Nonlinear optical microscopy for the invisible: Vibrational imaging of small molecules in live cells and electronic imaging of fluorophores into the ultra deep

Wei, Lu Columbia University 2015 해외박사(DDOD)

Nonlinear optical microscopy (NOM) has become increasingly popular in biomedical research in recent years with the developments of laser sources, contrast mechanisms, novel probes and etc. One of the advantages of NOM over the linear counterpart is the ability to image deep into scattering tissues or even on the whole animals. This is due to the adoption of near-infrared excitation that is of less scattering than visible excitation, and the intrinsic optical sectioning capability minimizing the excitation background beyond focal volume. Such an advantage is particularly prominent in two-photon fluorescence microscopy compared to one-photon fluorescence microscopy. In addition, NOM may provide extra molecular information (e.g. second harmonic generation and third harmonic generation) or stronger signal (e.g. stimulated Raman scattering and coherent anti-Stokes Raman scattering compared to spontaneous Raman scattering), because of the nonlinear interaction between strong optical fields and molecules. However, the merits of NOM are not yet fully exploited to tackle important questions in biomedical research. This thesis contributes to the developments of NOM in two aspects that correspond to two fundamental problems in biomedical imaging: first, how to non invasively image small functional biomolecules in live biological systems (Chapters 1-4); second, how to extend the optical imaging depth inside scattering tissues (Chapters 5-6). The ability to non-perturbatively image vital small biomolecules is crucial for understanding the complex functions of biological systems. However, it has proven to be highly challenging with the prevailing method of fluorescence microscopy. Because it requires the utilization of large-size fluorophore tagging (e.g., the Green Fluorescent Protein tagging) that would severely perturb the natural functions of small bio-molecules. Hence, we devise and construct a nonlinear Raman imaging platform, with the coupling of the emerging stimulated Raman scattering (SRS) microscopy and tiny vibrational tags, which provides superb sensitivity, specificity and biocompatibility for imaging small biomolecules (Chapters 1-4). Chapter 1 outlines the theoretical background for Raman scattering. Chapter 2 describes the instrumentation for SRS microscopy, followed with an overview of recent technical developments. Chapter 3 depicts the coupling of SRS microscopy with small alkyne tags (C≡C) to sensitively and specifically image a broad spectrum of small and functionally vital biomolecules (i.e. nucleic acids, amino acids, choline, fatty acids and small molecule drugs) in live cells, tissues and animals. Chapter 4 reports the combination of SRS microscopy with small carbon-deuterium (C-D) bonds to probe the complex and dynamic protein metabolism, including protein synthesis, degradation and trafficking, with subcellular resolution through metabolic labeling. It is to my belief that the coupling of SRS microscopy with alkyne or C-D tags will be readily applied in answering key biological questions in the near future. The remaining chapters of this thesis (Chapters 5-6) present the super-nonlinear fluorescence microscopy (SNFM) techniques for extending the optical imaging depth into scattering tissues. Unlike SRS microscopy that is an emerging technique, multiphoton microscopy (mainly referred as two-photon fluorescence microscopy), has matured over 20 years with its setup scheme and biological applications. Although it offers the deepest penetration in the optical microscopy, it still poses a fundamental depth limit set by the signal-to-background ratio when imaging into scattering tissues. Three SNFM techniques are proposed to extend such a depth limit: unlike the conventional multiphoton microscopy whose nonlinearity stems from virtual-states mediated simultaneous interactions between the incident photons and the molecules, the high-order nonlinearity of the SNFM techniques that we have conceived is generated through real-state mediated population-transfer kinetics. In particular, Chapter 5 demonstrates the multiphoton activation and imaging (MPAI) microscopy, which adopts a new class of fluorophores, the photoactivatable fluorophores, to significantly extend the fundamental imaging depth limit. Chapter 6 theoretically and analytically depicts two additional SNFM techniques of stimulated emission reduced fluorescence (SERF) microscopy and focal saturation microscopy. Both MPAI and focal saturation microscopies exhibit a fourth order power dependence, which is effectively a four-photon process. SERF presents a third order power dependence for a three-photon process.

