Single-Cell Optical Absorbance Characterization With High-Throughput Microfluidic Microscopy

Morphological changes in cells associated with disease states are often assessed using clinical microscopy. However, the changes in chemical composition of cells can also be used to detect disease conditions. Optical absorption measurements carried out on single cells using inexpensive sources, detectors can help assess the chemical composition of cells; thereby enable detection of diseases. In this article, we present a novel technique capable of simultaneously detecting changes in morphology and chemical composition of cells. The presented technique enables characterization of optical absorbance-based methods against microscopy for detection of disease states. Using the technique, we have been able to achieve a throughput of about 1000 cells per second. We demonstrate the proof-of-principle by detecting malaria in a given blood sample. The presented technique is capable of detecting very lower levels of parasitemia within time scales comparable to antigen-based rapid diagnostic tests.

Interacting single atoms with nanophotonics for chip-integrated quantum networks

Underlying matter and light are their building blocks of tiny atoms and photons. The ability to control and utilize matter-light interactions down to the elementary single atom and photon level at the nano-scale opens up exciting studies at the frontiers of science with applications in medicine, energy, and information technology. Of these, an intriguing front is the development of quantum networks where N >> 1 single-atom nodes are coherently linked by single photons, forming a collective quantum entity potentially capable of performing quantum computations and simulations. Here, a promising approach is to use optical cavities within the setting of cavity quantum electrodynamics (QED). However, since its first realization in 1992 by Kimble et al., current proof-of-principle experiments have involved just one or two conventional cavities. To move beyond to N >> 1 nodes, in this thesis we investigate a platform born from the marriage of cavity QED and nanophotonics, where single atoms at ~100 nm near the surfaces of lithographically fabricated dielectric photonic devices can strongly interact with single photons, on a chip. Particularly, we experimentally investigate three main types of devices: microtoroidal optical cavities, optical nanofibers, and nanophotonic crystal based structures. With a microtoroidal cavity, we realized a robust and efficient photon router where single photons are extracted from an incident coherent state of light and redirected to a separate output with high efficiency. We achieved strong single atom-photon coupling with atoms located ~100 nm near the surface of a microtoroid, which revealed important aspects in the atom dynamics and QED of these systems including atom-surface interaction effects. We present a method to achieve state-insensitive atom trapping near optical nanofibers, critical in nanophotonic systems where electromagnetic fields are tightly confined. We developed a system that fabricates high quality nanofibers with high controllability, with which we experimentally demonstrate a state-insensitive atom trap. We present initial investigations on nanophotonic crystal based structures as a platform for strong atom-photon interactions. The experimental advances and theoretical investigations carried out in this thesis provide a framework for and open the door to strong single atom-photon interactions using nanophotonics for chip-integrated quantum networks.

Chelation-enforced metal-arene interactions : insights into substrate binding and catalysis by late transition metal complexes

Understanding and catalyzing chemical reactions requiring multiple electron transfers is an endeavor relevant to many outstanding challenges in the field of chemistry. To study multi-electron reactions, a terphenyl diphosphine framework was designed to support one or more metals in multiple redox states via stabilizing interactions with the central arene of the terphenyl backbone. A variety of unusual compounds and reactions and their relevance toward prominent research efforts in chemistry are the subject of this dissertation.

Chapter 2 introduces the para-terphenyl diphosphine framework and its coordination chemistry with group 10 transition metal centers. Both mononuclear and dinuclear compounds are characterized. In many cases, the metal center(s) are stabilized by the terphenyl central arene. These metal–arene interactions are characterized both statically, in the solid state, and fluxionally, in solution. As a proof-of-principle, a dinickel framework is shown to span multiple redox states, showing that multielectron chemistry can be supported by the coordinatively flexible terphenyl diphosphine.

Chapter 3 presents reactivity of the terphenyl diphosphine when bound to a metal center. Because of the dearomatizing effect of the metal center, the central arene of the ligand is susceptible to reactions that do not normally affect arenes. In particular, Ni-to-arene H-transfer and arene dihydrogenation reactions are presented. Additionally, evidence for reversibility of the Ni-to-arene H-transfer is discussed.

Chapter 4 expands beyond the chelated metal-arene interactions of the previous chapters. A dipalladium(I) terphenyl diphosphine framework is used to bind a variety of exogenous organic ligands including arenes, dienes, heteroarenes, thioethers, and anionic ligands. The compounds are structurally characterized, and many ligands exhibit unprecedented bindng modes across two metal centers. The relative binding affinities are evaluated spectroscopically, and equilibrium binding constants for the examined ligands are determined to span over 13 orders of magnitude. As an application of this framework, mild hydrogenation conditions of bound thiophene are presented.

