Optical imaging of metabolic dynamics in animals (original) (raw)
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Raman microscopy for dynamic molecular imaging of living cells
Journal of Biomedical Optics, 2008
We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slitscanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that 532-nm excitation can be used to generate strong Raman scattering signals and to suppress autofluorescence that typically obscures Raman signals. With this technique, we reveal time-resolved distributions of cytochrome c and other biomolecules in living cells in the process of cytokinesis without the need for fluorescent labels or markers.
Nature Photonics, 2011
Label-free microscopy that has chemical contrast and high acquisition speeds up to video rates has recently been made possible using stimulated Raman scattering (SRS) microscopy. SRS imaging offers high sensitivity, but the spectral specificity of the original narrowband implementation is limited, making it difficult to distinguish chemical species with overlapping Raman bands. Here, we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species, based on tailored multiplex excitation of its vibrational spectrum. This is implemented by spectral modulation of a broadband pump beam at a high frequency (>1 MHz), allowing detection of the SRS signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live Caenorhabditis elegans.
Two-color vibrational imaging of glucose metabolism using stimulated Raman scattering
Chemical communications (Cambridge, England), 2017
A two-color vibrational imaging technique for simultaneously mapping glucose uptake and incorporation activity inside single living cells is reported. Heterogeneous patterns of glucose metabolism are directly visualized from the ratiometric two-color images for various cell types, cells undergoing epithelia-to-mesenchymal transitions and live mouse brain tissues. The two-color imaging of glucose metabolism here demonstrates the development of multi-functional vibrational probes for multicolor imaging of cellular metabolism.
Mammalian cell and tissue imaging using Raman and coherent Raman microscopy
Direct imaging of metabolism in cells or multicellular organisms is important for understanding many biological processes. Raman scattering (RS) micros-copy, particularly, coherent Raman scattering (CRS) such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has emerged as a powerful platform for cellular imaging due to its high chemical selectivity, sensitivity, and imaging speed. RS microscopy has been extensively used for the identification of subcellular structures, metabolic observation, and phenotypic characterization. Conjugating RS modalities with other techniques such as fluorescence or infrared (IR) spectroscopy, flow cytometry, and RNA-sequencing can further extend the applications of RS imaging in microbiology, system biology, neurology, tumor biology and more. Here we overview RS modalities and techniques for mammalian cell and tissue imaging, with a focus on the advances and applications of CARS and SRS microscopy, for a better understanding of the metabolism and dynamics of lipids, protein, glucose , and nucleic acids in mammalian cells and tissues. coherent Raman, glucose, lipid, mammalian cell, metabolism, nucleic acid, optical imaging, protein, Raman scattering, stimulated Raman 1 | INTRODUCTION Raman scattering (RS) is the inelastic scattering of visible monochromatic light, during which a quantum of energy from a photon induces vibration in molecular bonds. Given incident light of certain wavelength, different molecular bonds within an analyte will vibrate at different frequencies, allotting each molecular bond a distinct "fingerprint," and each molecular bond's vibrational frequency requires specific quanta of energy from incident photons. Since the light is not absorbed by the substance, the photons are then scattered, having an energy (wavelength) different from incident photons. The change in wavelength between the incident and scattered light is called Raman shift. A Raman shift may be either a negative or positive shift in energy, referred to as Stokes or anti-Stokes, respectively. The Raman shift is directly related to the energy required to vibrate the molecular bonds in the analyte. Higher Raman signal intensities
Cancers, 2021
Metabolic reprogramming is a common hallmark in cancer. The high complexity and heterogeneity in cancer render it challenging for scientists to study cancer metabolism. Despite the recent advances in single-cell metabolomics based on mass spectrometry, the analysis of metabolites is still a destructive process, thus limiting in vivo investigations. Being label-free and nonperturbative, Raman spectroscopy offers intrinsic information for elucidating active biochemical processes at subcellular level. This review summarizes recent applications of Raman-based techniques, including spontaneous Raman spectroscopy and imaging, coherent Raman imaging, and Raman-stable isotope probing, in contribution to the molecular understanding of the complex biological processes in the disease. In addition, this review discusses possible future directions of Raman-based technologies in cancer research.
