Title: Investigating Metabolic Activities and Phenotypes in Biological Systems with Vibrational Probes and Raman Techniques
Description: 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.