Chapter 5 studies nickel-mediated C–O bond cleavage of aryl alkyl ethers, a transformation with emerging applications in fields such as lignin biofuels and organic methodology. Other group members have shown the mechanism of C–O bond cleavage of an aryl methyl ether incorporated into a meta-terphenyl diphosphine framework to proceed through β-H elimination of an alkoxide. First, the electronic selectivity of the model system is examined computationally and compared with catalytic systems. The lessons learned from the model system are then applied to isotopic labeling studies for catalytic aryl alkyl ether cleavage under dihydrogen. Results from selective deuteration experiments and mass spectrometry draw a clear analogy between the mechanisms of the model and catalytic systems that does not require dihydrogen for C–O bond cleavage, although dihydrogen is proposed to play a role in catalyst activation and catalytic turnover.

Appendix A presents initial efforts toward heterodinuclear complexes as models for CO dehydrogenase and Fischer Tropsch chemistry. A catechol-incorporating terphenyl diphosphine is reported, and metal complexes thereof are discussed.

Appendix B highlights some structurally characterized terphenyl diphosphine complexes that either do not thematically belong in the research chapters or proved to be difficult to reproduce. These compounds show unusual coordination modes of the terphenyl diphosphine from which other researchers may glean insights.

Sensing and Actuation from Biology to Electronics

We introduce an in vitro diagnostic magnetic biosensing platform for immunoassay and nucleic acid detection. The platform has key characteristics for a point-of-use (POU) diagnostic: portability, low-power consumption, low cost, and multiplexing capability. As a demonstration of capabilities, we use this platform for the room temperature, amplification-free detection of a 31 bp DNA oligomer and interferon-gamma (a protein relevant for tuberculosis diagnosis). Reliable assay measurements down to 100 pM for the DNA and 1 pM for the protein are demonstrated. We introduce a novel "magnetic freezing" technique for baseline measurement elimination and to enable spatial multiplexing. We have created a general protocol for adapting integrated circuit (IC) sensors to any of hundreds of commercially available immunoassay kits and custom designed DNA sequences.

We also introduce a method for immunotherapy treatment of malignant gliomas. We utilize leukocytes internalized with immunostimulatory nanoparticle-oligonucleotide conjugates to localize and retain immune cells near the tumor site. As a proof-of-principle, we develop a novel cell imaging and incubation chamber for in vitro magnetic motility experiments. We use the apparatus to demonstrate the controlled movement of magnetically loaded THP-1 leukocytes.

Finally, we introduce an IC transmitter and power ampli er (PA) that utilizes electronic digital infrastructure, sensors, and actuators to self-heal and adapt to process, dynamic, and environmental variation. Traditional IC design has achieved incredible degrees of reliability by ensuring that billions of transistors on a single IC die are all simultaneously functional. Reliability becomes increasingly difficult as the size of a transistor shrinks. Self-healing can mitigate these variations.

星间激光通信中复合轴系统的带宽设计研究

星间激光通信中，由粗跟踪系统和精跟踪系统组成的复合轴系统完成了系统的大范围、高精度跟踪任务。对复合轴系统进行了基于Z变换的数字模拟以研究其带宽设计。根据计算的卫星轨迹和粗跟踪系统机械误差，得到了粗跟踪系统的带宽设计结果；给出了粗跟踪系统和精跟踪系统的配合工作关系；得到了精跟踪系统的带宽设计结果；最后研究了在中高频扰动下，复合轴系统的带宽设计。以上结果为星间激光通信中的复合轴系统控制系统设计提供了重要依据。

Optoelectronic quotient-selected modified signed-digit division

A novel optoelectronic quotient-selected modified signed-digit division technique is proposed. This division method generates one quotient digit per iteration involving only one shift operation, one quotient selection operation and one addition/subtraction operation. The quotient digit can be selected by observing three most significant digits of the partial remainder independent of the divisor. Two algorithms based on truth-table look-up and binary logic operations are derived. For optoelectronic implementation, an efficient shared content-addressable memory based architecture as well as compact logic array processor based architecture with an electron-trapping device is proposed. Performance evaluation of the proposed optoelectronic quotient-selected division shows that it is faster than the previously reported convergence division approach. Finally, proof-of-principle experimental results are presented to verify the effectiveness of the proposed technique. (C) 2001 Society of Photo-Optical Instrumentation Engineers.

