This dissertation focuses on laser-based noninvasive photoacoustic microscopy of subsurface structures in vivo. Photoacoustic microscopy is a hybrid imaging modality that combines the high-resolution advantage of ultrasonic imaging in deep tissue with the high-contrast advantage of optical imaging. It detects the short-pulsed laser-induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. The spatial resolution is determined by the ultrasonic focal ability and the ultrasonic bandwidth. The imaging depth is primarily limited by the acoustic attenuation within the reach of diffuse photons. The ratio of maximum imaging depth to axial resolution in photoacoustic microscopy is greater than 100, which is comparable to that of modern high-resolution optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography. However, the maximum imaging depth has been much enlarged by taking advantages of absorbed diffuse photons. Based on the intrinsic optical contrast, we have achieved in vivo volumetric imaging of subcutaneous microvasculature, skin melanoma, and acute thermal injuries in high spatial resolution. We have imaged physiological parameters in subcutaneous microvessels, such as total hemoglobin concentration and hemoglobin oxygen saturation, on a single vessel basis in small animals in vivo. We have also monitored changes of hemoglobin oxygen concentration between different systemic physiological states on a vessel-by-vessel basis. Moreover, we have demonstrated the feasibility of human imaging using photoacoustic microscopy by imaging finger tips and subcutaneous palm vessels.

This dissertation focuses on laser-based noninvasive photoacoustic microscopy of subsurface structures in vivo. Photoacoustic microscopy is a hybrid imaging modality that combines the high-resolution advantage of ultrasonic imaging in deep tissue with the high-contrast advantage of optical imaging. It detects the short-pulsed laser-induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. The spatial resolution is determined by the ultrasonic focal ability and the ultrasonic bandwidth. The imaging depth is primarily limited by the acoustic attenuation within the reach of diffuse photons. The ratio of maximum imaging depth to axial resolution in photoacoustic microscopy is greater than 100, which is comparable to that of modern high-resolution optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography. However, the maximum imaging depth has been much enlarged by taking advantages of absorbed diffuse photons. Based on the intrinsic optical contrast, we have achieved in vivo volumetric imaging of subcutaneous microvasculature, skin melanoma, and acute thermal injuries in high spatial resolution. We have imaged physiological parameters in subcutaneous microvessels, such as total hemoglobin concentration and hemoglobin oxygen saturation, on a single vessel basis in small animals in vivo. We have also monitored changes of hemoglobin oxygen concentration between different systemic physiological states on a vessel-by-vessel basis. Moreover, we have demonstrated the feasibility of human imaging using photoacoustic microscopy by imaging finger tips and subcutaneous palm vessels.

We present a novel concept to design apertureless plasmonic probes for near-field scanning optical microscopy (NSOM) with enhanced optical power throughput and near-field confinement. Specifically, we combine unidirectional surface plasmon polariton (SPP) generation along the tip lateral walls with nanofocusing of SPPs through adiabatic propagation towards an apertureless tip. Three probe designs are introduced with different light coupling mechanisms. Optimal design parameters are obtained with 2D analysis and realistic probe geometries with patterned plasmonic surfaces are proposed using the optimized designs. The electromagnetic properties of the designed probes are characterized in the near-field and compared to those of a conventional single-aperture probe with same pyramidal shape. The optimized probes feature enhanced light localization in near-field of tip apex and improved optical throughput. Our ideas effectively combine the resolution of apertureless probes with throughput levels much larger than those available even in aperture-based devices. / text

A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.