A Study on the Modernization Process of Urban Area and Architecture in Qingdao

Hong-Wei Lu 군산대학교 대학원 2008 국내석사

Historical urban planning and architecture are the memory of a city and play an important role in the future development of the city. Realizing the birth and historical changes of Qingdao city is essential to its future development. With this intension, the birth and historical changes of Qingdao city,which focus on the formation of modern urban planning and architecture, are clearly presented in this paper.

Understanding the Folding Mechanisms of Membrane Proteins through Molecular Simulations and Energy Landscape Analysis

Lu, Wei ProQuest Dissertations & Theses Rice University 2021 해외박사(DDOD)

Fabrication and characterization of a double torsional mechanical oscillator and its applications in gold micromass measurements

Lu, Wei The University of Texas at Austin 2008 해외박사(DDOD)

We report the design and fabrication of a micro-mechanical oscillator for use in extremely small force detection experiments such as transverse force measurements of a moving vortex and Nuclear Magnetic Resonance Force Microscopy (NMRFM). We study the basic physics of the double torsional mechanical oscillator, and pursue double torsional oscillators with small spring constants, high resonance frequencies, and high quality factors. Using a series of semiconductor manufacturing techniques, especially using the electron-beam lithography technique, we successfully micro-fabricate double torsional mechanical oscillators from silicon-on-insulator wafers. We conduct characterization experiments to extract important parameters of a mechanical oscillator, including the resonance frequencies, spring constants, and quality factors. We focus on the four typical resonance modes of these oscillators, and then compare the force detection sensitivity of each mode. Eventually we apply these force sensitive oscillators to gold micro-mass measurements, and achieve very small mass detection. In the future we are going to continue to micro-fabricate thinner oscillators to reduce the spring constants, and improve the quality factors by designing more suitable geometric shapes and by pursuing annealing studies. Thus, we might be able to achieve single nuclear spin measurements using NMRFM.

Self-organizing nanophases on solid surfaces

Lu, Wei Princeton University 2001 해외박사(DDOD)

Experimental evidence has accumulated in the recent decade that nanoscale patterns can self-assemble on solid surfaces. A two-component monolayer grown on a solid surface may separate into distinct phases, forming periodic stripes, triangular lattice of dots, or other regular patterns. The size of the phases may be in the range 1–100 nm, and stable against coarsening on annealing. This thesis develops a thermodynamic framework to study the remarkable phenomena. The thermodynamic forces that drive the self-assembly are identified. A double-welled, composition-dependent free energy drives phase separation. The phase boundary energy drives phase coarsening. The concentration-dependent surface stress drives phase refining. It is the competition between the coarsening and the refining that leads to size selection and spatial ordering. These thermodynamic forces are embodied in a nonlinear diffusion equation. The linear stability analysis gives the stable condition for a uniform concentration field, suggests the fastest growth wavelength, and provides an estimation of the pattern sizes. The numerical techniques to simulate the pattern formation process are developed. The simulations reveal rich dynamics. It is relatively fast for the phases to separate and select a uniform size, but exceedingly slow to order over a long distance, unless the symmetry of the system is suitably broken. The effects of inhomogeneity and anisotropy are investigated. Coarse pre-patterns, which can be introduced in the form of initial conditions or spatial dependent chemical potentials, may guide the self-assembly process and help to generate desired fine patterns. Anisotropy introduces directional preference. By varying the degree of surface stress anisotropy, a variety of patterns, such as serpentine stripes, parallel stripes, triangular lattice of dots, and herringbone structures are obtained. In addition, a surprising second order transition between the parallel stripes and the herringbone structures is discovered. The simulations reveal that patterns on an elastically anisotropic substrate with a cubic structure tend to arrange along the compliant directions. Patterns like square lattice of dots are obtained in this way.

