本论文介绍了一种基于扫描共聚焦显微镜和微流反应器用于研究化学振动子同步的系统。这个系统利用300微米的PNIPAm胶体颗粒作为振动子，Ru(vmbpy)(bpy)₂(PF₆)₂ 被参杂到振动子里充当BelousovZhabotinsky（BZ）振荡反应的催化剂。扫描共聚焦显微镜具有很高的灵敏度，可以给出高质量的图片以供研究分析。通过实验证明低于56W/cm²强度的扫描激光对BZ反应没有影响。这里所运用的微流反应器包括两个部分，PMMA材质的微池和PDMS材质的流道。此反应器可通过流道不停地补充BZ反应的反应物从而保证振荡的一致性。 / 通通过此系统，我们可以研究不同的两个振动子的同步问题。在实验中，振动子间的同步是由两者间的距离决定的。振动子在靠近时同步在分开至临界距离以外处不同步。另外，我利用COMSOL来模拟实验中的现象，发现模拟的结果和实验中的现象十分吻合。 / In this thesis, I present an experimental platform based on laser scanning confocal microscopy (LSCM) and continuous flow microreactor (CFMR) to study coupled chemical oscillators. PNIPAm gel particles around 300 micron were synthesized in the microfluidic device as the oscillators. Ru(vmbpy)(bpy)₂(PF₆)₂ was used as the catalyst of the BelousovZhabotinsky (BZ) reaction. The LSCM offers a good signal-to-noise ratio and better imaging quality. We demonstrated that the scanning laser with the power below 56W/cm² had no influence to BZ reaction. The CFMR, consisting of the PMMA microwell and the PDMS microchannel, can maintain the oscillation of the oscillators with a continuous supply of the BZ mixture. / The synchronization of the double heterogeneous oscillators was studied by the platform. The coupling intensity was controlled by changing the distance between the two oscillators. Results showed that the synchronization occurred as the oscillators were close and was lost as the oscillators were separated beyond a critical distance. The results of the numerical simulation by COMSOL agreed well with the experimental observation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Dameng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 40-41). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of contents --- p.iv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Synchronization of chemical oscillating systems --- p.1 / Chapter 1.2 --- The Belousov-Zhabotinsky reaction --- p.4 / Chapter 1.2.1 --- FKN model --- p.5 / Chapter 1.2.2 --- Effect of light illumination on ruthenium catalyzed BZ reaction --- p.7 / Chapter 1.2.3 --- Luminescence of ruthenium catalyzed in the BZ reaction --- p.8 / Chapter 1.3 --- Oscillators based on the BZ reaction --- p.9 / Chapter 1.4 --- Detection methods for the BZ oscillating systems --- p.10 / Chapter 1.4.1 --- Ion selective electrode and optical microscopy --- p.10 / Chapter 1.4.2 --- Objective of the research --- p.10 / Chapter Chapter 2 --- LSCM and continuous flow microreactor based platform --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.2 --- Experimental --- p.13 / Chapter 2.2.1 --- The fabrication of the oscillators --- p.13 / Chapter 2.2.2 --- The fabrication of the CFMR --- p.14 / Chapter 2.2.3 --- The detection method --- p.16 / Chapter 2.3 --- Results and discussions --- p.16 / Chapter 2.3.1 --- The comparison of PMMA and PDMS microreactors --- p.16 / Chapter 2.3.2 --- The flow rate of the BZ mixture to maintain the oscillation --- p.18 / Chapter 2.3.3 --- The size of the microreactor --- p.19 / Chapter 2.3.4 --- The factors to reduce the influence of the laser on the oscillators --- p.21 / Chapter 2.4 --- Conclusions --- p.23 / Chapter Chapter 3 --- The studying of the synchronization of the double oscillators --- p.24 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.1.1 --- Kuramoto model for illustrating the synchronization of double oscillators --- p.24 / Chapter 3.1.2 --- Transition from disorder to synchronization --- p.25 / Chapter 3.2 --- Experimental and simulation --- p.26 / Chapter 3.2.1 --- The dispensing and detection of the oscillators in the microreactor --- p.26 / Chapter 3.2.2 --- The simulation model --- p.27 / Chapter 3.3 --- Results and discussion --- p.28 / Chapter 3.3.1 --- The controlling of the coupling intensity --- p.28 / Chapter 3.3.2 --- The results of the synchronization --- p.30 / Chapter 3.3.2.1 --- Synchronization --- p.30 / Chapter 3.2.2.2 --- The critical distance for the synchronization and the transition --- p.31 / Chapter 3.3.3 --- The results of the simulation --- p.34 / Chapter 3.4 --- Conclusion --- p.35 / Chapter Chapter 4 --- Conclusion --- p.37 / Chapter 4.1 --- Summary --- p.37 / Chapter 4.2 --- Discussions and future perspectives --- p.38 / Reference --- p.40 / Appendix --- p.42 / Chapter 1. --- Code in Matlab for calculating the RGB value of ROI in the images from the LSCM --- p.42 / Chapter 2. --- The power of the laser --- p.43 / Chapter 2.1 --- The power of the laser in the LSCM --- p.43 / Chapter 2.2 --- The irradiation power on the oscillators --- p.44 / Chapter 3. --- The energy transferred to the oscillator --- p.44 / Chapter 4. --- The model in COMSOL --- p.45

