Stimulated Raman Scattering: a Biophysical Perspective for Imaging Cells and Tissues

Citation

Miao, Kun (2024) Stimulated Raman Scattering: a Biophysical Perspective for Imaging Cells and Tissues. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ch88-9173. https://resolver.caltech.edu/CaltechTHESIS:08312023-053642106

Abstract

This thesis explores the utilization of Stimulated Raman Scattering (SRS) microscopy as a novel imaging method in the biomedical field, aiming to overcome the limitations associated with traditional fluorescence-based techniques. Given the drawbacks of fluorescence imaging, such as photobleaching, auto-fluorescence, and the complexity of fluorophore labeling, SRS microscopy emerges as a promising solution. The optical imaging contrast in this method originates from bond vibrations of endogenous biomolecules. Grounded in the principle of Raman scattering, SRS amplifies weak spontaneous Raman transitions through stimulated emission, offering a target-specific, high-speed, and label-free imaging modality that can overcome the challenges of traditional bio-imaging techniques.

To tackle the interference from fluorescent proteins when imaging small proteins of interest, we demonstrated a combination of SRS with selective deuterium labeling for visualizing polyQ aggregates in Huntington's disease. We targeted the C-D vibration on deuterated glutamines, which are metabolically enriched in the polyQ sequence. This allowed us to image Huntingtin aggregates without using fluorescent labels. Our method enables, for the first time, the quantification of protein concentrations and compositional analyses of polyQ and non-polyQ proteins within native Huntingtin aggregates. This novel perspective suggests that aggregates have distinct biophysical roles at different stages of aggregation.

In addition to fluorescent proteins, immunofluorescence is the gold standard for visualizing the location and distribution of proteins within cells or tissues. However, the proper delivery of antibodies is slow and labor-intensive. To overcome this issue, we developed a novel method, Vibrational Imaging of Swelled Tissue and Analysis (VISTA), that combines SRS microscopy with sample expansion to enable label-free super-resolution volumetric imaging in tissues. We developed a unique fixation hydrogel chemistry to maximize protein retention, delipidation, and isotropic expansion in tissue samples. By targeting the bond vibrations from endogenous proteins, VISTA bypasses the limitations of antibody labeling and provides an efficient tool for high-throughput imaging that can be scaled to large-volume clinical samples. The addition of image segmentation methods to VISTA equips it with protein-level specificity similar to immunofluorescence. We further used this technique to study protein aggregates, such as amyloid-β plaques in Alzheimer's disease, revealing intricate aggregate structures and polymorphisms absent in conventional fluorescence methods.

Finally, as fluorescent biosensors are indispensable tools for studying intracellular dynamics, we worked on extending the utility of SRS microscopy into the realm of sensing. We employed hydrogen-deuterium exchange on alkyne substrates to develop a Raman-based sensing strategy sensitive to subtle variations in local microenvironments. The rate of hydrogen-deuterium exchange changes under different conditions, and the resulting frequency shift from alkyne to deuterated alkyne is captured by SRS microscopy. This new platform enhances the study of chemical environments in various biological structures, marking a pivotal step in integrating imaging and sensing in biophysical research.

Thesis Files