3D printing, also known as ”additive manufacturing”, indicates a set of fabrication techniques that build objects by adding material, typically layer by layer. The main advantages of 3D printing are unlimited shapes and geometry, fast prototyping, and cost-effective small scale production. Two-photon lithography (TPL) is a laserbased 3D printing technique with submicron resolution, that can be used to create miniaturized structures. One of the most compelling applications of TPL is the 3D printing of miniaturized optical elements with unprecedented complexity, small-scale and precision. This could be potentially beneficial in biophotonics, a multidisciplinary research field in which light-based techniques are used to study biological processes. My research has been aimed at demonstrating novel applications of 3D printing based on TPL to different biophotonic applications. In particular, here we show 3D printed micro-optical structures that enhance and/or enable novel functions in advanced biophotonics methods as two-photon microendoscopy, optical trapping and Stimulated Raman Scattering microscopy. Remarkably, the micro-optical structures presented in this thesis enable the implementation of advanced techniques in existing or simpler microscopy setups with little to no modification to the original setup. This possibility is essentially allowed by the unique miniaturization and in-situ 3D printing capabilities offered by TPL.

Angiogenesis is a central process to both physiological and pathological aspects of living organisms. Understanding the angiogenic system via the key mediator, vascular endothelial growth factor (VEGF), has led to the development of biophotonic models capable of monitoring how this process is programmed. The whole animal model tested here is based on the involvement of angiogenesis in a wound healing environment. This model proved to be functional as a system monitor but lacked the precision to yield significant results between the biological compounds tested (estrogen, methoxychlor, and relaxin). The in vitro model is based on angiogenesis in a cancer environment. This model proved to be both a valid and functional way of monitoring the biological compounds tested (CoCl2, epinephrine, and norepinephrine).

Using silicon based microfabrication and materials, a photonic platform, capable of single bioparticle analysis, has been developed. This platform combines liquid and hollow core waveguides on the micron-scale (5 µm x 12 µm) to isolate femtoliter sized sample volumes. Fluorescence excitation and signals in the visible range are directed into and out of the sample volume at an orthogonal angle to maximize signal-to-noise. In order to guide light in a low-index material antiresonant reflecting optical waveguides (ARROWs) were incorporated into the platform. This thesis reveals the development path of these structures over several device generations including innovations in material, geometries, and fabrication techniques to increase detection sensitivity. As a result of these developments, this photonic platform has shown to successfully detect virus samples and other particles. This thesis also presents a new idea for increasing the signal to noise ratio (SNR) by incorporating Y-splitter devices into the design. Specifically, the 1 x 2 and 1 x 4 splitter structures can be used as orthogonal excitation points to the liquid core waveguide. When fluorescently tagged particles are introduced into the hollow core, these points create an optical signal that is correlated in time and space. The data collected by a photodetector can then be processed by an algorithm to increase SNR. Such advancements have shown to increase the SNR by 175 times.

Recent, cooperative advances in chemistry, biology, computing, photophysics, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed 'biophotonics'. The integrative and interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research through the clinical and pharmaceutical industries. This holds true for the development and application of the novel imaging modality utilizing multiphoton absorption and its extraordinary contribution to recent advances in bioimaging. Intimately involved in the revolution of nonlinear bioimaging has been the development of optical probes for probing biological function and activity. The focus of this dissertation is in the area of probe development, particularly conjugated organic probes, optimized for efficient two-photon absorption followed by upconverted fluorescence for nonlinear, multiphoton bioimaging applications. Specifically, [pi]-conjugated fluorene molecules, with enhanced two-photon absorbing (2PA) properties and high photostability, were prepared and characterized. Contemporary synthetic methods were utilized to prepare target fluorene derivatives expected to be highly fluorescent for fluorescence imaging, and, in particular, exhibit high two-photon absorptivity suitable for two-photon excitation (2PE) fluorescence microscopy. The flexibility afforded through synthetic manipulation to integrate hydrophilic moieties into the fluorophore architecture to enhance compatibility with aqueous systems, more native to biological samples, was attempted. Incorporation of functional groups for direct covalent attachment onto target biomolecules was also pursued to prepare fluorene derivatives as efficient 2PA reactive probes. Linear and two-photon spectroscopic characterizations on these novel compounds reveal they exhibit high 2PA cross-sections on the order of ~100 GM units, nearly an order of magnitude greater than typical, commonly used fluorophores utilized in nonlinear, multiphoton microscopy imaging of biological samples. Photostability studies of representative fluorene derivatives investigated and quantified indicate these derivatives are photostable under one- and two-photon excitation conditions, with photodecomposition quantum yields on the order of 10[super-5]. Preliminary cytotoxicity studies indicate these fluorene derivatives exhibit minimal cytotoxic effects on proliferating cells. Finally, their ultimate utility as high-performance, 2PA fluorescent probes in 2PE fluorescence microscopy imaging of biological samples was demonstrated in both fixed and live cells. Due to the low cytotoxicity, high photostability, efficient 2PA, and high fluorescence quantum yield, the probes were found suitable for relatively long-term, two-photon fluorescence imaging of live cells, representing a significant advance in biophotonics.

