We've established a nonlinear multimodal imaging system that incorporates stimulated Raman Scattering (SRS), multiphoton fluorescence (MPF), and second harmonic generation (SHG) to explore the connections between metabolic activities and the distribution of metabolites in cells and tissues. Furthermore, we've devised the Adam-based Pointillism Deconvolution (A-PoD) and Correlation Coefficient Mapping (CoCoMap) algorithms, enabling a deeper insight into the simultaneous recording and regulation of various metabolic processes within super-resolved images of nanoscale Regions of Interest (ROIs). In our pursuit of specifically identifying signals originating from distinct subcellular organelles, we've introduced a pioneering clustering algorithm known as Multi-SRS reference matching (Multi-SRM). This approach has the potential to improve early disease detection, prognosis, the evaluation of therapeutic effects, and our comprehension of the mechanisms underpinning aging and biomedicine.
Lipid and protein metabolism play important roles in the aging process of ovary. High resolution in situ optical imaging provides a powerful approach to study the metabolic dynamics of macro-molecules such as lipids and proteins. Here, we applied a multimodal imaging platform integrated with D2O probing stimulated Raman scattering (DO-SRS), multiphoton fluorescence (MPF), and second harmonic generation (SHG) to explore metabolic changes of biomolecules in Drosophila ovaries during aging process. In this study, the sub-cellular spatial distribution of de novo protein synthesis, lipogenesis and redox ratio in ovaries are quantitatively imaged and examined in different ages. The regulation of diets on aging-dependant changes of ovary metabolism are investigated. Our results provide valuable insights for the underlying mechanism of ovary aging and how to intervene the aging process to achieve a better health
KEYWORDS: Second harmonic generation, Molecules, Breast cancer, Multimodal imaging, Proteins, Mode conditioning cables, Biological samples, Super resolution, Signal detection
In this study, we developed a new technique to analyze spatial correlation between metabolic activities of multiple biomolecules. Using a multimodal imaging platform integrating Stimulated Raman Scattering (SRS), Multiphoton Fluorescence (MPF), and Second Harmonic Generation (SHG), together with an image deconvolution algorithm, we obtained super resolution images of biomolecular metabolism and investigated the correlations between metabolic activities and distributions of metabolites in tissues such as breast cancer tissues. Further, we developed a Pearson’s Correlation Coefficient based algorithm to examine the co-registration and co-regulation of metabolic activities in multiple channels of super-resolved images of nanoscopic Regions of Interest (ROIs). The multimodal imaging platform and Pearson’s correlation coefficient-based algorithm potentially facilitate early-stage breast cancer detection, and mechanistic understanding of breast cancer.
We use D2O probed stimulated Raman scattering (DO-SRS) and multiphoton fluorescence (MPF) microscopy to visualize metabolic changes in cells and tissues during aging processes and diseases. We further developed deconvolution algorithm to enhance the spatial resolution to generate super resolution metabolic imaging. The cellular spatial distribution of de novo lipogenesis, protein synthesis, NADH, Flavin, unsaturated lipids, and cholesterol were all imaged and quantified with hyperspectral detection of various macromolecules as multiplex imaging. Our study shows that the integrated metabolic imaging and spectroscopy platform can be applied to study nutrition regulated metabolic dynamics in cancer and during aging processes.
Quantification of molecular colocalization is an essential issue in understanding many biological processes in living organisms. To measure the spatial distribution of multiple biomolecules, an ideal way is to image them one by one in the exact same region of interest and the same focus plane. To achieve this goal, we integrated multiple optical imaging modalities including stimulated Raman scattering (SRS), multiphoton fluorescence (MPF), and second harmonic generation (SHG) all together into one multimodal microscopy. We further combined deuterium oxide probing with stimulated Raman scattering (DO-SRS) for visualizing newly synthesized protein and lipid molecules, in addition to macromolecules (protein and lipid, NADH and Flavin, collagen) imaged with label free SRS, MPF, and SHG. We quantitatively measured the metabolic dynamics in cells and animals under various conditions, including HeLa cells grew in different serine concentrations, Drosophila ovaries in young and old individuals, and two different types of breast cancer tissues from xenograft mouse models. The results show the capabilities and advantages of this multimodal imaging system in accessing the spatial distributions of multiple molecules quantitatively.
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