Two-photon microscopy is often used in biological imaging due to its optical sectioning and depth penetration capabilities. These characteristics have made two-photon microscopy especially useful for neurobiological studies where imaging a volume at single cell resolution is typically required. This dissertation focuses on the optimization of two-photon excited fluorescence for volumetric imaging of biological samples, with special attention to imaging the mouse brain. Chapter 2 studies wavefront manipulation as a way of optimizing two-photon excited fluorescence. We show, through numerical simulations and experiments, that the magnitude of the two-photon fluorescence signal originating from cell-sized objects can be used as a metric of beam quality. We also show that the cranial window used in mouse experiment is a major source of aberrations, which can readily be represented in the Zernike basis. Finally, we implement a modal wavefront optimization scheme that optimizes the wavefront based entirely on the magnitude of the fluorescence. Along with this scheme, Zernike functions are found to be a useful basis for correcting aberrations encountered in mouse brain imaging while the Hadamard basis is found to be useful for scattering compensation. Corrections performed in mouse brain using Zernike functions are found to be valid over hundreds of microns, allowing a single correction to be applied to a whole volume. Finally, we show that the wavefront correction system can double as a wavefront encoding system for experiments that require custom point-spread-functions. Chapter 3 aims to significantly improve the volume imaging rate of two-photon microscopy. The imaging speed is improved by combining two-photon excitation with scanning confocally-aligned planar excitation microscopy (SCAPE). Numerical simulations, analytical arguments, and experiments reveal that the standard method of combining nano-joule pulses with 80 MHz repetition rates is inadequate for two-photon light-sheet excitation. We use numerical simulations and experiments to explore the possibility of achieving fast volumetric imaging using line and sheet excitation and find that the sheet excitation scheme is more promising. Given that two-photon excitation requires high photon-flux-densities near the focus, achieving high enough fluorescence has to be balanced with restrictions placed by saturation, photodamage, photobleaching and sample heating effects. Finally, we experimentally study light sheet excitation at various pulse repetition rates with femtosecond pulses and find that repetition rates near 100 kHz allow imaging of nonbiological samples of ~200x300x300 μm^3 volume at 20 volumes per second while balancing the above constraints. This work paves the way for achieving fast, volumetric two-photon imaging of the mouse brain.

