Fiber Laser Based Nonlinear Spectroscopy

To date, nonlinear spectroscopy has been considered an expensive technique and confined mostly to experimental laboratory settings. Over recent years, optical-fiber lasers that are highly reliable, simple to operate and relatively inexpensive have become commercially available, removing one of the major obstacles to widespread utilization of nonlinear optical measurement in biochemistry. However, fiber lasers generally offer relatively low output power compared to lasers traditionally used for nonlinear spectroscopy, and much more careful design is necessary to meet the excitation power thresholds for nonlinear signal generation. On the other hand, reducing the excitation intensity provides a much more suitable level of user-safety, minimizes damage to biological samples and reduces interference with intrinsic chemical processes. Compared to traditional spectroscopy systems, the complexity of nonlinear spectroscopy and imaging instruments must be drastically reduced for them to become practical. A nonlinear spectroscopy tool based on a single fiber laser, with electrically controlled wavelength-tuning and spectral resolution enhanced by a pulse shaping technique, will efficiently produce optical excitation that allows quantitative measurement of important nonlinear optical properties of materials. The work represented here encompasses the theory and design of a nonlinear spectroscopy and imaging system of the simplest architecture possible, while solving the difficult underlying design challenges. With this goal, the following report introduces the theories of nonlinear optical propagation relevant to the design of a wavelength tunable system for nonlinear spectroscopy applications, specifically Coherent Anti-Stokes Spectroscopy (CARS) and Förster Resonance Energy Transfer (FRET). It includes a detailed study of nonlinear propagation of optical solitons using various analysis techniques. A solution of the generalized nonlinear Schrödinger equation using the split-step Fourier method is demonstrated and investigation of optical soliton propagation in fibers is carried out. Other numerical methods, such as the finite difference time domain approach and spectral-split step Fourier methods are also described and compared. Numerical results are contrasted with various measurements of wavelength shifted solitons. Both CARS and FRET test-bed designs and experiments are presented, representing two valuable biochemical measurement applications. Two-photon excitation experiments with a simplified calibration process for quantitative FRET measurement were conducted on calmodulin proteins modified with fluorescent dyes, as well as modified enhanced green fluorescent protein. The resulting new FRET efficiency measurements showed agreement with those of alternative techniques which are slower and can involve destruction of the sample. In the second major application of the nonlinear spectroscopy system, CARS measurement with enhanced spectral resolution was conducted on cyclohexane as well as on samples of mouse brain tissue containing lipids with Raman resonances. The measurements of cyclohexane verified the ability of the system to precisely determine its Raman resonances, thus providing a benchmark within a similar spectral range for biological materials which have weaker Raman signal responses. The improvement of spectral resolution (resonance frequency selectivity), was also demonstrated by measuring the closely-spaced resonances of cyclohexane. Finally, CARS measurements were also made on samples of mouse brain tissue which has a lipids-based Raman signature. The CARS spectrum of the lipid resonances matched well with other cited studies. The imaging of mouse brain tissue with Raman resonance contrast was also partially achieved, but it was hindered by low signal to noise ratio and limitations of the control hardware that led to some dropout of the CARS signal due to power coupling fluctuations. Nevertheless, these difficulties can be straightforwardly addressed by refinement of the wavelength tuning electronics. In conclusion, it is hoped that these efforts will lead to greater accessibility and use of CARS, FRET and other nonlinear spectral measurement instruments, in line with the promising advances in optics and laser technology

Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

Dynamics and interactions of nuclear proteins revealed by quantitative photobleaching microscopy