To better understand the facets of nutrient utilization, a series of in vivo and in vitro studies were undertaken to elucidate the effect of supplementation on utilization of moderate-quality bermudagrass hay and to identify mechanisms to evaluate the role of rumen bacterial populations on feedstuff utilization. A digestion trial was conducted using 6 ruminally cannulated steers receiving bermudagrass hay supplemented with soybean hulls (HULLS), cracked corn (CORN), or soybean hulls and cracked corn (MIX; 75% and 25%, respectively) in a 3x3 Latin Rectangle arrangement. Additionally, ruminal fluid was continuously cultured using the BioFlo® 110 fermentation system to evaluate the in vitro fermentive parameters of ground moderate-quality bermudagrass hay either alone (HAY; 20 g DM L-1 d-1) or supplemented (7 g DM L-1 d-1) with corn (CORN), soybean hulls (SBH), or both (25:75; MIX) in a randomized complete block. Genomic DNA from continuous culture as well as from pure bacterial culture samples were sought to differentially enumerate select bacterial strains via real-time PCR using specie-specific DNA primers. The information is to be used for elucidating responses in ruminal digestibility of varying feed-types. Finally, as an alternative to PCR, bioluminescence of transformed Escherichia coli was evaluated by measuring extent of photonic emission with and without antibiotic selection over time. Evaluations were also made of photonic emission by E. coli grown in ruminal fluid with and without additional feed particles. Data seem to indicate that replacing a portion of corn with soybean hulls may successfully improved fiber digestion and improved ruminal N-utilization. Real-time PCR shows potential for evaluating ruminal bacteria where as biophotonics may need further modification before meaningful in situ evaluations of live ruminants can be employed.

<p>Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.</p><p>Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions. </p><p>To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.</p><p>To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology. </p><p>Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy. </p><p>Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation. </p><p>Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone. </p><p>Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted. </p><p>In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.</p> / Dissertation

Optical manipulation covers a wide range of techniques to guide and trap cells using only the forces exerted by light. Another optical tool is photoporation, the technique of injecting membrane-impermeable molecules using light, which has become an important alternative to other injection techniques. Together they provided sterile tools for manipulation and molecule delivery at the single-cell level. In this thesis, the properties of low Reynolds fluid flows are exploited to guide cells though a femtosecond Bessel beam. This design allows for high-throughput optical injection of cells without the need to individually target cells. A method of 'off-chip' hydrodynamic focusing was evaluated and was found to confine 95.6% of the sample within a region which would receive a femtosecond dose compared to 20% without any hydrodynamic focusing. The system was tested using two cell lines to optically inject the membrane-impermeable dye, propidium iodide. This resulted in an increase of throughput by an order of magnitude compared to the previous microfluidic design (to up to 10 cells per second). Next optical trapping and photoporation were combined to create a multimodal workstation. The system provides 3D beam control using spatial light modulators integrated into a custom user interface. The efficiency of optical injection of adherent cells and trapping capabilities were tested. The development of the system provides the groundwork for exploration of the parameters required for photoporation of non-adherent cells. Finally optical trapping is combined with temporally focused multiphoton illumination for scanless imaging. The axial resolution of the system was measured using different microscope objectives before imaging cells stained with calcein. Both single and a pair of recently trypsinised cells were optically trapped and imaged. The position of the trapped cells was manipulated using a spatial light modulator in order to obtain a z-stack of images without adjusting the objective position.

Optical cell manipulation allows precise and non-invasive exploration of mammalian cell function and physiology for medical applications. Plants, however, represent a vital component of the Earth's ecosystem and the knowledge gained from using optical tools to study plant cells can help to understand and manipulate useful agricultural and ecological traits. This thesis explores the potential of several biophotonic techniques in plant cells and tissue. Laser-mediated introduction of nucleic acids and other membrane impermeable molecules into mammalian cells is an important biophotonic technique. Optical injection presents a tool to deliver dyes and drugs for diagnostics and therapy of single cells in a sterile and interactive manner. Using femtosecond laser pulses increases the tunability of multiphoton effects and confines the damage volume, providing sub-cellular precision and high viability. Extending current femtosecond photoporation knowledge to plant cells could have sociological and environmental benefits, but presents different challenges to mammalian cells. The effects of varying optical and biological parameters on optical injection of a model plant cell line were investigated. A reconfigurable optical system was designed to allow easy switching between different spatial modes and pulse durations. Varying the medium osmolarity and optoinjectant size and type affected optoinjection efficacy, allowing optimisation of optical delivery of relevant biomolecules into plant cells. Advanced optical microscopy techniques that allow imaging beyond the diffraction limit have transformed biological studies. An ultimate goal is to merge several biophotonic techniques, creating a plant cell workstation. A step towards this was demonstrated by incorporating a fibre-based optical trap into a commercial super-resolution microscope for manipulation of cells and organelles under super-resolution. As proof-of-concept, the system was used to optically induce and quantify an immunosynapse. The capacity of the super-resolution microscope to resolve structure in plant organelles in aberrating plant tissue was critically evaluated.

