bioorthogonal-chemical-imaging-cell-metabolism-regulated-aromatic

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

University of California San Diego

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS) Here, we use DO (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of  lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in  lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells' nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution .

Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy.

Triple negative breast cancer (TNBC) is a particularly aggressive cancer subtype that is difficult to diagnose due to its discriminating epidemiology and obscure metabolome. For the first time, 3D spatial and chemometric analyses uncover the unique lipid metabolome of TNBC under the tandem modulation of two key metabolites - insulin and methionine - using non-invasive optical techniques. By conjugating heavy water (DO) probed Raman scattering with label-free two-photon fluorescence (TPF) microscopy, we observed altered  lipogenesis, 3D lipid droplet morphology, and lipid peroxidation under various methionine and insulin concentrations. Quantitative interrogation of both spatial and chemometric lipid metabolism under tandem metabolite modulation confirms significant interaction of insulin and methionine, which may prove to be critical therapeutic targets, and proposes a powerful optical imaging platform with subcellular resolution for metabolic and cancer research.

Imaging Sub-Cellular Methionine and Insulin Interplay in Triple Negative Breast Cancer Lipid Droplet Metabolism.

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (DO)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.

Article	Year
Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy. Frontiers in molecular biosciences\| PubMed ID: 34977157	2021
Imaging Sub-Cellular Methionine and Insulin Interplay in Triple Negative Breast Cancer Lipid Droplet Metabolism. Frontiers in oncology\| PubMed ID: 35359364	2022
Super-resolution SRS microscopy with A-PoD. Nature methods\| PubMed ID: 36797410	2023

Khang Hoang

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