Uncovering the "Shape" of Intracellular Water by Hyperspectral Stimulated Raman Scattering Microscopy

Abstract

The biochemical environment of the cellular interior is extremely complex andplays a critical role in the function of all biomolecules necessary to life. As the most abundant molecules in cytomatrix, intracellular water actively participates in a wide range of biochemical processes, thus the local water structure and solvation in different cellular compartments should strongly impact the local biochemistry. Despite the fundamental nature, it remains unclear to which extent the intracellular water differs from the familiar “bulk” water and there is a dearth of experimental evidence regarding the spatial variation of solvation inside the cell. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering technique with a water-sensing nitrile probe. This dissertation contributes to a complete picture of understanding the hydrogen bonding network and heterogenous solvation at the subcellular level, which has a profound impact on interpreting biochemical studies conducted outside of the native biological environment. This dissertation is structured as the following chapters. Chapter 1 provides an overview of the structure of intercellular water and the evolvement of this field. Chapter 2 introduces the basics of stimulated Raman scattering and elaborates the construction of a hyperspectral stimulated Raman scattering (hsSRS) microscope. Chapter 3 demonstrates the application of hsSRS to probe solvation heterogeneity at the microscopic level by coupling with vibrational solvatochromism of an environmentally-sensitive nitrile probe. The sensing ability is validated in the solution phase, microscopic droplets, and cellular environments. Finally, the subcellular solvation variance between the cytoplasm and the nucleus is quantitatively measured and the origin of the intracellular solvation heterogeneity is explored. To study protein-associated water, different nitrile probes with various sizes and hydrophobicity are investigated in the solution phase measurement. Chapter 4 illustrates a preliminary attempt to develop a real-time chemical imaging technique called Diffusive Enhanced Raman Nano-spectrometer (DERNS) by combining 3D real-time tracking technique with the advanced Raman techniques. The main focus is synthesizing the near-field fluorescent probes coupled with plasmonic structure and its pump-probe behaviors. Chapter 5 summarizes the key findings from the previous chapter and proposes a few future projects along vibrational-shift imaging.