Studies on photoblastic germination in lettuce seed

It was shown, with the aid of osmotic inhibition of germination, that the action of the far-red-absorbing form of phytochrome (Pf) in promoting germination can be completed even if the seed is held under conditions where germination is not possible. An effect of the continuing action of Pf beyond the point of complete germination promotion was demonstrated by enhancement of germination rate after removal of the osmotically active solute.

Previous reports that the rate of growth in water of seeds freed from the expansion-restricting endosperm is independent of the state of phytochrome were confirmed. However, a marked, phytochrome-mediated enhancement of the growth potential of such seeds was demonstrated through restricting water uptake by incubation in an osmoticum.

An experimental system, utilizing the appearance of a geotropic curvature in the radicle of the excised axial portion of the seed, was developed for more detailed studies of the phytochrome-enhanced growth potential. It was possible to demonstrate the light effect in water as well as in osmotica; this apparently is not possible with de-endospermed entire seeds. As in intact seeds, the effect of the continuing action of Pf is to enhance the rate of the response. Secretion of a chemical inhibitor of growth by the endosperm as a possible mechanism of induction of light sensitivity has been ruled out.

The phytochrome-dependent rate of appearance of geotropic curvature in osmotica is paralleled in time by a similar dependence of the rate of early extension growth of the embryonic axis. Only the first small increment of growth is a differentially responsive to red (R) and far-red (F); the rate of later increase in length is independent of the light regime.

It was shown that the high concentrations of gibberellic acid required for germination promotion in the intact seed are due at least in part to a diffusion barrier in the endosperm, and that the occasional reports in the literature of the ineffectiveness of kinetin are probably due to the same phenomenon. It was shown that gibberellin, like red light, enhances the growth potential of the axis, but kinetin does not. The difference in rates of response obtained after R-irradiation or gibberellin treatment, together with other results reported in the literature, strongly suggests that gibberellic acid and red light promote germination by different means. The idea that kinetin promotes germination by yet another mechanism, probably operating in the cotyledons, was supported through two different experimental approaches.

The phenomenon of temperature-dependent dark germination was examined in detail, using a wide range of both temperatures and incubation times. With the aid of the half-seed system, it was demonstrated that the promotive effect of low temperature on germination could not be due to a low optimum temperature for early growth of the radicle, since the rate of that process increased with increasing temperature, up to the highest temperature used.

It was shown that phytochrome does not function at high temperatures. This fact is of considerable importance in interpreting the phenomenon of thermodormancy, since in the literature only a small part of the effect of high temperature has been ascribed to an effect on phytochrome, and at that, only to an acceleration of dark reversion of Pf to the red-absorbing form of phytochrome (Pr). Partial denaturation of phytochrome may also make some contribution.

It was shown that the germination-promoting effect of low temperature depends on the presence of Pf, and concluded that low temperatures act by delaying or preventing transformation of Pf. Support for the assumption that Pf, not Pr, is the active form of phytochrome in lettuce seeds was drawn from the same evidence.

Attempts to stimulate germination by repeated irradiation with F over relatively prolonged incubation times resulted in failure, as have similar attempts reported in the literature. However, an enhancement of growth potential in the half-seed system by the maintenance of a small amount of Pf over long periods at ordinary temperatures by repeated irradiation with F was demonstrated.

It was observed that cold storage of the dry seed prevents or delays loss of dark dormancy during post-harvest storage. No change in the response of the half-seed in osmoticum to R and F was observed in seeds that has lost dark dormancy; that is, no internal change took place to measurably increase the growth potential of the embryonic axis. This suggests that the endosperm is the seat of changes responsible for after-ripening of photoblastic lettuce seed.

Design for Survival: Sustainability for Planet and Structure

Buildings in Port Aransas encounter drastic environmental challenges: the potential catastrophic storm surge and high winds from a hurricane, and daily conditions hostile to buildings, vehicles, and even most vegetation. Its location a few hundred feet from the Gulf of Mexico and near-tropical latitude expose buildings to continuous high humidity, winds laden with scouring sand and corrosive salt, and extremes of temperature and ultraviolet light. Building construction methods are able to address each of these, but doing so in a sustainable way creates significant challenges. The new research building at the Marine Science Institute has been designed and is being constructed to meet the demand for both survivability and sustainability. It is tracking towards formal certification as a LEED Gold structure while being robust and resistant to the harsh coastal environment. The effects of a hurricane are mitigated by elevating buildings and providing a windproof envelope. Ground-level enclosures are designed to be sacrificial and non-structural so they can wash or blow away without imposing damage on the upper portions of the building, and only non-critical functions and equipment will be supported within them. Design features that integrate survivability with sustainability include: orientation of building axis; integral shading from direct summer sunlight; light wells; photovoltaic arrays; collection of rainwater and air conditioning condensate for use in landscape irrigation; reduced impervious cover; xeriscaping and indigenous plants; recycling of waste heat from air conditioning systems; roofing system that reflects light and heat; long life, low maintenance stainless steel, high-tensile vinyl, hard-anodized aluminum and hot-dipped galvanized mountings throughout; chloride-resistant concrete; reduced visual impact; recycling of construction materials.

