Understanding brain lipid metabolism is vital for unraveling brain aging and neurodegeneration mechanisms. Traditional imaging methods lack chemical specificity and resolution. Stimulated Raman scattering (SRS) imaging provides high specificity, resolution, and deep penetration. We utilize a multimodal imaging platform integrating deuterium isotope probed SRS microscopy (DO-SRS), multiphoton fluorescence (MPF), and second harmonic generation (SHG) to visualize lipid dynamics in animal brains. Deuterium incorporation generates detectable carbon-deuterium bonds in lipids, revealing turnover. We find decreased lipid activity during aging and neurodegeneration in Drosophila brains, with interventions like dietary restriction and pathway modulation enhancing turnover. Our advanced imaging and analysis methods offer high-resolution insights applicable to diverse biomedical studies.
Diabetic nephropathy, a common diabetes complication, affects millions globally and requires precise biomarker detection without added surgical risks. Our emphasis on label-free multi-modal imaging removes the need for sample preparation and destruction, allowing analysis of smaller biopsies. This approach offers a comprehensive and accurate assessment of structural and biochemical changes in 2D and 3D, improving our understanding of kidney disease using a single microscopy setup. Crucially, it remains compatible with conventional methods, providing a holistic view without requiring extra tissue availability. Preserving this flexibility allows us to uncover hidden insights by visualizing previously quantified data.
The extensively employed technique of Stimulated Raman Scattering (SRS) has found widespread application in the domain of biomedical imaging. Typically, the CH band is harnessed for the identification of biomolecules. Nonetheless, the task of distinguishing between CH2 and CH3 bonds presents an ongoing challenge. In the present investigation, Circularly Polarized Stimulated Raman Scattering (CP-SRS) illumination was employed. This was carried out to explore the impact of polarization on the differentiation of CH vibrational modes. Intriguingly, it was observed that circular polarization notably repressed CH2 bonds in contrast to CH3 bonds. This selective effect facilitated the visualization of pure protein, eschewing the complexities posed by protein-lipid compounds. This unique unmixing approach significantly enhances the chemical specificity of SRS imaging. As a result, it holds remarkable implications for applications such as metabolic imaging and beyond.
Stimulated Raman scattering (SRS) technique has been widely used in biomedical imaging, in which the CH band is usually used for detecting biomolecules. However, Raman peaks have the nonlinear identity limitation in which a Raman peak can be comprised of multiple molecules and a molecule can manifest itself in multiple Raman peaks. In this study, we applied a circularly polarized light for SRS (CP-SRS) imaging to examine the effect of polarization on differentiating CH vibrational modes. We observed that CP exerted a significant suppression on CH2 bonds compared to CH3 bonds, allowing for visualizing pure protein instead of protein-lipid complexes. This distinctive physics-informed unmixing method has the potential to enhance the chemical sensitivity of SRS imaging, displaying important implications in label-free biomedical imaging field.
We use D2O probed stimulated Raman scattering (DO-SRS) and Multiphoton Fluorescence (MPF) microscopy to visualize metabolic changes in HeLa cells under excess AAA of phenylalanine or tryptophan. The cellular spatial distribution of de novo lipogenesis, protein synthesis, NADH, Flavin, unsaturated lipids, and cholesterol were all imaged and quantified in this experiment. Our studies reveal the increase in NADH to Flavin ratio by 10% and unsaturated lipids to saturated by 50% in cells treated with excess phenylalanine and tryptophan. Our study shows that DO-SRS can be used to as a high resolution imaging platform to study AAA regulated metabolic activities in cells.
Emerging studies have shown that oxidative imbalance is critical in disease progression such as cancer and Alzheimer’s [1, 2]. This variation can lead to the upregulation of certain metabolic pathways inducing diseases and disorders. Aromatic amino acids (AAA) are involved with the production of Reactive Oxygen Species (ROS), resulting in the increase of oxidative stress [3]. AAA studies typically rely on gas chromatography (GC) or mass spectroscopy (MS)-based imaging techniques to study lipids; however, these methods lack the ability to show the cell’s lipid spatial distribution or require fluorescent dyes that can interfere with the cell’s molecular activities [4, 5]. Here, we established an optical imaging approach that combines D2O (heavy water) probed Stimulated Raman scattering (DO-SRS) and Multiphoton Fluorescence (MPF) microscopy to directly visualize metabolic activities in situ in cancer cells under the regulation of excess AAA, specifically Phenylalanine and Tryptophan. The cellular spatial distribution of de novo lipogenesis, unsaturated and saturated lipids, NADH, Flavin, and new protein synthesis were quantitatively imaged and examined. We discovered an increase in de novo lipogenesis, Flavin/(Flavin + NADH), and unsaturated to saturated lipids in the cancer cells treated with excess AAAs. Decrease of protein turnover rate occurred in the same treated cells with observations of higher lipid droplet content. These observed metabolic activities are signs of mitochondrial dysfunction and oxidative stress. Our study demonstrates that DO-SRS can be used as a high-resolution imaging platform to study AAA regulated metabolic activities in cells and elucidates the linkage between lipid metabolism and cancer.
Two breast cancer subtypes with contrasting aggressiveness and prognoses - triple negative and normal-like - had their lipid droplets interrogated under methionine and insulin manipulation. Raman scattering results suggest that excess methionine increases droplet volume and de novo lipogenesis but decreases droplet number and alters distribution in both subtypes. These effects are significantly augmented in the triple-negative subtype with the presence of insulin. Flavin autofluorescence increases with insulin but decreases with excess methionine, in which an abundance of unsaturated lipids was also observed. This suggests interplay between methionine and insulin related lipid homeostasis and demonstrates the utility of optical techniques.
Breast cancer is a diverse disease rife with numerous subtypes with material impact on prognoses. Current methods may lack accuracy or be cost and time prohibitive, but optical techniques such as Isotope Probed Stimulated Raman Scattering microscopy (ip-SRS), and two photon excitation fluorescence (TPEF) microscopy can reveal spatial biomolecular information useful in distinguishing cell subtypes and phenotypic states rapidly and accurately. In the present study, we used heavy water and L-methionine to probe the enzymatic incorporation during scavenging and de novo biosynthesis of macromolecules in MCF10A, MCF7, and MDA-MB-231 breast cancer cells with spontaneous Raman spectro-microscopy and SRS microscopy, as well as their effects on cellular respiration and organelle health by imaging the NADH and flavin pools and labeled organelles with TPEF. This will enhance diagnostic efficacy and illustrate specific biochemical effects of manipulated nutrition and targeted therapies.
Early diagnosis of Triple Negative Breast Cancer (TNBC) is essential to implementing early, life-saving treatment before the development of metastases. Traditional methods for detecting TNBC is difficult, being both tedious and vulnerable to false positive results. Here we combine optical imaging techniques—deuterium probed resonance Raman spectroscopy (RRS) and multiphoton fluorescence (MPF) to detect TNBC metabolism in early stage. These hallmarks such as glucose and lipid metabolism are revealed through chemical bond vibrational modes with RRS and morphological changes with MPF at subcellular scale.
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