<p>This thesis considers the effect of manufacturing conditions and polymer ratio on water permeability and morphology of free-films. A novel spray method for producing ethyl cellulose (EC) and hydroxypropyl cellulose (HPC) free-films was developed where several process parameters was controlled. The process was optimised by pre-spraying solvent until the system reached a steady-state temperature. This minimised the variation of outlet air temperature to < 2.5 °C. Coating time was approximately 4 minutes excluding drying.</p><p>Free-films were produced using 94 wt% solvent (95 %-ethanol) and 6 wt% polymer. The amount of HPC in the films was varied (wt% HPC defined as HPC/(HPC+EC)*100). Films with 30-40-50-57 wt% HPC were studied. Phase diagrams was constructed to study the phase transformation of polymer mixtures. Results show that all polymer mixtures with HPC content above 30 wt% were phase separated prior to film manufacturing. Temperature had an effect on the polymer phase transformation. In the phase diagram, the 2-phase area was larger for temperatures above 40 °C.</p><p>The investigated manufacturing conditions were outlet air temperature (°C) and spray rate (g/min). Outlet air temperature was controlled by adjusting the inlet air temperature. The films were characterized by measuring water permeability (m²/s). Cross section structure of the films was analyzed with confocal laser scanning microscopy (CLSM). FITC-HPC was added for enhanced contrast between the domains.</p><p>Higher outlet air temperature gave higher water permeability of the film whereas higher spray rate gave lower water permeability. The outlet air temperature had an impact on evaporation rate. The evaporation rate together with spray rate affected the solidification and hence the structure of the film. Images show that longer solidification time smeared the domains into larger domains. Lower water permeability was caused by less connectivity between the pores.</p><p>In conclusion, experiments show that water permeability of EC/HPC free-films was highly dependent on the manufacturing conditions.</p><p></p>

This thesis considers the effect of manufacturing conditions and polymer ratio on water permeability and morphology of free-films. A novel spray method for producing ethyl cellulose (EC) and hydroxypropyl cellulose (HPC) free-films was developed where several process parameters was controlled. The process was optimised by pre-spraying solvent until the system reached a steady-state temperature. This minimised the variation of outlet air temperature to &lt; 2.5 °C. Coating time was approximately 4 minutes excluding drying. Free-films were produced using 94 wt% solvent (95 %-ethanol) and 6 wt% polymer. The amount of HPC in the films was varied (wt% HPC defined as HPC/(HPC+EC)*100). Films with 30-40-50-57 wt% HPC were studied. Phase diagrams was constructed to study the phase transformation of polymer mixtures. Results show that all polymer mixtures with HPC content above 30 wt% were phase separated prior to film manufacturing. Temperature had an effect on the polymer phase transformation. In the phase diagram, the 2-phase area was larger for temperatures above 40 °C. The investigated manufacturing conditions were outlet air temperature (°C) and spray rate (g/min). Outlet air temperature was controlled by adjusting the inlet air temperature. The films were characterized by measuring water permeability (m2/s). Cross section structure of the films was analyzed with confocal laser scanning microscopy (CLSM). FITC-HPC was added for enhanced contrast between the domains. Higher outlet air temperature gave higher water permeability of the film whereas higher spray rate gave lower water permeability. The outlet air temperature had an impact on evaporation rate. The evaporation rate together with spray rate affected the solidification and hence the structure of the film. Images show that longer solidification time smeared the domains into larger domains. Lower water permeability was caused by less connectivity between the pores. In conclusion, experiments show that water permeability of EC/HPC free-films was highly dependent on the manufacturing conditions.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [140]-152).

Traditional laser scanning systems, such as those used for microscopy, typically image objects of finite thickness. If the depth-of-focus of such systems is low, as is the case when a simple clear pupil is used, the object must be very thin or the image will be distorted. Several methods have been developed to deal with this problem. A microscope with a thin annular pupil has a very high depth-of-focus and can image the entire thickness of a sample, but most of the laser light is blocked, and the image shows poor contrast and high noise. In confocal laser microscopy, the depth-of-focus problem is eliminated by using a small aperture to discard information from all but one thin plane of the sample. However, such a system requires scanning passes at many different depths to yield an image of the entire thickness of the sample, which is a time-consuming process and is highly sensitive to registration errors. In this thesis, a novel type of scanning system is considered. The sample is simultaneously scanned with a combination of two Gaussian laser beams of different widths and slightly different temporal frequencies. Information from scanning with the two beams is recorded with a photodetector, separated electronically, and processed to form an image. This image is similar to one formed by a system using a difference-of-Gaussians pupil, except no light has been blocked or wasted. Also, the entire sample can be scanned in one pass. The depth-of-focus characteristics of this synthesized difference-of-Gaussians pupil are examined and compared with those of well-known / Master of Science