Full-range parallel Fourier-domain optical coherence tomography using sinusoidal phase-modulating interferometry

We demonstrate a full-range parallel Fourier-domain optical coherence tomography (FD-OCT) in which a tomogram free of mirror images as well as DC and autocorrelation terms is obtained in parallel. The phase and amplitude of two-dimensional spectral interferograms are accurately detected by using sinusoidal phase-modulating interferometry and a two-dimensional CCD camera, which allows for the reconstruction of two-dimensional complex spectral interferograms. By line-by-line inverse Fourier transformation of the two-dimensional complex spectral interferogram, a full-range parallel FD-OCT is realized. Tomographic images of two separated glass coverslips obtained with our method are presented as a proof-of-principle experiment.

Towards in situ Single Cell Systems Biology

Systems-level studies of biological systems rely on observations taken at a resolution lower than the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled measurements of the transcriptomes in single cells excised from their environment, but it remains a daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In this thesis I develop methods for the routine, quantitative in situ measurement of gene expression using fluorescence microscopy.

The number of molecular species that can be measured simultaneously by fluorescence microscopy is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally limited to the simultaneous measurement of only five labeled biomolecules at a time. The two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent strategies for overcoming this limitation to monitor expression of many genes in a single cell. Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically increases the optical space in a cell, allowing a large numbers of barcodes to be visualized simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium stress response. The second method, sequential barcoding, reads out a temporal barcode through multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of sequential barcoding increases exponentially with the number of rounds of hybridization, allowing over a hundred genes to be profiled in only a few rounds of hybridization.

The utility of sequential barcoding was further demonstrated by adapting this method to study gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction), was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated in combination with light sheet microscopy and background reducing tissue clearing technology, enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact mammalian tissue.

The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed transcriptional observations in any tissue of interest. These technologies will serve as a general platform for future transcriptomic studies of complex tissues.

洛仑兹脉冲光束的复振幅包络解和复解析信号解的比较研究

使用复振幅包络表示式和复解析信号表示式推导了洛仑兹脉冲光束的传输公式．通过具体数值计算对脉冲光束的复振幅包络解和复解析信号解在不同带宽时的传输进行了对比研究．数值结果表明脉冲光束为窄带时，在传输方向轴中心的一定范围内两种解是一致的，而对于宽带脉冲光束，复振幅包络解在轴中心较近的距离即表现出奇异性，复解析信号解才是符合物理意义的表示式．由数值计算得出了选择脉冲光束研究方法的条件，并从所得公式对复振幅包络解出现奇异性的现象进行了解释．

部分端面抽运板条激光器腔镜倾斜问题研究

部分端面抽运的板条激光器是一种新型固体激光器．配合混合腔可以实现在大功率下保持高光束质量的激光振荡输出。谐振腔的腔镜倾斜是影响激光器输出特性的重要因素，快速傅里叶方法是一种快捷有效的计算方法，利用此方法模拟了腔镜倾斜对近场相位分布和远场光强分布的影响，并分析了光束质量的变化。理论分析表明，腔镜的小角度倾斜对近场相位影响较大，但对远场光强空间分布影响不大；随着倾斜角度不断增大，远场发散角和光束腰宽度也增大，光束质量虽然存在恶化的趋势，但光束质量因子肝值仍然较小，离轴非稳腔仍能保持高光束质量的输出。

新型板条激光器的离轴混合腔模场计算

报道了一种新型双板条离轴混合腔激光器。这种激光器结构通过改变传统的冷却方式和采用特殊的谐振腔设计，将使从第一块介质板条高温一侧出射的激光对称地进入另一块板条的低温一侧，从而可对由于温度分布不均匀造成的波面畸变进行一定程度的自校正，减少热效应的影响，可望提高激光器的输出功率和光束质量。利用快速傅里叶变换（FFT）对这种激光器的近场、远场以及相位等模场特性进行了数值计算。分析了波面畸变对输出光束质量的影响，并与常规双板条激光器进行了比较，结果表明这种新型双板条离轴混合腔激光器可以实现一定程度的波面畸变自补偿，