Gong, Xiangjun = 新型全內反射顯微鏡的搭建及其在膠體研究中的應用 / 龔湘君. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Abstracts in English and Chinese. / Gong, Xiangjun = Xin xing quan nei fan she xian wei jing de da jian ji qi zai jiao ti yan jiu zhong de ying yong / Gong Xiangjun. / Abstract (Chinese) --- p.i / Abstract --- p.iii / Contents --- p.v / Acknowledgement --- p.vii / Chapter Chapter 1 --- Introduction to Total Internal Reflection Microscopy (TIRM) / Chapter 1.1 --- History of TIRM --- p.1 / Chapter 1.2 --- Instrumentation --- p.1 / Chapter 1.2.1 --- Apparatus --- p.1 / Chapter 1.2.2 --- Optical Tweezer --- p.3 / Chapter 1.3 --- The principle of the technique --- p.5 / Chapter 1.3.1 --- Total Internal Reflection --- p.5 / Chapter 1.3.2 --- Details on Scattering of the Evanescent Wave --- p.7 / Chapter 1.3.3 --- Data analysis --- p.14 / Chapter 1.3.4 --- A typical potential energy profile --- p.17 / Chapter 1.4 --- Noise Analysis and Removal Method --- p.19 / Chapter 1.4.1 --- Noise analysis --- p.19 / Chapter 1.4.2 --- Noise removal: low-pass filtering --- p.22 / Chapter 1.5 --- Other techniques on force measurement --- p.25 / Chapter 1.6 --- References --- p.27 / Chapter Chapter 2 --- Experiments on TIRM Calibration / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Pre-Experimental Section --- p.30 / Chapter 2.2.1 --- Glass Surface Preparation Methods --- p.30 / Chapter 2.2.2 --- Experimental Setup --- p.33 / Chapter 2.3 --- Calibration Results and Discussion --- p.36 / Chapter 2.4 --- Conclusions --- p.42 / Chapter 2.5 --- References --- p.43 / Chapter Chapter 3 --- TIRM investigation on polymer-induced collodial interactions / Chapter 3.1 --- Introduction to polymer-induced forces --- p.45 / Chapter 3.1.1 --- Non-absorbing case - depletion force --- p.46 / Chapter 3.1.2 --- Attached polymer layers --- p.49 / Chapter 3.2 --- Applications of TIRM in polymer-mediated colloidal interactions --- p.54 / Chapter 3.2.1 --- Measurements on depletion force --- p.54 / Chapter 3.2.2 --- Measurements on Steric Forces --- p.56 / Chapter 3.3 --- Direct measurement of thermosensitive Poly(N-isopropylacrylamide)-mediated colloidal interactions with TIRM --- p.56 / Chapter 3.3.1 --- Introduction --- p.56 / Chapter 3.3.2 --- Experimental Section --- p.57 / Chapter 3.3.3 --- Results and Discussion --- p.60 / Chapter 3.3.4 --- Conclusions --- p.73 / Chapter 3.4 --- References --- p.74 / Publication List --- p.77

Gong, Xiangjun = 基於磁鑷和全反射顯微鏡的單粒子追踪微流變方法 / 龔湘君. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Gong, Xiangjun = Ji yu ci nie he quan fan she xian wei jing de dan li zi zhui zong wei liu bian fang fa / Gong Xiangjun.

Através de um hamiltoniano efetivo baseado no tratamento de NAMBU POT.90 para o BCS, no qual incluímos além do emparelhamento monopolar o emparelhamento quadrupolar, discutimos a relação entre esse modelo e o modelo de bósons interagentes. Uma aplicação e feita a cadeia o (6) do IBM, que corresponde ao limite GAMA-instável do modelo de bohr. As dificuldades encontradas e uma possível generalização para o modelo são discutidas. / The nambu mechanism for BCS theory is extended with inclusion of quadrupole pairing in adition to the usual monopole pairing. An effective Hamiltonian is constructed and its relation to the IBM is discussed. We discussed the faced difficulties and a possible generalization of the model.

