Vibrational Imaging for Chemical Biology: from Label-Free to Molecular probes

Citation

Du, Jiajun (2024) Vibrational Imaging for Chemical Biology: from Label-Free to Molecular probes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/dm1y-r078. https://resolver.caltech.edu/CaltechTHESIS:11032023-041429778

Abstract

Since the invention of stimulated Raman scattering (SRS) microscopy in 2008, vibrational imaging is increasingly recognized as a powerful tool for biological investigation. As the most suitable far field vibrational imaging modality for live biological studies, SRS microscopy is taking the lead role within its vibrational counterparts with desired sensitivity and image quality. The totally different mechanism of generating vibration signals from fluorescence signals determines the special features of vibrational imaging. Bond vibration originating signals provide inherent optical contrast for every molecule and the quantitative manner allows straightforward quantification. Since the inception, SRS microscopy has achieved large success in label-free imaging. Label-free imaging avoids tedious labeling step and has the least perturbation to the biological samples but with limited sensitivity and specificity. The introducing of labeling starting about 10 years ago opens up a new avenue for SRS microscopy to tackle the fundamental limitations of label-free approaches. Whether to use label-free or molecular probes for SRS microscopy depends on the specific studies. This thesis aims to utilize SRS microscopy (both label-free and minimally labeling) for metabolic study and develop new molecular probes for SRS microscopy.

We start from comparing different vibrational imaging modality and fluorescence imaging and conclude that SRS is the best vibrational imaging technique for biological samples. Then we discuss the features of label-free, bioorthogonal labeling and super-multiplexed SRS imaging. The minimally perturbative triple bond tagging and isotope labeling makes SRS especially suitable for tracking metabolites and accessing metabolic pathways. Furthermore, we also summarize the design principles for functional Raman imaging probe development based on their spectroscopic signatures. (Chapter 1).

Non-invasively probing metabolites within single live cells is highly desired but challenging. We explored Raman spectro-microscopy towards spatially-resolved single cell metabolomics, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. The chemical composition analysis of single cell and single organelle lipid droplets identified the fatty acid synthesis pathway and lipid mono-unsaturation as druggable susceptibility. More importantly we revealed that inhibiting lipid mono-unsaturation leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. (Chapter 2).

Next, we established a first-in-class design of multi-color photoactivatable Raman probes for subcellular imaging and tracking. The fast photochemically generated alkynes from cyclopropenones enable background-free Raman imaging with desired photocontrollable features. After necessary molecule engineering to improve the biocompatibility and sensitivity, we generated organelle-specific probes for targeting mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. Multiplexed photoactivated imaging and tracking at both subcellular and single-cell levels was also demonstrated to monitor the dynamic migration and interactions of the cellular contents. (Chapter 3).

Further improvement of the Raman signal with molecular probes is a central topic for Raman imaging. Recently developed electronic preresonance (epr) probes boost Raman signals and pushed SRS sensitivity close to that offered by confocal fluorescence microscopy. To guide the development of even stronger Raman probes and fill the final gap between epr-SRS probes and single molecule imaging, the structure-function relationship of epr-SRS probes is indispensable. We therefore used ab initio approach employing the displaced harmonic oscillator (DHO) model for calculating the epr-SRS signals, which proves to provide a consistent agreement between simulated and experimental SRS intensities of various triple-bond bearing epr-SRS probes. The theory also allows us to illustrate how the observed intensity differences between molecular scaffolds stem from the coupling strength between the electronic excitation and the targeted vibrational mode. Utilizing the discovered structure-function relationship of epr-SRS probes, we engineered MARS palette for higher sensitivity. With chemical modification to improve Raman mode displacement or enhance transition dipole moment or adjust detuning, we enhance the signal of alkynyl pyronins and nitrile pyronins, setting the current sensitivity records for small molecule far-field Raman probes. (Chapter 4 and 5).