Regulation of protein tyrosine phosphatase SHP-2 by tyrosine phosphorylation

Lu, Wei The Johns Hopkins University 2003 해외박사(DDOD)

SHP-2 is a cytoplasmic protein tyrosine phosphotase (PTPase) that has been implicated to be an essential component in several signaling pathways. The regulation of SHP-2 is proposed to involve tyrosine phosphorylation on two C-terminal tail tyrosine residues. However, the detailed mechanism of how tail tyrosine phosphorylation affects SHP-2 function is lacking due to its strong tendency to undergo autodephosphorylation. Using ‘expressed protein ligation’, we successfully incorporated two different non-hydrolyzable phosphotyrosine analogues into two known phosphorylation sites in SHP-2 individually and/or simultaneously. We systematically studied the impact of these analogues on the activities of SHP-2s. We found that a phosphonate at Tyr-542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase; whereas a phosphonate at Tyr-580 stimulates the PTPase activity by engaging the C-terminal SH2 domain. We also found that phosphonates at both sites have a synergistic effect on stimulating PTPase activity. In order to understand the physiological significance of tyrosine phosphorylation of SHP-2 in cell signaling, we microinjected phosphonate-containing SHP-2s into REF-52 cells and found that a single phosphorylation of Tyr-542 is sufficient to activate the MAP kinase pathway. We also characterized the dephosphorylation reaction of tyrosine phosphorylated SHP-2 and found that intramolecular interaction between SH2 domains and phosphotyrosines renders the phosphotyrosine resistant to hydrolysis. These data support a novel mechanism in which elevated enzymatic activity of tyrosine phosphorylated SHP-2 plays an active role in MAP kinase signaling pathway. This study also provides a general method for investigating the role of tyrosine phosphorylation on other tyrosine phosphatases.

