Coherent anti-Stokes Raman scattering (CARS) microscopy allows fast, label-free and chemically selective imaging of condensed-phase samples thanks to its high signal sensitivity. It also offers several other advantages such as intrinsic three-dimensional sectioning capability, longer penetration depth and high spatial resolution. In conventional CARS microscopy, two synchronized narrowband laser pulses are typically used to generate signals at a single vibrational resonance, from which vibrational images are constructed. Although this type of CARS methods has been proven to be an excellent visualizing tool for lipid in biological samples, it has two serious problems. First, the ubiquitous nonresonant background smears out vibrational signals, which makes quantitative image analysis very difficult. Second, the chemical information obtained in this method is seriously limited since only a single vibrational resonance is measured, which is far less information than full vibrational spectrum can offer. In the past few years, we have developed several novel CARS imaging techniques that can overcome these issues. All our methods require only a single broadband laser and produce background-free vibrational spectrum by combining the laser pulse shaping and various signal detection schemes. The first one obtains a vibrational spectrum over 800 ~ 1800 cm-1 in a single measurement by simultaneous excitation of multiple vibrational resonances and analysis of spectral interferences between the resonant and nonresonant signals. The second method adopts the spectral focusing mechanism, where stretched broadband pulses are used to excite a single vibrational resonance with great sensitivity. A novel frequency modulation (FM) scheme is invented to eliminate the non-resonant background. Complimentary spectral analysis algorithm is also developed to obtain quantitative CARS signals at the CH stretching region (2800 ~ 3100 cm-1). In this dissertation, the fundamental mechanisms, experimental implementations and various imaging applications of the above CARS methods are described in detail. / text

<p>The materials and working method of a painting can reveal important information about our cultural history, as well as lend the conservator the necessary knowledge for treatment options. The removal of a cross-section sample reveals the three-dimensional (3d) structure of the painting and can be used to identify materials. However, cross-section samples are destructive and provide only local information. Nonlinear optical ultrafast pump-probe microscopy, originally developed for biomedical imaging, can provide high resolution 3d images with chemical contrast. In this dissertation, I adapt pump-probe microscopy to multiple materials and applications in cultural heritage research. Pump-probe dynamics were found to be sensitive to the ratio of the two chromophores present in the precious blue pigment lapis lazuli and its synthetic analogs, ultramarines blue and violet. Virtual pump-probe cross-sections were combined with nonlinear fluorescence contrast to study differences between the interactions of paper supports with inorganic crystalline pigments and organic dyes. Multiple early Italian paintings (The Crucifixion by Puccio Capanna, The Martyrdom of St. Alexander and The Body of Christ Supported by Angels attributed to Lorenzo Lotto) were imaged in-situ, in conjunction with traditional conservation science methods, as a part of a technical case study. Thus, pump-probe microscopy offers an important new tool for gaining fundamental insights into our cultural heritage.</p> / Dissertation

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC’s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.

Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC’s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

Nonlinear microscopy has become an indispensable tool in the study of biological systems. It includes many nonlinear contrast mechanisms, each sensitive to different biological structures. However, interpretation of the images generated in nonlinear microscopy is a complex matter due to factors such as the structural complexity of the sample, phase relationships between the excitation beam and the detected signal and the nonlinear interactions in the focal volume of the microscope. This thesis contains a new theoretical and numerical framework that describes the focusing of an excitation beam in a nonlinear microscope, the nonlinear optical interactions with the material in the focal volume, and the resulting nonlinear optical signal in the far field. The framework is the first to include reflection and refraction of the excitation beam and nonlinear signals by an arbitrary number of interfaces in the focal volume, which is especially significant for the interpretation of third harmonic generation (THG). It also uses the chirp-z transform to speed up calculations by orders of magnitude compared to numerical integration techniques. The framework is used to investigate second harmonic generation (SHG) by collagen. Focusing effects alter polarization-dependent SHG measurements of collagen properties compared to the plane wave approximation, and this is verified experimentally. Furthermore, a technique of imaging the far field SHG radiation from collagen fibres is proposed, which can be used to extract the orientation of collagen fibres unambiguously. The framework is then applied to analyze the influence of interfaces on THG. Reflection effects at interfaces significantly affect THG, which leads to the development of a new super-resolution THG imaging technique based on backward-propagating THG. This super-resolution technique is experimentally demonstrated by imaging surface profiles with tens of nanometers resolution, which is the first time that such resolution is obtained in coherent nonlinear microscopy. Therefore, this imaging technique shows promise to become an important tool in high-resolution imaging of (biological) samples. The theoretical and numerical framework provides a foundation for future research on the origin of nonlinear microscopy signals. The new imaging techniques based on this framework have great potential in quantifying fibrillar structures and interfaces in biological samples.