激光二极管阵列的窄线宽、可调谐输出

外腔反馈的激光二极管阵列(LDA)可获得窄线宽、可调谐的光谱输出。外腔由快轴准直镜、准直光学系统和闪耀光栅组成。由于阵列中各发光单元的排列弯曲导致不同波长的光原路返回,引起谱线展宽,在输出光路中加入光谱滤波器,使激光二极管阵列的线宽进一步窄化。这样,激光二极管阵列的输出光谱由自由运转时的2 nm压缩到0.12 nm,在恒定温度23 ℃时,实现了激光在806～818 nm的调谐,调谐范围达12 nm。

Uma abordagem integrativa do conceito de Eco-eficiência: elementos para a concepção de Sistemas de Gestão de Resíduos Sólidos Industriais em Arranjos Produtivos Locais Têxtil-Vestuário

A identificação e avaliação das vulnerabilidades e potencialidades socioambientais e socioecológicas, ao considerar as múltiplas realidades de ecossistemas, territórios e lugares, podem revelar novos caminhos, ações coletivas, solidárias, bem como auxiliar na tomada de decisão estratégica em sistemas de gestão de resíduos sólidos. Tal perspectiva, qualificaria princípios e conceitos metodológicos e técnicos, como aqueles direcionados a responder aos problemas que surgem da interação Humanidade-Sociedade-Natureza, com vistas à sustentabilidade. A Eco-eficiência, ao final do século XX, apresentou-se como solução para os problemas ambientais corporativos. Idealizada para ser aplicada em um empresas isolada, evoluiu para uma categoria central da Ecologia Industrial, ganhou contornos de Princípio e Filosofia para a Gestão Ambiental Empresarial, passando a orientar a geopolítica das nações no trato das questões ambientais supranacionais e políticas públicas das nações. A recente Política Nacional de Resíduos Sólidos apresenta a Eco-eficiência como um de seus princípios que deverão orientar gestores públicos e privados na elaboração de seus sistemas de gestão de resíduos sólidos. Devido aos impactos sociais, ambientais e ecológicos resultantes da geração e destinação inadequada dos resíduos sólidos, estes sistemas de gestão apresentam interfaces com outras políticas e sistemas de gestão públicos e privados nacionais. Estas interações conferem ao sistema de gestão de resíduos sólidos um caráter complexo, aberto, dinâmico e inclusivo, no qual o conceito de Eco-eficiência, baseado na alocação de recursos naturais, pode ser ressignificado em um outro nível de realidade, o nível coletivo, apresentando-se, como um conceito potencial voltado para a criação de recursos. Para corroborar esta hipótese apresenta-se uma abordagem integrativa com base na perspectiva socioecológica e no pensamento e metodologia transdisciplinar, na qual o conceito será contextualizado, problematizado e complexificado em seis níveis de realidade: Nível Genus-Global, Nível Político, Nível Acadêmico, Nível do Ecossistema, Território e Lugares, Nível Operacional e Nível Coletivo. Considera-se nesta abordagem os Arranjos Produtivos (indústrias e a Cadeia de Reciclagem), Arranjos Sociais (Comunidade localizadas e áreas de risco e precárias), Ecossistemas e Territórios, como Teia de Lugares, como Unidade Transdisciplinar perceptiva, cognitiva e analítica. A aplicação desta metodologia no Arranjo Produtivo Local Têxtil- Vestuário de Petrópolis, localizado na Região Serrana Fluminense, que se insere em um Ecossistema de Montanha, permitiu revelar outro Arranjo, invisível ao sistema de gestão público. Neste Arranjo de Retalhos e Estopas, cerca de 240 mulheres, residentes em comunidades precárias e de risco, beneficiam os resíduos sólidos gerados pelas indústrias do arranjo formal, conhecidos como retalhos, com os quais fabricam estopas. Conclui-se que esta abordagem integrativa, como proposta de ressignificação do conceito de Eco-eficiência, permitiu revelar, não somente uma nova realidade conceitual para a sua adoção e prática, como novos aspectos e variáveis para a construção de sistemas de gestão de resíduos sólidos industriais que tenham por foco a inclusão social, produtiva, cientifica e tecnológica de novos atores e recursos ao sistema.