Absorption and fluorescence spectroscopy techniques were employed to study the spectral properties of these dyes. Polarized NSOM was used to determine the ThT or BTA-2's orientation with an individual fibril. Understanding how these dyes bind to fibrils will enable researchers to use spectroscopy to study the early stages of fibril formation. / text