Investigating Metabolic Activities and Phenotypes in Biological Systems with Vibrational Probes and Raman Techniques

In this dissertation, the emerging stimulated Raman scattering (SRS) microscopy in combination with various vibrational tags was extensively used to explore various aspects of biological systems. New techniques as well as new Raman active materials were also developed to facilitate the applications of SRS in biology. Chapter one introduces and comprehensively reviews vibrational tags that have been developed to date in combination with imaging techniques and their applications in biological sciences to investigate metabolism in living organisms. Chapter two studies lipotoxicity, a phenomenon that is well known but poorly understood. The study found phase separation can form on ER membrane in cells treated with long chain fatty acids due to the high transition temptation of their metabolites. It was also found that the phase separation severely disturbs normal distribution of ER membrane proteins because of hydrophobic mismatching. As the result, ER normal structure is disrupted, luminal space is collapsed, and interconnectivity of ER that ensures normal ER functions is lost. Additionally, ER stress sensor IRE1α was found to be activated directly by the formation of phase separation, which triggers apoptosis and ultimately leads to cell death. Chapter three describes the development of a new method termed as metabolic activity phenotyping (MAP) that acquires quantitative measurements of metabolic activities of individual cells, which is essential to understanding questions in diverse fields in biology. To achieve the goal, an automatic system was designed and built that improves the acquisition speed by more than 100 times compared to commercially available instruments. A set of vibrational probes with deuterium labeling was also carefully selected to enable accurate measurement of metabolic flux. Combining the merits of high throughput measurements and vibrational tags, MAP was applied to investigate the metabolic activity differences among various cancer cells, to study the heterogeneity of drug efficacy, and to facilitate breast cancer subtyping. Chapter four describes the development and application of a new class of Raman active nanoparticles, or Rdots. These Rdots were generated by non-covalently incorporating small molecule Raman probe into polymeric nanoparticles. The resulted Rdots are of compact size (~20 nm) and preserve all Raman spectral features of the small molecule probes used. Rdots were compared to other existing Raman active materials including SERS nanoparticles, and Rdots surpass all the other materials in terms of brightness. In addition, Rdots also possess narrow spectral linewidth (< 3 nm), making them ideal for multiplexed imaging. In the study, Rdots were used as immunostaining reporters to visualize cytoskeleton networks and surface markers in cell and tissue samples

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy

Metabolic activity induces membrane phase separation in endoplasmic reticulum

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell

Energy storage properties in Nd doped AgNbTaO3 lead-free antiferroelectric ceramics with Nb-site vacancies

It is crucial to discover lead-free materials with ultrahigh recoverable energy density (Wrec) that can be employed in future pulse power capacitors. In this work, a high Wrec of 4.51 J/cm3 was successfully obtained in lead-free Nd-doped AgNb0.8Ta0.2O3 antiferroelectric ceramics at an applied electric field of 290 kV/cm. It is discovered that Nd doping paired with Nb-site vacancies could stabilize the antiferroelectric phase by lowering the temperatures of the M1–M2 and M2–M3 phase transitions, which leads to higher energy storage efficiency. Furthermore, Nd and Ta co-doping will contribute to the electrical homogeneity and low electrical conductivity, resulting in large breakdown strengths. Aliovalent doping in Ag-site with Nb-site vacancies serves as a novel strategy for the construction of AgNbO3-based ceramics with excellent energy storage performance

Microstructure and electrical properties of Nb‐doped SrTiO3‐BiFeO3 based lead‐free ceramics