Nonlinear microscopy has become an indispensable tool in the study of biological systems. It includes many nonlinear contrast mechanisms, each sensitive to different biological structures. However, interpretation of the images generated in nonlinear microscopy is a complex matter due to factors such as the structural complexity of the sample, phase relationships between the excitation beam and the detected signal and the nonlinear interactions in the focal volume of the microscope. This thesis contains a new theoretical and numerical framework that describes the focusing of an excitation beam in a nonlinear microscope, the nonlinear optical interactions with the material in the focal volume, and the resulting nonlinear optical signal in the far field. The framework is the first to include reflection and refraction of the excitation beam and nonlinear signals by an arbitrary number of interfaces in the focal volume, which is especially significant for the interpretation of third harmonic generation (THG). It also uses the chirp-z transform to speed up calculations by orders of magnitude compared to numerical integration techniques. The framework is used to investigate second harmonic generation (SHG) by collagen. Focusing effects alter polarization-dependent SHG measurements of collagen properties compared to the plane wave approximation, and this is verified experimentally. Furthermore, a technique of imaging the far field SHG radiation from collagen fibres is proposed, which can be used to extract the orientation of collagen fibres unambiguously. The framework is then applied to analyze the influence of interfaces on THG. Reflection effects at interfaces significantly affect THG, which leads to the development of a new super-resolution THG imaging technique based on backward-propagating THG. This super-resolution technique is experimentally demonstrated by imaging surface profiles with tens of nanometers resolution, which is the first time that such resolution is obtained in coherent nonlinear microscopy. Therefore, this imaging technique shows promise to become an important tool in high-resolution imaging of (biological) samples. The theoretical and numerical framework provides a foundation for future research on the origin of nonlinear microscopy signals. The new imaging techniques based on this framework have great potential in quantifying fibrillar structures and interfaces in biological samples.

<p>Nonlinear optical microscopy serves as a great tool for biomedical imaging due to its high resolution, deep penetration, inherent three dimensional optical sectioning capabilities and superior performance in scattering media. Conventional nonlinear optical microscopy techniques, e.g. two photon fluorescence and second harmonic generation, are based on detecting a small light signal emitted at a new wavelength that is well separated from the excitation light. However, there are also many other nonlinear processes, such as two-photon absorption and self-phase modulation, that do not generate light at new wavelengths and that have not been extensively explored for imaging. This dissertation extends the accessible mechanisms for contrast to the later nonlinear optical processes by combining femtosecond laser pulse shaping and homodyne detection. We developed a rapid pulse shaper with a relatively simple and compact instrument design that modifies the spectrum of individual laser pulses from an 80 MHz mode-locked laser. The pulse shaper enables simultaneous two-photon absorption and self-phase modulation imaging of various nanoparticles in-vitro with high sensitivity. We also applied this imaging technique to study the nonlinear optical response in graphene. Because our technology detects the nonlinear signature encoded within the laser pulse itself, we achieve intrinsic contrast of biological and non-biological samples in highly scattering media. These capabilities have significant implications in biomedical imaging and nanophotonics.</p> / Dissertation

<p>Imaging based on nonlinear processes takes advantage of the localized excitation to achieve high spatial resolution, optical sectioning, and deeper penetration in highly scattering media. However, the use of nonlinear contrast for imaging has conventionally been limited to processes that create light of wavelengths that are different from the wavelengths used for excitation. Intrinsic nonlinear contrasts that do not generate light at distinct wavelengths are generally difficult to measure because of the overwhelming background from the excitation light. This dissertation focuses on extension of nonlinear microscopy to these new intrinsic processes by using femtosecond pulse shaping to encode the nonlinear information as new frequency components in the spectrum. We will present a pump-probe microscopy technique based on pulse train shaping technology to sensitively access nonlinear transient absorption or gain processes. This technique has recently been used to uniquely identify a variety of biological pigments with high spatial resolution. Here, we extend this technique to image and characterize several inorganic and organic pigments used in historical artwork. We also present a spectral reshaping technique based on individual femtosecond pulse shaping to sensitively access nonlinear refractive contrasts in scattering media. We will describe an extension of this technique to utilize two distinct wavelengths and discuss its application in biological imaging. This two-color implementation would allow the extension of widely employed phase contrast to the nonlinear regime.</p> / Dissertation