Functional Stimulated Raman Imaging for Quantitative Cell Biology with Small Bioorthogonal Tags

Citation

Bi, Xiaotian (2025) Functional Stimulated Raman Imaging for Quantitative Cell Biology with Small Bioorthogonal Tags. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/w2hc-fa62. https://resolver.caltech.edu/CaltechTHESIS:05282025-224557158

Abstract

The development of imaging techniques, particularly optical imaging, has significantly advanced the field of cell biology. Compared to conventional fluorescence imaging, vibrational imaging leverages the intrinsic chemical bond information of molecules, providing multidimensional insights into molecular structures and local environments. So far, stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool for quantitative measurements in biological research. It overcomes several fundamental limitations associated with fluorescence-based techniques and offers high spatial and temporal resolution along with excellent compatibility for live-cell imaging.

In this thesis, I mainly focus on utilizing small bioorthogonal vibrational tags for quantitative investigations in cell biology. These tags, such as alkyne and carbon-deuterium (C-D) bonds, are absent in endogenous biomolecules and smaller than 1 nm in size, enabling minimally perturbative labeling with high molecular specificity. Another key advantage is that the SRS signal scales linearly with bond concentrations, allowing for robust and quantitative analysis. Moreover, because their vibrational modes distinctly reside in the cellular silent region (1800-2700 cm⁻¹), they provide a high signal-to-background ratio, making them particularly well-suited for quantitative applications in complicated cellular environments.

In Chapter 2, we explored the potential of alkyne-tagged probes to serve as environment-sensitive vibrational sensors, extending their utility beyond imaging markers. We developed a generalizable sensing platform based on hydrogen-deuterium exchange (HDX) at terminal alkynes. This subtle isotopic substitution induces a detectable shift in the alkyne vibrational frequency, allowing for real-time monitoring of exchange kinetics. These kinetics, in turn, provide insight into the chemical structures and local environments. We conducted a comprehensive study of the HDX process through both theoretical analysis and experimental validation. This platform was further applied to detect structural changes in DNA and to indicate pH within live cells, demonstrating the broader applicability of alkyne-tagged Raman probes for local environmental sensing in complex biological systems.

In Chapter 3, we utilized deuterated glutamine to label and study polyglutamine (polyQ) aggregates, a pathological hallmark of Huntington’s disease, in neurons. Traditional imaging approaches typically rely on tagging with bulky fluorescent proteins such as EGFP, which can perturb aggregation behavior with their non-negligible sizes. Through deuterium labeling, we achieved EGFP-free imaging of polyQ aggregates, allowing for a more native characterization with live-cell compatibility. This strategy facilitated quantitative analysis of the aggregate composition and growth dynamics of polyQ aggregates in live neurons. Our results revealed significant variations in polyQ aggregates depending on cell types, subcellular localizations, aggregate sizes, and protein constructs. Notably, we identified a previously unknown type of nuclear aggregates, shedding light on the heterogeneity of polyQ pathology.

In Chapter 4, we applied deuterium-labeled small molecules to study neuronal metabolism and its dynamic interactions with neuronal activity. As neuronal firing requires high and tightly regulated metabolic input, it is critical to understand the coupling between neuronal activity and metabolism for elucidating brain function. Using deuterated glucose and fatty acids, we were able to track their downstream metabolites for metabolism studies with high spatial and temporal resolution via SRS microscopy. In parallel, we employed optogenetic stimulation through Channelrhodopsin to achieve precise control of neuronal activity and also used neurotransmitters for longer-term modulation. By correlating different states of neuronal activation with metabolic flux changes, we gained valuable insights into how neuronal activity dynamically regulated glucose and lipid metabolism, advancing our understanding of neuroenergetic mechanisms in live neurons.

Through these studies, I demonstrate that the integration between small bioorthogonal vibrational tags and the advanced vibrational imaging technique, SRS microscopy, can provide powerful, minimally invasive, and highly quantitative tools for tackling fundamental questions in cell biology with high spatial and temporal resolution.

Thesis Files