Coherent anti-Stokes Raman scattering spectromicroscopy in biomedical and climate research

Abstract

Coherent anti-Stokes Raman scattering spectromicroscopy (CARS SM) is a powerful label-free technique based on intrinsic molecular contrast from nuclear vibrations that provides detailed information on the chemical composition and structure in microscopic objects and samples. Following the technological development of pulsed lasers, optomechanical and optical elements, CARS SM systems have experienced substantial advances during recent decades. However, many applications of the technique were shown mainly as proof-of-concept experiments and were not developed further by the scientists in the field.

The work presented in this thesis focuses on further development of CARS SM applications to promote it for research outside the nonlinear optics community by addressing biomedical and climate science questions, specifically in the area of protein biophysics and aerosol microdroplets.

The data retrieval process of CARS SM is complicated, which has restricted its wider application. Therefore, an easy-to-use way for removing the modulation error was developed for cases where the measured spectra are highly corrupted by the nonresonant background contribution.

In the context of biomedical research for this thesis, CARS SM was applied to in-depth studies of the protein structure for the low complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC). Firstly, CARS SM was applied to investigate molecular interactions that stabilize the liquid phase separated FUS LC droplets. The results gained from CARS SM along with NMR, computer simulations, and mutagenesis support a model in which FUS LC forms dynamic, multivalent interactions via multiple residue types and remains disordered in the densely packed liquid phase. Secondly, the G156E mutation in FUS LC, linked to neurological disease, was investigated. My work showed that aggregates formed by G156E FUS LC are fibrillar and β-sheet rich, and its aggregation behavior may be important for pathogenesis. Thirdly, kinetically trapped condensates formed at a low temperature by the wild type FUS LC were characterized. A distinct interfacial molecular structure, higher protein concentration, and slower molecular motion inside kinetically trapped condensates were found in comparison with annealed (untrapped) condensates, thereby demonstrating two unique, liquid demixed states of FUS LC. Lastly, the effect of membrane phospholipids on the phase separation behavior of FUS LC was probed. The findings included various morphologies due to the different lipid packing and different protein ordering in phase separated FUS LC in the presence of two membrane phospholipids.

In the context of climate research, CARS SM was used to study aerosol composition and pH, which is very important in atmospheric multiphase chemistry. The measurements showed that aerosol microdroplets have pH values indistinguishable from the corresponding bulk solutions and the pH is constant across the microdroplets. The observation of the spatially homogeneous pH inside microdroplets, determined simply by the parent solution, resolves fundamental questions about aerosol pH and offers CARS SM as an artifact-free method to further measure aerosol chemistry.