COVID-19 감염병 위기 상황에서 유학생 안전 인식 분석 -중국 유학생을 중심으로-

Lu Wei 충북대학교 2021 국내석사

본 연구는 중국 유학생들을 중심으로 유학생들의 안전 의식과 COVID-19로 인한 위기를 연구하고 있다. 중국과 한국 간의 외교 관계 수립과 교류가 점점 더 가까워지고 있고, 이로 인해 한국에서 유학하는 학생들의 수가 계속해서 증가하고 있다. 하지만, 해외 유학생들과 관련된 사고와 부상 또한 해외에서 공부하는 동안 자주 발생하고 있다. 중국 유학생들의 해외 안전이 각계각층의 관심을 끌고 있다. COVID-19 공공 위기 사건은 유학생들의 생활, 공부, 건강 및 안전 교육의 부족한 점과 결함을 드러냈고, 이로 인해 유학생들은 학업, 생활 및 다양한 측면에서 다양한 영향을 받게 되었다. 이 연구의 내용은 크게 다섯 부분으로 나뉜다. 첫 번째는 유학생 안전의 연구 배경, 한국, 중국 등의 문헌을 설명하고 이 주제의 연구 목적과 중요성을 설명한다. 두 번째는 이론적인 분석입니다. 첫째, 이 정보에 따르면, 이것은 코로나19와 새로운 코로나19로 인한 위기, 유학생들이 안전의식 그리고 유학생들의 현존하는 안전 문제들을 설명한다. 둘째, 참고인과 함께 COVID-19로 인한 유학생 위기를 분류하는데, 이는 주로 생활 위기, 학업 위기, 심리 건강 및 개인 안전 위기로 나뉜다. 이어 중국 내 COVID-19 사태 이후 위기관리 과정을 정리하고 유학생 지원을 분석했다. 마지막으로, 유학생들의 안전의식 강화와 유학 시 주의해야 할 점들을 분석한다. 세 번째는 선행 연구와 참고 자료를 결합하여 유학생과 COVID-19의 안전 인식에 대한 설문지를 작성한다. 설문지에는 인구사회학의 기본적 특성, 기본적인 안전의식, 코로나19로 인한 위기, 유학생을 위한 중국이 채택한 보호조치의 인식, 수용 및 만족도, 향후 유학생의 방향과 방법 등이 포함되어 있다. 중국인 유학생 대상 설문조사 결과에 따르면 2021년 3월 24일부터 2021년 3월 30일까지 COVID-19 예방의 필요성으로 인터넷을 통해 설문조사를 실시했다. 총 261개의 설문지가 집계되었고 261개의 유효한 설문지가 집계되었다. SPSS26을 사용하여 이 설문조사의 신뢰성과 유효성을 테스트한다. 빈도와 백분율의 통계 분석은 샘플의 기본 상황을 설명하는 데 사용된다. 인구통계학적 사회학적 특성과 변수의 관계에 따라 교차분석을 채택한다. T 검정 및 일원 분산 분석을 통해 그룹 간의 평균값을 비교하여 평균값의 차이가 있는지 확인한다. 네 번째는 설문조사 결과를 분석한다. 이 연구는 유학생들의 안전 의식이 인구사회학적 특성에 따라 학교 안전과 생활 안전, 그리고 COVID-19의 영향에 더 집중되어 있다는 것을 발견했다. COVID-19 발생 이후 중국은 해외 유학생에 대한 영사보호 활동을 적극적이고 효과적으로 수행하였으나 설문조사와 온라인 인터뷰를 통해 유학생 영사보호 메커니즘 운영에 중점을 두었으며, 영사보호에 대한 인기가 부족한 것으로 나타났다. 여러 주제 간의 높고 불충분한 조정과 같은 문제는 여전히 있다. COVID-19 발생 이후, 유학생들은 보건 안전과 심리 건강에 대해 더 많이 알게 되었고 직접 경험에게 안전 교육 방법을 선호하게 되었다. 다섯 번째는 설문지 조사 결과를 통해 유학생들에게 미치는 COVID-19의 영향을 분석하고 유학생 안전보호의 장단점을 짚어본다. 유학생에 대한 영사보호 훈련 강화, 대사관 업무 효율성 향상, 유학생 단체 역할 중시, 다방면 참여 활성화 등이 필요하다. This study is centered on Chinese students, studying the basic safety awareness of international students and the crisis caused by COVID-19. The establishment of diplomatic relations and exchanges between China and South Korea have become increasingly close, which has led to the continuous increase in the overall number of students studying in Korea. However, accidents and personal injuries involving overseas students have also occurred frequently while studying abroad. The overseas safety of Chinese overseas students has attracted great attention from all walks of life. The COVID-19 public crisis incident exposed the vacancies and deficiencies in current life, health and safety education, which caused international students to be affected to varying degrees in their studies, life, and various aspects. The study content is mainly divided into five parts. The first part describes the research background of international student safety, literature reviews from South Korea, China and other countries, and explains the purpose and significance of this topic. The second part is a conceptual and theoretical analysis. First, according to the information, it explains the crisis caused by the new coronary pneumonia and the new coronary pneumonia, the basic safety awareness and existing problems of international students. Second, combined with references, first sort out the international student crisis caused by COVID-19, which is mainly divided into life crisis, academic crisis, mental health and personal safety crisis. Subsequently, the crisis management in China after the outbreak of COVID-19 was sorted out, and the assistance provided to international students was analyzed. Finally, it analyzes the need for international students to strengthen their safety awareness and the aspects that need to be paid attention to when studying abroad. The third part combines advanced research and references to compile a questionnaire on basic safety awareness of international students and COVID-19. The questionnaire includes the basic characteristics of population sociology, basic safety awareness, the crisis caused by the new crown pneumonia, the awareness, acceptance and satisfaction of the protective measures adopted by China for the new crown pneumonia for international students, and the direction and method of future international students' focus on safety. According to the survey subjects of Chinese students studying in South Korea, during the period from March 24, 2021 to March 30, 2021, due to the need for COVID-19 prevention, a questionnaire survey was conducted through the Internet. A total of 261 questionnaires were counted and 261 valid questionnaires were counted. Use SPSS26 to test the reliability and validity to illustrate the reliability and validity of this questionnaire survey. A statistical analysis of frequency and percentage is used to describe the basic situation of the sample. According to the relationship between demographic sociological characteristics and variables, a cross-analysis is adopted. Through independent sample T test and one-way analysis of variance, the average values between groups are compared to see if there is a difference in the average value. The fourth part analyzes the survey results. The study found that the basic safety awareness of international students is more concentrated on campus safety and life safety, and the impact of COVID-19 according to demographic and sociological characteristics. Since the outbreak of COIVID-19, although China has actively and effectively carried out consular protection for overseas students, it has focused on the operation of my country’s consular protection mechanism for overseas students through questionnaire surveys and online interviews, and found that there is a lack of popularity of consular protection information. Problems such as high and insufficient coordination among multiple subjects. Since the outbreak of COVID-19, international students have become more aware of health safety and mental health, and prefer offline experience-based safety education methods. The fifth part analyzes the impact of COVID-19 on international students based on the results of the questionnaire survey, and points out the advantages and disadvantages of the safety protection of international students. It is necessary to further strengthen the training of consular protection for overseas students, improve the efficiency of embassies, attach importance to the role of international student organizations, and promote Multiple forces participate.

