Biomolecular imaging is one of the rapidly developing areas of research in biomedical sciences. Since the discovery of Raman scattering by Raman and Krishnan and especially in the last few decades, significant progress has been made in developing Raman scattering-based imaging tools and their applications to studying biomolecules. Raman spectroscopy with enhancement and advances especially applied on lipid imaging is reviewed and discussed in the present study. Different Raman modalities and their performances are evaluated and compared in different aspects. Experimental performance of Raman imaging on lipid study is achieved with justified outcome. Raman spectroscopy could be justified as a powerful technique for biological imaging and lipid study.

Macromolecules, such as lipids and proteins, are key players in metabolic pathways related to cancer diseases. The synthesis of these macromolecules is related to the levels of amino acids in cells. Both essential and nonessential amino acids are considered to affect cancer cell survival and growth. Optical techniques that are both high resolution and non-destructive on the sample can aid in the understanding of metabolic changes in cancer cells. The objectives of this thesis are to provide insights into the effects of aromatic amino acids and nonessential amino acid (serine) on metabolic activities in cancer cells, by using Raman scattering microscopy for in situ subcellular imaging. The first chapter of this thesis presents a study on the influence of aromatic amino acids on lipid and protein metabolism in cancer cells using deuterium-oxide probed stimulated Raman scattering (SRS) and two-photon fluorescence microscopy. The second chapter presents work investigating the effects of the nonessential amino acid (serine) on lipid and protein metabolism in cancer cells using SRS imaging. The data and results presented in this thesis provide insights into the implications of essential and nonessential amino acids manipulations in cancer metabolism and application of Raman imaging techniques as a potential early diagnostic tool for cancers.

Aging is a natural process involving a gradual decline in physiological condition of most living organisms. This decline in physiological condition can lead to many age-related illnesses like Alzheimer’s disease, immunosenescence, and cancer. Metabolic processes play a crucial role in aging due to energy production, nutrition sensing, and signaling pathways being tightly regulated by cellular metabolism. Macromolecules such as lipids and proteins are key players in metabolic pathways for age-related decline and illnesses. High resolution and non-destructive optical techniques can be used to track metabolic and structural changes of these macromolecules. The objectives of this thesis are to provide insights into the effects of aging, genetic manipulation, and drug treatment on cellular metabolism in Drosophila Melanogaster using Raman scattering microscopy for in situ tissue imaging. The first chapter of this thesis presents a study on the influence of aging and amyloid precursor protein (APP) overexpression on protein metabolism in Drosophila antennal lobe by using spontaneous Raman spectroscopy. The second chapter presents the work investigating effects of the senolytic drug on lipid and protein metabolism and lifespan in Drosophila fat body tissue using spontaneous Raman spectroscopy, stimulated Raman scattering (SRS) and two-photon fluorescence microscopy. The data and results presented in these studies provides insights on the implications of aging, genetic manipulation, and senolytic drug administration on metabolism in Drosophila. These studies also demonstrate the broad applications of Raman imaging techniques in detection and diagnosis of aging and age-related illnesses.

In instances where tissue availability is limited or surgical risks to the patient are significant, the application of multiplexed biomedical imaging can be a challenge due to limitations in spectral bandwidth of fluorophores and histological stain chroma. This dissertation presents a label-free multimodal optical imaging platform that combines stimulated Raman scattering (SRS), second harmonic generation (SHG), and multiphoton fluorescence (MPF) in a single microscopy setup. Using this home built multimodal microscopy, the dissertation investigates subcellular organelle morphology, their molecular composition, and metabolic dynamics in triple negative breast cancer cells under tandem nutrient modulation, as well as assesses various biomarkers such as 3D mesangial expansion, collagen fiber thickening, oxidative stress, and lipid dysregulation in diabetic kidney disease. From subcellular to tissue levels, the platform provides spatial, biomolecular, and metabolic insight across disease models without relying on exogenous labels or serial sectioning. Developing and employing analytical techniques such as chemometric spectro-microscopy such as relative entropy, penalized reference matching, and stimulated Raman histology, this work advances quantitative bioimaging while preserving the flexibility of traditional methods through its non-destructive properties. Ultimately, this custom platform optimizes informatics while minimizing sample utilization and disruption, unlocking novel biological insights that were previously reliant on more intricate sample preparation, expensive reagents, and abundant tissue availability.

Digital light processing (DLP)-based 3D printing is an emerging 3D printing technology that allows for the fabrication of tissue-engineered scaffolds with complex microarchitecture mimicking that of native tissue with high fidelity and resolution in a matter of a few seconds to minutes. This doctoral dissertation explores the application of light-based 3D printing, specifically the fabrication of poly(glycerol sebacate) acrylate (PGSA) scaffolds, for treating volumetric muscle loss (VML) and muscle-tendon junction (MTJ) ruptures. First, the scaffold’s mechanical properties were tuned to match those of native skeletal muscle by adjusting light intensity used during the 3D printing process. Cellular response to the tissue-engineered scaffold in terms of muscle progenitor cell infiltration, alignment, proliferation, and differentiation was evaluated both in vitro and in vivo. Next, a novel light-based 3D printing workflow to create a complex, multi-scale elastomeric scaffold with user-defined regional stiffness control through the adjustment of 3D printing parameters was explored. This developed 3D printing workflow was demonstrated to have great potential in fabricating elastomer-based scaffolds for interfacial tissue engineering applications, which demand high spatial control of 3D printed scaffold’s mechanical properties. The final section of this dissertation applied DLP-based 3D printing to fabricate PGSA-based muscle-tendon scaffolds featuring regional mechanical variations for MTJ repair. These scaffolds, exhibiting microstructure and mechanical properties akin to native muscle-tendon tissue, showed high cell viability post-seeding and expressed MTJ-related markers. This dissertation sets the stage for further investigations into the utilization of 3D printed elastomer-based scaffolds, potentially transforming treatment for patients with muscle-related injuries and degenerative conditions.