archives@tulane.edu / This dissertation is rooted in clinical pathology research, and the main character is addressing limitations in current pathology evaluation workflows. Diagnostic procedures for cancer are typically conducted via core needle biopsy procedures; however, tissue sampling limitations often result in a low yield of samples containing cancer – there are no reliable intraoperative methods to determine if the “lesion is in the needle”. If biopsy procedures result in a diagnosis of cancer, surgical removal of the tumor is often the frontline curative therapy for many cancers. Importantly, histologic evaluation following the whole resected organ is necessary to determine the presence of residual cancer, yet current methods do not allow efficient determination of tumor removal completeness intraoperatively. To address limitations of current histopathology methods, what is critically needed is a point-of-procedure fresh tissue evaluation system that facilitates 1) rapid on-site imaging and evaluation, 2) less destruction, and 3) more complete assessment of tumor content in fresh specimens. A novel microscopy system using video-rate structured illumination (VR-SIM), has been developed with the intent of rapid, point-of-procedure histological screening of intact biopsy and whole surgical specimens. VR-SIM leverages widefield imaging, rapid acting fluorescent stains, and optical sectioning to provide high contrast digital images of tissue with histological relevance. The method is to replicate the standard approach as closely as possible, but replace the physical section with an optically sectioned digital image. The overall goal of this work is to perform technological and methodological refinements necessary to translate VR-SIM as a clinical tool for histologic evaluation of fresh tissue in diagnostic procedures, biobanking, and tumor margin assessment. This project will lay the groundwork for quantitative evaluation of VR-SIM as a clinical tool – with the goal of leading toward industrial design of a VR-SIM as a medical device for hospital use. Developing a new framework for integration of high throughput microscopy into the clinical and research workflow, as well as developing new methods for quantification and evaluation of clinical effectiveness these tools will be presented and discussed in the context of patient outcomes and economic impact. / 1 / David Tulman

A program was written in 'C' to control the functions of an already existing Scanning Tunneling Microscope (STM). A DAS-1601 data acquisition card (from Keithley Data Acquisition) was installed together with its drivers for 'C' on a computer with a 486-DX motherboard. The computer was interfaced to the electronics of the STM. Images taken of HOPG (highly oriented pyrolitic graphite) were of a reasonable quality and showed atomic resolution.

acase@tulane.edu / Current microscopy-based tissue diagnostics, particularly hematoxylin and eosin (H&E) histology, requires multiple complex tissue processing steps: fixation, paraffin embedding, microtome sectioning, dying the tissue, and imaging individual slides through a bright field microscope. The time and labor-intensive result of this process makes it unsuitable for patient point-of-care evaluation. Therefore, many bedside procedures are completed without efficient real-time analysis of tissue adequacy and diagnostic results are unnecessarily delayed. Additionally, research experiments that require information regarding changes to tissue morphology or function before proceeding to the next experimental phase are severely interrupted by histology processing in their workflow. Fluorescence histology, which relies on rapid fluorescent staining of tissue, optical sectioning microscopy, and image processing for digital viewing, can provide an inexpensive, non-destructive, 3-dimensional, and fast alternative to traditional histology and point-of-care screening protocols. The objective of this work is to further advance the concept of “fluorescence histology,” in which traditional histopathology preparation methods are replaced by optical-sectioning (in lieu of physical sectioning), sensitive and flexible fluorescence-based contrast (in lieu of chromophore-based contrast), and computational strategies to replicate traditional color-schemes. In this work, we demonstrate the development and use of a fluorescent analogue to H&E on fixed and frozen tissue sections and fresh human biopsies. This fluorescent analogue, DRAQ5 & eosin, is compared against the current single-agent, monochrome fluorescence histology system, and their effects on diagnostic downstream molecular analyses, including quantitative-PCR and immunohistochemistry, is evaluated. We create a methodology to develop and characterize fluorescent analogues for any histological stain, with demonstration using Masson’s Trichrome and Periodic Acid-Schiff, enabling the expansion of fluorescence histology for multiple applications. This work demonstrates the ability to improve point-of-care pathology and research by replacing destructive, incomplete, and time-consuming histology with fluorescence histology, which preserves the tissue for later analysis or experiments while providing accurate and rapid histology assessment. / 1 / Katherine Elfer