The membrane aerated bioreactor (MABR) is a unique technological innovation where a gas permeable membrane is applied to biological processes. In an MABR, oxygen and other substrates diffuse from the opposite directions into a biofilm, and thus simultaneous chemical oxygen demand (COD) and nitrogen removal can be achieved. However, controlling biofilm thickness, stability, and attachment is challenging. The objectives of this research were to study the effect of oxygen partial pressure on process performance with respect to nitrogen removal and examine the biomass properties in MABRs at different oxygen partial pressures and COD loadings. The conditions within the bioreactors were based on a low hydrodynamic condition (average fluid velocity 22 cm/min along the membrane surface), with the intention of minimizing the impact of the hydrodynamic shear on biomass properties. Simultaneous nitrification and denitrification were achieved in the reactors, and increasing oxygen partial pressure enhanced the total nitrogen removal. The biomass at the membrane-biofilm interface was more porous at a loading of 11.3 kg COD/1000 m2/day (areal porosity about 0.9) as compared with a loading of 22.6 kg COD/1000 m2/day (areal porosity about 0.7), indicating carbon substrate was limiting near the membrane. Long-term (over 30 days) experimental results showed that at the loading of 11.3 kg COD/1000 m2/day, the oxygen partial pressures of 0.59 atm and 0.88 atm caused over 80% of the biomass to become suspended in the bulk phase while at 0.25 atm and 0.41 atm oxygen over 97% of the biomass was immobilized on the membrane. There is a critical oxygen partial pressure that can sustain the biofilm, which increases with an increasing COD loading. The nitrifying population in the reactors was examined by applying fluorescence in situ hybridization (FISH). At the loading of 22.6 kg COD/1000 m2/day, there were 12% beta-proteobacterial ammonia oxidizing bacteria (AOB) and 17%Nitrobacter in homogenized biofilm biomass at 0.59 atm oxygen while there were 7% beta-proteobacterial AOB and 4% Nitrobacter at 0.25 atm oxygen. The ratio of protein to carbohydrate in extracellular polymeric substances (EPS) of the homogenized biomass in the reactor decreased with increasing oxygen partial pressure. Surface characterization of the biomass revealed that the higher the oxygen partial pressure, the lower the biomass hydrophobicity and surface charge. The ratio of EPS protein to carbohydrate in a membrane aerated biofilm decreased when approaching the membrane-biofilm interface. The distribution of nitrifiers and dissolved oxygen profiles inside the biofilm suggested that dual substrate limitations exist, and it was concluded that the membrane aerated biofilm had an aerobic region in the inner layer and an anoxic region in the outer layer. It is proposed that the loss of EPS due to secondary substrate consumption, especially the loss of EPS proteins, at the bottom of the biofilm was responsible for biofilm detachment subjected to a critical oxygen partial pressure.

This dissertation presents techniques of three-dimensional microscopy. First, an economical method of microscopic image formation that employs a raster-scanning laser beam focused on a sample, while non-imaging detector receives the scattered light is presented. The images produced by this method are analogous to the scanning electron microscopy with visible effects of shadowing and reflection. Compared to a conventional wide-field imaging system, the system allows for a greater flexibility, as the variety of optical detectors, such as PMT and position-sensitive quadrant photodiode can be used to acquire images. The system demonstrates a simple, low-cost method of achieving the resolution on the order of a micron. A further gain in terms of resolution and the depth of focus by using Bessel rather than Gaussian beams is discussed. Then, a phase-imaging technique to quantitatively study the three-dimensional structure of reflective and transmissive microscopic samples is presented. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of porous coal samples and SKOV-3 ovarian cancer cells are presented.