Understanding and identifying critical protein-protein interactions is just one of the key outcomes in biological research. It can help to confirm key cellular interactions, which in some fields, such as cancer research, can result in a greater understanding of disease pathogenesis, elucidate mechanisms of therapeutic resistance and aid in the development of new specific targets, leading to new methods of prevention and treatment. Time-correlated single photon counting fluorescence lifetime imaging microscopy is just one of the tools used to carry out this line of research. Here we demonstrate a direct interaction between two proteins involved in gene regulation and expression; p21 and FMN2. Furthermore, we also show the capability of this system to measure chromatin compaction in three dimensions. However, fluorescence lifetime imaging has some drawbacks, acquisition times on such a system can range from the tens of seconds to minutes, which is often too long to comprehensively measure many biological events. But microscopy is always developing, aided by new techniques and, perhaps even more so, new technological developments. This thesis also demonstrates two new methods of light sheet microscopy, that use both new equipment made available because of technological developments to allow time resolved imaging and traditional microscopic aspects to form a light sheet system based on polarisation. It outlines the design and how to build these systems and presents their function to show their great promise. Both techniques presented in this thesis utilise aspects of light not conventionally used in light sheet microscopy. Further development of these systems and application of emerging technologies will yield a system capable of outperforming current light sheet fluorescence microscopy-based fluorescence lifetime imaging techniques. The implementation of polarisation control into such a system would enable three-dimensional anisotropy based SPIM-FLIM measurements, an indispensable tool in researching molecular orientation and mobility at a macroscopic level in developing organisms.

Multicellular development in fungi is fundamentally different from that of animals or plants. In filamentous fungi, multicellular structures are formed by aggregation and adhesion of hyphae, followed by septation and specialisation of hyphal compartments within the aggregate. The perithecium, a flask-shaped sexual fruitbody produced by both Neurospora crassa and Sordaria macrospora, provides a model system in which to study fungal multicellular development. This study presents a detailed description of the morphological stages of perithecial morphogenesis in N. crassa and S. macrospora and its early stages, the ascogonial and protoperithecial stages, using a range of microscopical techniques. Details of the development of several mutants impaired in perithecial development are described, including: gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; and three mutants pro22, pro40 and pro41 of S. macrospora, and their corresponding gene-deletions in N. crassa. The results confirm that all three MAP kinase cascades are required for sexual development. However, only the pheromone response and cell-wall integrity MAP kinase pathways, but not the osmoregulatory MAP kinase pathway, are essential for hyphal cell fusion. Evidence of cell fusion-related processes, regulated through MAP kinase signalling, have been identified as novel features important for the construction of fertilisable protoperithecia. These cell-fusion related processes include extracellular matrix deposition, hyphal attachment and envelopment. A novel phenotype of S. macrospora with defective ascogonial septation is presented. This pro22 mutant also has impaired hyphal cell fusion and produces only small, defective protoperithecia. The pro22 gene encodes a protein that is highly conserved throughout eukaryotes. Live-cell imaging revealed that this PRO22 protein is localised in the dynamic tubular and vesicular vacuolar-network of the colony periphery and in ascogonia. PRO22 is absent from the large spherical vacuoles in the vegetative hyphae of the sub-peripheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar-network. Furthermore, the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

Tip Enhanced Near-field Optical Microscopy (TENOM) is a method for optically imaging at resolutions far below the diffraction limit. This technique requires optical nano-probes with very specialized geometries, in order to obtain large, localized enhancements of the electromagnetic field, which is the driver behind this imaging method. Traditional methods for the fabrication of these nano-probes involve electrochemical etching and subsequent FIB milling. However, this milling process is non-trivial, requiring multiple cuts on each probe. This requires multiple rotations of the probe within the FIB system, which may not be possible in all systems, meaning the sample must be removed from vacuum, rotated by hand and placed back under vacuum. This is time consuming and costly and presents a problem with reproducibility. The method presented here is to replace multiple cuts from a side profile with a small number of cuts from a top down profile. This method uses the inherent imaging characteristics of the FIB, by assigning beam dwell times to specific locations on the sample, through the use of bitmap images. These bitmaps are placed over the sample while imaging and provide a lookup table for the beam while milling. These images are grayscale with the color of each pixel representing the dwell time at that pixel. This technique, combined with grayscale gradients, can provide probes with a symmetric geometry, making the system polarization independent.

An atomic force microscopy beam-like cantilever is combined with an electrical tuning fork to form a shear force probe that is capable of generating an acoustic response from the mesoscopic water layer under ambient conditions while simultaneously monitoring force applied in the normal direction and the electrical response of the tuning fork shear force probe. Two shear force probes were designed and fabricated. A gallium ion beam was used to deposit carbon as a probe material. The carbon probe material was characterized using energy dispersive x-ray spectroscopy and scanning transmission electron microscopy. The probes were experimentally validated by demonstrating the ability to generate and observe acoustic response of the mesoscopic water layer.

Fatigue crack growth in ±-Zirconium phosphate/epoxy nanocomposites was investigated. A new fatigue testing technique was implemented for miniature samples. Two different methods “strength of materials and Rayleigh-Ritz - were used in determining the bending stress. The fatigue stress and fatigue life of different nanocomposite specimens were plotted in a traditional stress-life (S-N) curve. It was inferred from the S-N plot that the values obtained from both the methods compare well. The experimental results showed that fatigue life of filled epoxy nanocomposite is more than that of the unfilled epoxy composite. A model for bending stresses, ultimate strength and the number of cycles to failure was obtained to predict a component service life without conducting elaborate tests. Scanning electron examination of the fractured surfaces revealed that the crack takes a tortuous path during its propagation course, indicating crack blunting and crack deflection roles of ZrP and CSR nanofillers, which consequently improve the fracture resistance. In case of the M-ZrP-epoxy systems, delamination of ZrP platelets from surrounding epoxy matrix was proposed as the reason behind crack growth. The improved fracture resistance of these nanocomposites was attributed to the delamination of ZrP platelets and deflection of crack direction. The superior behavior of CSR-ZrP-epoxy composites was attributed to the cavitation process and void coalescence due to CSR particles delamination.

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.

A detailed understanding of the corneal biomechanical response is an important prerequisite to understanding corneal diseases such as keratoconus and for placing the empirical equations used in refractive surgery on a physical basis. We have assembled a combined nonlinear optical microscopy (NLOM) and optical coherence microscopy (OCM) imaging system to simultaneously capture coregistered volumetric images of corneal morphology and biochemistry. Fudicial markers visible in the OCM volume enabled the calculation of strains for multiple depth layers in rabbit cornea. The results revealed a depth dependent strain distribution, with smaller strains in the anterior stroma and larger strains in the posterior stroma. The stress-strain curves can be grouped readily by depth into three groups: anterior (~20%), transitional mid (~40%), and posterior (~40%). Cross-sectional images of collagen lamellae, visible in NLOM, showed inhomogeneous collagen structure and its response to intraocular pressure along the anterior-posterior direction. The inhomogeneities correlate well with the noted heterogeneous corneal mechanical properties. The combined NLOM-OCM system can measure corneal microstructure and mechanical response uniquely, thus providing a microstructural understanding of corneal response to changes of collagen structure.