Tese de doutoramento em Biofísica (Biofísica), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007The nucleus is a complex cellular organelle, exhibiting a high degree of organization and also a highly dynamic nature. Live cell imaging using fluorescent proteins (FPs) as molecular tags and photobleaching techniques have been essential in revealing the dynamic nature of the cell nucleus. In this thesis, these tools were used to study molecular dynamics and interactions inside this cellular compartment. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) were used to analyze the kinetic behavior of spliceosome components SmE, U2AF65, U2AF35, SF1 and SC35 in the nucleus of living cells. The recruitment mechanism of splicing factors (SFs) to the sites of transcription is still poorly understood. Our results rule out the hypothesis that a transcription specific signal recruits SFs from the speckles. They also suggest the formation of multi-protein complexes distinct from the spliceosome. The existence of these complexes was confirmed by Fluorescence Resonance Energy Transfer (FRET) techniques, which revealed that SFs could interact with each other even in the absence of active splicing. A novel U2AF65 self-interaction was also detected, suggesting altogether that levels of SFs in speckles are consistent with self-organization mechanisms. The intranuclear mobility of mRNPs was studied using two GFP-tagged mRNA-binding proteins, PABPN1 and TAP, as mRNA markers. A novel FLIP method was devised to quantify the mobility of the RNA-bound and unbound pools of molecules and used to test whether myosin motors were implicated in mRNP movement. We show that this is not the case and that myosin inhibition appears to affect transcription instead. A novel FLIP after Photoactivation method was developed to study the nucleocytoplasmic exchange dynamics of nuclear proteins, yielding the permanence times of molecules inside the nucleus. The method was used to study the role of the structural domains of TAP in its shuttling activity.O núcleo celular é um organito complexo, dotado de um elevado grau de organização mas também uma natureza extremamente dinâmica. A utilização de proteínas fluorescentes como marcadores moleculares para visualização em células vivas, bem como as técnicas de photobleaching, têm sido essenciais na descoberta da natureza dinâmica do núcleo. Neste trabalho, estas ferramentas foram aplicadas no estudo da dinâmica e interacções moleculares dentro deste compartimento celular. As técnicas de Fluorescence Recovery After Photobleaching (FRAP) e Fluorescence Loss In Photobleaching (FLIP) foram utilizadas na análise do comportamento cinético dos componentes do spliceosoma SmE, U2AF65, U2AF35, SF1 e SC35 no interior do núcleo de células vivas. O mecanismo de recrutamento dos factores de splicing (SFs) para os locais de transcrição é ainda pouco conhecido. Os nossos resultados excluem a hipótese de haver um sinal associado à transcrição que seja responsável por este recrutamento. Sugerem ainda a formação de complexos multi-proteicos distintos do spliceosoma. A existência destes complexos foi confirmada por técnicas de Fluorescence Resonance Energy Transfer (FRET), que mostraram que os SFs podiam interagir uns com os outros mesmo na ausência de splicing activo. Foi ainda descoberta uma nova auto-interacção para o factor U2AF65, sugerindo os resultados no seu conjunto que a distribuição de SFs no núcleo é compatível com mecanismos de auto-organização. A mobilidade de mRNPs no núcleo foi estudada utilizando como marcadores moleculares duas proteínas que se ligam ao mRNA marcadas com GFP, PABPN1 e TAP. Foi desenvolvido um método de FLIP para quantificação da mobilidade das fracções ligadas e não ligadas ao mRNA e usado para testar a possibilidade de motores de miosina estarem envolvidos no movimento de mRNPs. Mostramos que tal não acontece e que a inibição de miosina parece antes afectar a transcrição. Um novo método de FLIP após foto-activação foi desenvolvido para estudar a dinâmica de trocas entre o núcleo e o citoplasma de proteínas nucleares, permitindo a estimação do tempo de permanência de moléculas dentro do núcleo. O método foi utilizado para investigar o papel dos diferentes domínios estruturais da proteína TAP na sua actividade de exportação nuclear.Fundação para a Ciência e Tecnologia (BD/21518/99); European Commission (“RNOMICS” QLG2-CT-2001-01554 and “Integrated Technologies for in vivo Molecular Imaging” LSHG-CT-2003-503259

Optically Micro-fabricated Linear and Freeform 3-D Extracellular Matrix Scaffolds for Tissue Engineering