The necessity to explore nanoscopic systems is ever increasing in the world of science and technology. This evolving need to study such physically small systems demands new experimental techniques and methodologies. Atomic force microscopy (AFM) is a versatile technique that can overcome many nanoscopic size limitations. AFM has been utilized in the world of nanotechnology to study physiochemical properties of particles, materials, and biomolecules through characterization of morphology, electrical and mechanical properties, binding interactions, and surface tension, among others. The work discussed herein is largely a report of several novel AFM methodologies that were developed to allow new characterization techniques of individual submicrometer particles and single biomolecular interactions. The effects of atmospheric aerosols on the radiative budget of the earth and climate are largely unknown. For this reason, characterizing the physiochemical properties of aerosols is vital. Since the particles that have relatively long lifetimes in the atmosphere are smaller than one micrometer in size, high resolution microscopy techniques are required to study them. AFM is a suitable technique for single particle studies because it has nanometer spatial resolution, can perform experiments under ambient pressure and variable relative humidity and temperature. These advantages were utilized here and AFM was used to study morphology, organic volume fraction, water uptake, and surface tension of nascent sea spray aerosol (SSA) particles as well as laboratory generated aerosols composed of relevant chemical model systems. The morphology of SSA was found, often times, to be composed of core-shell structure. With complementary microscopy techniques, the composition of the core and the shell was found to be inorganic and organic in nature, respectively. Novel methodology to measure water uptake and surface tension of single substrate deposited particles with AFM was established using chemical model systems. Furthermore, these methodologies were employed on nascent chemically complex SSA particles collected from a biologically active oceanic waveflume experiment. Finally, phase imaging was used to measure organic volume fraction on a single particle basis and was correlated with biological activity. Overall, this suite of single (submicrometer) particle AFM analysis techniques have been established, allowing future systematic studies of increasing complexity aimed at bridging the gap between the simplicity of laboratory generated particles and the complexity of nature. Another nanotechnology topic of interest is studying single biomolecular interactions. Virtually every biological process involves some amount of minute forces that are required for the biomolecular system to function properly. For example, there are picoNewton forces associated with enzymatic motions that are important for enzyme catalysis. The AFM studies reported here use a model enzyme/drug system to measure the forces associated with single molecule adhesion events. Escherichia Dihydrofolate Reductase (DHFR) is a target of cancer therapeutic studies because it can be inhibited by drugs like methotrexate (MTX) that are structurally similar to the natural folate binder but have much higher binding affinity. One of the obstacles of single molecular recognition force spectroscopy (MRFS) studies is the contribution of non-specific forces that create a source of uncertainty. In this study, DHFR and MTX are bound to the surface and the AFM tip, respectively, using several different linking molecules. These linking molecules included polyethylene glycol (PEG) and double stranded DNA (dsDNA) and the distribution of forces was compared to scenarios were a linker was not employed. We discovered that dsDNA and PEG both allow identification and removal of non-specific interaction forces from specific forces of interest, which increases the accuracy of the measurement compared to directly bound constructs. Traditionally, the linker of choice in the MRFS community is PEG. Here, we introduce dsDNA as a viable linker that offers more rigidity than PEG, which may be desirable in future molecular constructs. The majority of the work and data presented in this dissertation supports the establishment of new AFM methodologies that can be used to better explore single biomolecular interactions and individual submicrometer particles on the nanoscale.

Traditionally, adaptive optics (AO) systems for microscopy have focused on AO at the pupil plane, however this produces poor performance in samples with both spatially-variant aberrations, such as non-flat sample interfaces, and spatially-invariant aberrations, such as spherical aberration due to a difference between the sample index of refraction and the sample for which the objective was designed. Here, we demonstrate well-corrected, wide field-of-view (FOV) microscopy by simultaneously correcting the two types of aberrations using two AO loops. Such an approach is necessary in wide-field applications where both types of aberration may be present, as each AO loop can only fully correct one type of aberration. Wide FOV corrections are demonstrated in a trans-illumination microscope equipped with two deformable mirrors (DMs), using a partitioned aperture wavefront (PAW) sensor to directly control the DM conjugated to the sample interface and a sensor-less genetic algorithm to control the DM conjugated to the objective’s pupil.