Sputtering deposition and characterization of ultrathin amorphous carbon films

Lu, Wei University of California, Berkeley 1999 해외박사(DDOD)

This dissertation focuses on experimental investigations of ultrathin, ultrasmooth amorphous carbon (a-C) films deposited on Si(100) substrates by radio frequency (RF) sputtering and characterization of the nanomechanical and nanotribological properties and thermal stability of the films. Ultrathin a-C films of thickness 5–100 nm and typical root-mean-square roughness of 0.15–1 nm were deposited on ultrasmooth Si(100) substrates using pure argon as the sputtering gas. A low-pressure RF argon discharge model was used to analyze the plasma parameters in the film growth environment. These plasma parameters correlate the deposition conditions with the film growth processes. Atomic force microscopy (AFM) and surface force microscopy (SFM) were used to characterize the nanomechanical and nanotribological properties of the a-C films. X-ray photoelectron spectroscopy (XPS) was used to investigate the compositions and microstructures of the films. Sputter-etching measurements of the a-C films by energetic argon ion bombardment were used to study the surface binding energy of carbon atoms in a-C films deposited under different conditions. The dependence of film properties on deposition conditions was studied, and relations between nanomechanical and nanotribological properties were discussed in terms of a modified deformation index. The deformation and nanotribology mechanisms of the a-C films were compared with those of other films, such as TiC and Cr films (both 100 nm thick), and bulk Si(100). Reactive RF sputtering of nitrogenated amorphous carbon (a-CN_x) films was investigated by introducing nitrogen into the a-C films during film growth by using an argon-nitrogen gas mixture as the sputtering gas. The alloying effect of nitrogen on the film growth and properties, such as hardness and surface energy, was studied and interpreted in terms of the changes in the plasma environment induced due to differences in the composition of the sputtering gas mixture. The thermal stability of the a-C films and the role of residual stresses in the a-C films were studied in high-vacuum annealing experiments performed in the XPS vacuum chamber. The changes in the film composition and microstructure during sequential annealing cycles at 495°C were analyzed in light of XPS spectra. A new method was proposed to quantify the compressive stresses in ultrathin films (less than 10 nm) using implanted argon atoms as stress-sensing probes in conjunction with XPS analysis.

Synthesis of Carbon Nanomaterials and Their Applications in the Oilfield

Lu, Wei Rice University 2013 해외박사(DDOD)