This work was aimed at advancing multi-photon excited, freeform fabrication technology with nano-scale and sub-micron precision as an enabler for tissue engineers to investigate cellular response to a biomimetic, bio-active extracellular matrix. We demonstrated that sub-micron and micron scale Collagen and Fibronectin structures can be fabricated via multi-photon excited photochemistry using a modified Benzophenone dimer and Rose Bengal while maintaining the biomimetic ECM structures’ bioactivity. We confirmed that three-photon excitation produces significantly smaller features at comparable excitation wavelengths as a consideration to better approach focal adhesion size. Bioactivity of MPE cross-linked FN and Collagens I and II was established via immunofluorescence and fibroblast adhesion. Additionally, the relative rates of degradation in these cross-linked matrices are consistent with the known activities of these enzymes. Morphology measurements of fibroblasts grown on these proteins include log(Area), Perimeter, Area/Perimeter2 were considered as proxies for cell response. Fibroblast perimeters are statistically different when associated with the Collagen I microenvironment. Among fibroblasts grown on MPE structures of Collagen I, Fibronectin, BSA and the BSA Monolayer, the stress fiber distributions on Collagen I (all fiber lengths) are highly significantly different (p \u3c 1x10-4) than the distribution of stress fibers of cells on BSA Lines. This suggests contact guidance only for cells on BSA Lines but yet a combination of contact guidance and chemical signaling (RGD) with cells on Collagen I Lines. This supports additional overall orientation findings based on fibroblasts’ fitted ellipse major axis direction for Collagens I, II and Fibronectin. Stress fiber distribution on BSA Monolayer differed significantly from those on BSA structures (p = 0.01). This underscores the effects of pure contact guidance alone provided by the BSA fibers compared to the combined contact guidance and ECM cues provided by the FN, and collagen structures. A method similar to rapid prototyping or three-dimensional printing was accomplished to resolve cellular response at the submicron level by fabricating biomimetic, bioactive extracellular matrices in a freeform three-dimensional (3D) manner. To the best of our knowledge, simultaneous 3D spatial and chemical control of collagen scaffold synthesis at the micrometer and sub-micrometer size scales has not been fully demonstrated

Roadmap on digital holography [Invited]

This Roadmap article on digital holography provides an overview of a vast array of research activities in the field of digital holography. The paper consists of a series of 25 sections from the prominent experts in digital holography presenting various aspects of the field on sensing, 3D imaging and displays, virtual and augmented reality, microscopy, cell identification, tomography, label-free live cell imaging, and other applications. Each section represents the vision of its author to describe the significant progress, potential impact, important developments, and challenging issues in the field of digital holography

Charged Particle Optics Theory

Charged Particle Optics Theory: An Introduction identifies the most important concepts of charged particle optics theory, and derives each mathematically from the first principles of physics. Assuming an advanced undergraduate-level understanding of calculus, this book follows a logical progression, with each concept building upon the preceding one. Beginning with a non-mathematical survey of the optical nature of a charged particle beam, the text: Discusses both geometrical and wave optics, as well as the correspondence between them Describes the two-body scattering problem, which is essential to the interaction of a fast charged particle with matter Introduces electron emission as a practical consequence of quantum mechanics Addresses the Fourier transform and the linear second-order differential equation Includes problems to amplify and fill in the theoretical details, with solutions presented separately Charged Particle Optics Theory: An Introduction makes an ideal textbook as well as a convenient reference on the theoretical origins of the optics of charged particle beams. It is intended to prepare the reader to understand the large body of published research in this mature field, with the end result translated immediately to practical application

Mapping Atomic Motions with Electrons: Toward the Quantum Limit to Imaging Chemistry

Recent advances in ultrafast electron and X-ray diffraction have pushed imaging of structural dynamics into the femtosecond time domain, that is, the fundamental time scale of atomic motion. New physics can be reached beyond the scope of traditional diffraction or reciprocal space imaging. By exploiting the high time resolution, it has been possible to directly observe the collapse of nearly innumerable possible nuclear motions to a few key reaction modes that direct chemistry. It is this reduction in dimensionality in the transition state region that makes chemistry a transferable concept, with the same class of reactions being applicable to synthetic strategies to nearly arbitrary levels of complexity. The ability to image the underlying key reaction modes has been achieved with resolution to relative changes in atomic positions to better than 0.01 Å, that is, comparable to thermal motions. We have effectively reached the fundamental space-time limit with respect to the reaction energetics and imaging the acting forces. In the process of ensemble measured structural changes, we have missed the quantum aspects of chemistry. This perspective reviews the current state of the art in imaging chemistry in action and poses the challenge to access quantum information on the dynamics. There is the possibility with the present ultrabright electron and X-ray sources, at least in principle, to do tomographic reconstruction of quantum states in the form of a Wigner function and density matrix for the vibrational, rotational, and electronic degrees of freedom. Accessing this quantum information constitutes the ultimate demand on the spatial and temporal resolution of reciprocal space imaging of chemistry. Given the much shorter wavelength and corresponding intrinsically higher spatial resolution of current electron sources over X-rays, this Perspective will focus on electrons to provide an overview of the challenge on both the theory and the experimental fronts to extract the quantum aspects of molecular dynamics