Le transfert d'énergie par résonance de type Förster (FRET) permet de mesurer des distances nanométriques grâce à la dépendance critique de l'efficacité du transfert avec la séparation entre un donneur et un accepteur d'énergie. Le phénomène se produit quand le fluorophore donneur dans l'état excité transfère son énergie d'excitation à un accepteur à proximité de façon non-radiative avec une interaction dipôle-dipôle de champ proche. Les structures nanophotoniques sont capables de contrôler cette interaction grâce à la modification de la densité local d'états électromagnétiques (LDOS) d'un émetteur quantique. Nous avons démontré clairement l'exaltation du transfert d'énergie des paires FRET individuelles sous l'influence des nano-ouvertures percées en or et en aluminium et aussi à l'aide des designs plus complexes comme la `` antenna-in-box ". Notamment, nous avons dévoilé l'importance essentielle de l'orientation relative entre les dipôles sur les possibilités d'exaltation du transfert d'énergie par le biais des nanostructures. Également, nous avons utilisé des nanofils en argent pour démontrer un transfert d'énergie de long-distance entre deux nanoparticles séparées de plus d'un micromètre. Nos résultats éclairent le chemin de l'exploration du FRET, qui est largement utilisé dans les sciences du vivant et la biotechnologie. Les nanostructures optiques ouvrent de plus des perspectives d'applications innovantes pour la construction de biocapteurs, de sources de lumière ou dans l'industrie photovoltaïque. / The technique of Förster resonance energy transfer (FRET) determines the separation between two molecules at the nanometer scale, where molecular interactions can take place. The phenomenon requires a donor fluorophore transferring its energy in a non-radiative way, through a near-field dipole-dipole interaction, to an acceptor. Nanophotonics achieves accurate control over these interactions by modifying the local density of optical states (LDOS) of a single quantum emitter. We have clearly demonstrated enhanced energy transfer within single FRET pairs confined in single nanoapertures made of gold and also aluminum or in more complex structures like the antenna-in-box design. In particular, we have revealed the strong influence of the mutual dipole orientation on the FRET enhancement using nanostructures. Also, by means of silver nanowires, we have demonstrated a long-range plasmon-mediated fluorescence energy transfer between two nanoparticles separated by micrometer distance. Our results are clearing a new path to improve the energy transfer process widely used in life sciences and biotechnology. Optical nanostructures open up many potential applications for biosensors, light sources or photovoltaics.

Carbon quantum dots (CQDs) are the latest generation of carbon nanomaterials in applications where fullerenes, carbon nanotubes, and graphene are abundantly used. With several attractive properties such as tunable optical property, edge-functionalization, and defect-rich chemical structure, CQDs have the potential to revolutionize optoelectronics, electro- and photocatalysis, and biomedical applications. Chemical modifications through the addition of heteroatoms, chemical reduction, and surface passivation are found to alter the band gap, spectral position, and emission pathways of CQDs. Despite extensive studies, fundamental understanding of structure-property relationship remains unclear due to the inhomogeneity in chemical structure and a complex emission mechanism for CQDs. This dissertation outlines a series of works that investigate the structure-property relationship of CQDs and its impact in a variety of applications. First, this relationship was explored by modifying specific chemical functionalities of CQDs and relating them to differences observed in optical, catalytic, and pharmacological performance. While a number of scientific articles reported that top-down or bottom-up synthesized CQDs yielded similar properties, the results herein present dissimilar chemical structures as well as photoluminescent and metal sensing properties. Second, the role of nitrogen heteroatoms in top-down synthesized CQD was studied. The effect of nitrogen atoms on spectral position and fluorescence quantum yield was considerably studied in past reports; however, thorough investigation to differentiate various nitrogen related chemical states was rarely reported. By finely tuning both the quantity of nitrogen doping and the distribution of nitrogen-related chemical states, we found that primary amine and pyridine induce a red-shift in emission while pyrrolic and graphitic nitrogen produced a blue-shift in emission. The investigation of nitrogen chemical states was extended to bottom-up synthesized CQDs with similar results. Finally, top-down, bottom-up, nitrogen-doped and chemically reduced CQDs were separately tested for their ability to act as photodynamic anti-cancer agents. This series of experiments uncovered the distribution of reactive oxygen species produced during light exposure which elucidated the photodynamic mechanisms of cancer cytotoxicity. The results presented in this dissertation provide key insight into engineering finely-tailored CQDs as the ideal nanomaterial for a broad range of applications.