High-speed molecular spectral imaging of tissue with stimulated Raman scattering
Nature Photonics, 2012
Recent development of stimulated Raman scattering (SRS) microscopy offers unprecedented capability for label-free imaging such as high-speed imaging at up to the video rate [4] and chemical contrast based on vibrational spectroscopy without spectral distortion . Fast imaging is important because it allows us to successively observe samples in different fields of view, and
arXiv (Cornell University), 2019
In situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destruct the sample or introduce strong perturbations to the functions of target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a labelfree single-cell analysis platform with chemical specifity, and high-throughput capabilities. Cellular compartments such as lipid droplets, endoplasmic reticulum, and nuclei are seperated from the cytoplasm. Based on these chemical segmentations, 260 features from both morphology and molecular composition were generated and analyzed for each cell. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapy drug treatments. We unveiled lipid-facilitated protrusion as a metabolic marker for stress-resistant cancer cells through statistical analysis of thousands of cells. Our findings also demonstrate the potential of targeting lipid metabolism for selective treatment of starvation-resistant and chemotherapy-resistant cancers. These results highlight our SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity. Altered cell metabolism is recognized as one of the hallmarks of cancer (Hanahan and Weinberg, 2011). The reprogrammed metabolism, which is deployed by cancer cells to fulfill the demands of fast proliferation , offers new opportunities for diagnosis and treatment of cancer . However, challenges remain in the quantification of cell metabolism, one of which is the inter-or intratumoral heterogeneous metabolic profiles of cancer cells. Although the ensemble measurement of large number of cancer cells identifies specific metabolic features under certain condition, individual cancer cells might show significantly different metabolic response . This cell heterogeneity is considered one of the major causes of incomplete tumor remission and relapse . Such an inhomogeneous response of cells to environments and drugs cannot be addressed using conventional methods, such as biochemical assays or mass spectrometry, which are based on ensemble measurement of a cell population. High-efficiency imaging tools which can quantify metabolic features of individual cells with subcellular information for a large number of cells become essential. Flow cytometry is one of the commonly used technologies for high-throughput single-cell analysis, which generates statistical information of a cell population .
GEN Biotechnology
Studies have shown that brain lipid metabolism is associated with biological aging and influenced by dietary and genetic manipulations; however, the underlying mechanisms are elusive. High-resolution imaging techniques propose a novel and potent approach to understanding lipid metabolic dynamics in situ. Applying deuterium water (D 2 O) probing with stimulated Raman scattering (DO-SRS) microscopy, we revealed that lipid metabolic activity in Drosophila brain decreased with aging in a sex-dependent manner. Female flies showed an earlier occurrence of lipid turnover decrease than males. Dietary restriction (DR) and downregulation of insulin/IGF-1 signaling (IIS) pathway, two scenarios for lifespan extension, led to significant enhancements of brain lipid turnover in old flies. Combining SRS imaging with deuterated bioorthogonal probes (deuterated glucose and deuterated acetate), we discovered that, under DR treatment and downregulation of IIS pathway, brain metabolism shifted to use acetate as a major carbon source for lipid synthesis. For the first time, our study directly visualizes and quantifies spatiotemporal alterations of lipid turnover in Drosophila brain at the single organelle (lipid droplet) level. Our study not only demonstrates a new approach for studying brain lipid metabolic activity in situ but also illuminates the interconnection of aging, dietary, and genetic manipulations on brain lipid metabolic regulation.
Chemphyschem : a European journal of chemical physics and physical chemistry, 2018
Retinoids play critical roles in development, immunity and lipid metabolism, and their deficiency leads to various human disorders. Yet, tools for sensing retinoids in vivo are lacking, which limits the understanding of retinoid distribution, dynamics and functions in living organisms. Here, using hyperspectral stimulated Raman scattering microscopy, we discover a previously unknown cytoplasmic store of retinoids in Caenorahbditis elegans. Following the temporal dynamics of retinoids, we reveal that their levels are positively correlated with fat storage, and their supplementation slows down fat loss during dauer starvation. We also discover that retinoids promote fat unsaturation in response to high-glucose stress, and improve organism survival. Together, our studies report a new method for tracking the spatiotemporal dynamics of retinoids in living organisms, and suggest the crucial roles of retinoids in maintaining metabolic homeostasis and enhancing organism fitness upon develop...