Development of Swept Source Optical Coherence Tomography and Adaptive Optics Scanning Laser Ophthalmoscopy: Improved Imaging Speed and Handheld Applications

Optical coherence tomography (OCT) is a noninvasive three-dimensional interferometric imaging technique capable of achieving micrometer scale resolution. It is now a standard of care in ophthalmology, where it is used to improve the accuracy of early diagnosis, to better understand the source of pathophysiology, and to monitor disease progression and response to therapy. In particular, retinal imaging has been the most prevalent clinical application of OCT, but researchers and companies alike are developing OCT systems for cardiology, dermatology, dentistry, and many other medical and industrial applications.

Adaptive optics (AO) is a technique used to reduce monochromatic aberrations in optical instruments. It is used in astronomical telescopes, laser communications, high-power lasers, retinal imaging, optical fabrication and microscopy to improve system performance. Scanning laser ophthalmoscopy (SLO) is a noninvasive confocal imaging technique that produces high contrast two-dimensional retinal images. AO is combined with SLO (AOSLO) to compensate for the wavefront distortions caused by the optics of the eye, providing the ability to visualize the living retina with cellular resolution. AOSLO has shown great promise to advance the understanding of the etiology of retinal diseases on a cellular level.

Broadly, we endeavor to enhance the vision outcome of ophthalmic patients through improved diagnostics and personalized therapy. Toward this end, the objective of the work presented herein was the development of advanced techniques for increasing the imaging speed, reducing the form factor, and broadening the versatility of OCT and AOSLO. Despite our focus on applications in ophthalmology, the techniques developed could be applied to other medical and industrial applications. In this dissertation, a technique to quadruple the imaging speed of OCT was developed. This technique was demonstrated by imaging the retinas of healthy human subjects. A handheld, dual depth OCT system was developed. This system enabled sequential imaging of the anterior segment and retina of human eyes. Finally, handheld SLO/OCT systems were developed, culminating in the design of a handheld AOSLO system. This system has the potential to provide cellular level imaging of the human retina, resolving even the most densely packed foveal cones.

In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Stochastic switching in infinite dimensions with applications to random parabolic PDE

© 2015 Society for Industrial and Applied Mathematics.We consider parabolic PDEs with randomly switching boundary conditions. In order to analyze these random PDEs, we consider more general stochastic hybrid systems and prove convergence to, and properties of, a stationary distribution. Applying these general results to the heat equation with randomly switching boundary conditions, we find explicit formulae for various statistics of the solution and obtain almost sure results about its regularity and structure. These results are of particular interest for biological applications as well as for their significant departure from behavior seen in PDEs forced by disparate Gaussian noise. Our general results also have applications to other types of stochastic hybrid systems, such as ODEs with randomly switching right-hand sides.

Emulsion-Based RIR-MAPLE Deposition of Conjugated Polymers: Primary Solvent Effect and Its Implications on Organic Solar Cell Performance.

Emulsion-based, resonant infrared matrix-assisted pulsed laser evaporation (RIR-MAPLE) has been demonstrated as an alternative technique to deposit conjugated polymer films for photovoltaic applications; yet, a fundamental understanding of how the emulsion target characteristics translate into film properties and solar cell performance is unclear. Such understanding is crucial to enable the rational improvement of organic solar cell (OSC) efficiency and to realize the expected advantages of emulsion-based RIR-MAPLE for OSC fabrication. In this paper, the effect of the primary solvent used in the emulsion target is studied, both experimentally and theoretically, and it is found to determine the conjugated polymer cluster size in the emulsion as well as surface roughness and internal morphology of resulting polymer films. By using a primary solvent with low solubility-in-water and low vapor pressure, the surface roughness of deposited P3HT and PCPDTBT polymer films was reduced to 10 nm, and the efficiency of P3HT:PC61BM OSCs was increased to 3.2% (∼100 times higher compared to the first MAPLE OSC demonstration [ Caricato , A. P. ; Appl. Phys. Lett. 2012 , 100 , 073306 ]). This work unveils the mechanism of polymer film formation using emulsion-based RIR-MAPLE and provides insight and direction to determine the best ways to take advantage of the emulsion target approach to control film properties for different applications.

Active Surface Deformation Technology for Management of Marine Biofouling

Biofouling, the accumulation of biomolecules, cells, organisms and their deposits on submerged and implanted surfaces, is a ubiquitous problem across various human endeavors including maritime operations, medicine, food industries and biotechnology. Since several decades, there have been substantial research efforts towards developing various types of antifouling and fouling release approaches to control bioaccumulation on man-made surfaces. In this work we hypothesized, investigated and developed dynamic change of the surface area and topology of elastomers as a general approach for biofouling management. Further, we combined dynamic surface deformation of elastomers with other existing antifouling and fouling-release approaches to develop multifunctional, pro-active biofouling control strategies.

This research work was focused on developing fundamental, new and environment-friendly approaches for biofouling management with emphasis on marine model systems and applications, but which also provided fundamental insights into the control of infectious biofilms on biomedical devices. We used different methods (mechanical stretching, electrical-actuation and pneumatic-actuation) to generate dynamic deformation of elastomer surfaces. Our initial studies showed that dynamic surface deformation methods are effective in detaching laboratory grown bacterial biofilms and barnacles. Further systematic studies revealed that a threshold critical surface strain is required to debond a biofilm from the surface, and this critical strain is dependent on the biofilm mechanical properties including adhesion energy, thickness and modulus. To test the dynamic surface deformation approach in natural environment, we conducted field studies (at Beaufort, NC) in natural seawater using pneumatic-actuation of silicone elastomer. The field studies also confirmed that a critical substrate strain is needed to detach natural biofilm accumulated in seawater. Additionally, the results from the field studies suggested that substrate modulus also affect the critical strain needed to debond biofilms. To sum up, both the laboratory and the field studies proved that dynamic surface deformation approach can effectively detach various biofilms and barnacles, and therefore offers a non-toxic and environmental friendly approach for biofouling management.

Deformable elastomer systems used in our studies are easy to fabricate and can be used as complementary approach for existing commercial strategies for biofouling control. To this end, we aimed towards developed proactive multifunctional surfaces and proposed two different approaches: (i) modification of elastomers with antifouling polymers to produce multifunctional, and (ii) incorporation of silicone-oil additives into the elastomer to enhance fouling-release performance.

In approach (i), we modified poly(vinylmethylsiloxane) elastomer surfaces with zwitterionic polymers using thiol-ene click chemistry and controlled free radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionalities. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. In approach (ii), we incorporated silicone-oil additives in deformable elastomer and studied synergistic effect of silicone-oils and surface strain on barnacle detachment. We hypothesized that incorporation of silicone-oil additive reduces the amount of surface strain needed to detach barnacles. Our experimental results supported the above hypothesis and suggested that surface-action of silicone-oils plays a major role in decreasing the strain needed to detach barnacles. Further, we also examined the effect of change in substrate modulus and showed that stiffer substrates require lower amount of strain to detach barnacles.

In summary, this study shows that (1) dynamic surface deformation can be used as an effective, environmental friendly approach for biofouling control (2) stretchable elastomer surfaces modified with anti-fouling polymers provides a pro-active, dual-mode approach for biofouling control, and (3) incorporation of silicone-oils additives into stretchable elastomers improves the fouling-release performance of dynamic surface deformation technology. Dynamic surface deformation by itself and as a supplementary approach can be utilized biofouling management in biomedical, industrial and marine applications.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.