In this work, Nb-doped 0.75SrTiO3-0.25BiFeO3 (ST-BF) lead-free ceramics are designed and synthesized using a conventional solid-state reaction method. The influence of Nb doping on the microstructure, dielectric, and electrical properties are systematically investigated. With the increase of Nb concentration, the crystal structure of ST-BF remains pseudo-cubic as exhibited in the X-ray diffraction patterns. The grain size is found to increase from 0.33 to 6.23 μm, and then decrease to 1.88 μm by Nb doping, along with a clear heterogeneous core–shell microstructure. A relatively low dielectric loss (∼0.1, at 1 kHz) and a stable dielectric constant (∼700, at 1 kHz) are obtained for the 0.03 Nb-doped ST-BF composition at room temperature. Impedance spectroscopy analysis shows that Nb doping in ST-BF increases the total resistivity, forming an electrically conductive core and a nonconductive shell, with enhanced activation energy. The results may provide a feasible approach to develop novel ST-based lead-free dielectric ceramics for capacitor application

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy

Neutron Spectroscopy Evidence for a Possible Magnetic-Field-Induced Gapless Quantum-Spin-Liquid Phase in a Kitaev Material α-RuCl3

As one of the most promising Kitaev quantum-spin-liquid (QSL) candidates, α-RuCl3 has received a great deal of attention. However, its ground state exhibits a long-range zigzag magnetic order, which defies the QSL phase. Nevertheless, the magnetic order is fragile and can be completely suppressed by applying an external magnetic field. Here, we explore the evolution of magnetic excitations of α-RuCl3 under an in-plane magnetic field, by carrying out inelastic neutron scattering measurements on high-quality single crystals. Under zero field, there exist spin-wave excitations near the M point and a continuum near the Γ point, which are believed to be associated with the zigzag magnetic order and fractional excitations of the Kitaev QSL state, respectively. By increasing the magnetic field, the spin-wave excitations gradually give way to the continuous excitations. On the verge of the critical field μ0Hc = 7.5 T, the former ones vanish and only the latter ones are left, indicating the emergence of a pure QSL state. By further increasing the field strength, the excitations near the Γ point become more intense. By following the gap evolution of the excitations near the Γ point, we are able to establish a phase diagram composed of three interesting phases, including a gapped zigzag order phase at low fields, possibly gapless QSL phase near μ0Hc, and gapped partially polarized phase at high fields. These results demonstrate that an in-plane magnetic field can drive α-RuCl3 into a long-sought QSL state near the critical field

Probabilistic Seismic Assessment of CoSPSW Structures Using Fragility Functions

The corrugated steel plate shear wall (CoSPSW) is a new type of steel plate shear wall, in which corrugated wall plates instead of flat wall plates are adopted. The lateral stiffness and shear buckling capacity of the shear wall system could be significantly enhanced, and then, wall plate buckling under gravity loads would be mitigated. This paper presents a study on the probabilistic assessment of the seismic performance and vulnerability of CoSPSWs using fragility functions. The damage states and corresponding repair states of CoSPSWs were first established from experimental data. Then, incremental dynamic analyses were conducted on the CoSPSW structures. The structural and nonstructural fragility functions were developed, based on which the seismic performance and vulnerability of the CoSPSWs were obtained and compared with the conventional steel plate shear walls (SPSWs). It was shown that for various repair states, the 25th percentile PGA of the CoSPSW was always higher than the SPSWs with the same wall thickness and boundary frame, which indicated that the CoSPSW has a lower damage potential and better seismic performance than the SPSW

Probing the structure of water in individual living cells

Abstract Water regulates or even governs a wide range of biological processes. Despite its fundamental importance, surprisingly little is known about the structure of intracellular water. Herein we employ a Raman micro-spectroscopy technique to uncover the composition, abundance and vibrational spectra of intracellular water in individual living cells. In three different cell types, we show a small but consistent population (~3%) of non-bulk-like water. It exhibits a weakened hydrogen-bonded network and a more disordered tetrahedral structure. We attribute this population to biointerfacial water located in the vicinity of biomolecules. Moreover, our whole-cell modeling suggests that all soluble (globular) proteins inside cells are surrounded by, on average, one full molecular layer (about 2.6 Angstrom) of biointerfacial water. Furthermore, relative invariance of biointerfacial water is observed among different single cells. Overall, our study not only opens up experimental possibilities of interrogating water structure in vivo but also provides insights into water in life