This dissertation explores the potential applications of nanotechnology in the oilfield including poly(vinyl alcohol) stabilized carbon black nanoparticles for oil exploration and temperature-responsive carbon black nanoparticles for enhanced oil recovery. Also, it describes the rational design of graphene nanoribbons via intercalating reactive metals into multi-walled carbon nanotubes followed by addition of vinyl monomers or haloalkanes. Efficient production and modification of these aforementioned nanomaterials will make them more attractive for applications in the oilfield and electronics materials. A method is reported for detecting the hydrocarbon in the porous media with stabilized nanoparticles that are capable of efficiently transporting hydrophobic molecules through oil-containing rocks and selectively releasing them when a hydrocarbon is encountered. Nano-sized carbon black was oxidized and then functionalized with poly(vinyl alcohol) via a coupling reaction between the polymer's hydroxyl groups and the carboxylic groups on oxidized carbon black. Breakthrough curves show that poly(vinyl alcohol)-coated oxidized carbon black was stable in synthetic sea brine at room temperature and could carry the 14C-labeled radioactive tracer 2,2',5,5 '-tetrachlorobiphenyl through rocks and then released the tracer upon exposure to hydrocarbon. Due to the temperature-sensitivity of hydrogen bonds, higher molecular weight poly(vinyl alcohol) was used to improve the stability of carbon black nanoparticles in synthetic sea brine at higher temperatures. After sulfation, high molecular weight poly(vinyl alcohol) could stabilized carbon black nanoparticles in American Petroleum Institute standard brine at high temperatures. Those nanoparticles could efficiently transport mass-tagged probe molecules through a variety of oil-field rock types and selectively released the probe molecules into the hydrocarbon-containing rocks. Those proof-of-concept chemical nanoreporters can potentially be used under conditions commonly observed in the reservoir, and aid in the recovery of oil that remains in place. Amphiphilic carbon nanoparticles have been prepared that are capable of reversibly transferring across the water/oil interface in a temperature-controlled manner. Nano-sized carbon black was oxidized and then functionalized with amphiphilic diblock polyethylene-b-poly(ethylene glycol) copolymers that were water-soluble at low-to-moderate temperatures but oil-soluble at higher temperatures. The correlation between the phase transfer temperature and the melting temperature of the hydrophobic block of the copolymers and the weight percent of hydrophilic block were investigated. The amphiphilic nanoparticles were used to stabilize oil droplets for demonstrating potential applications in reducing the water/oil interfacial tension, a key parameter in optimizing crude oil extraction from downhole reservoirs. Graphene nanoribbons free of oxidized surfaces can be prepared in large batches and 100% yield by splitting multi-walled carbon nanotubes with potassium vapor. If desired, exfoliation is attainable in a subsequent step using chlorosulfonic acid. The low-defect density of these GNRs is indicated by their electrical conductivity, comparable to that of graphene derived from mechanically exfoliated graphite. Additionally, cost-effective and potentially industrially scalable, in situ functionalization procedures for preparation of soluble graphene nanoribbons from commercially carbon nanotubes are presented. To make alkane-functionalized graphene nanoribbons, multi-walled carbon nanotubes were intercalated by sodium/potassium alloy under liquid-phase conditions, followed by addition of haloalkanes, while polymer-functionalized graphene nanoribbons were prepared via polymerizing vinyl monomers using potassium-intercalated graphene nanoribbons. The correlation between the splitting of MWCNTs, the intrinsic properties of the intercalants and the degree of graphitization of the starting MWCNTs has also been demonstrated. Those functionalized graphene nanoribbons could have applications in conductive composites, transparent electrodes, transparent heat circuits, and supercapcitors.

Learning Guarantees for Graph Convolutional Networks in the Stochastic Block Model

Lu, Wei ProQuest Dissertations & Theses Brandeis Universit 2022 해외박사(DDOD)

An abundance of neural network models and algorithms for diverse tasks on graphs have been developed in the past five years. However, very few provable guarantees have been available for the performance of graph neural network models. In this paper we present the first provable guarantees for one of the best-studied families of graph neural network models, Graph Convolutional Networks (GCNs), for semi-supervised community detection tasks. We show that with high probability over the initialization and training data, a GCN will efficiently learn to detect communities on graphs drawn from a stochastic block model.The thesis starts with general introduction to machine learning and machine learning on graphs, in particular graph neural network models. Then we introduce PAC learning framework, which is a mathematical framework to analyze the performance of a machine learning model. Afterwards, we present a detailed description of stochastic block model and Graph Convolutional Networks, then the main results on learning guarantee. We demonstrate the idea of our proof before giving the detailed proofs. Our proof relies on a fine-grained analysis of the training dynamics in order to overcome the complexity of a non-convex optimization landscape with many poorly-performing local minima. We also show some experimental results and visualization to verify our results.