Long-Term Single-Cell Imaging of Live Microbes by Correlative Fluorescence and Raman Microscopy & Time-Resolved Stark Effect Spectroscopy of Protein Crystals

Abstract

Understanding the behavior of a microbe requires not only an understanding of molecular mechanisms, e.g. DNA replication, transcription, and translation, but also knowledge of the cell’s chemical composition over time. There are powerful tools for probing the mechanisms of core cell-biological processes. However, the existing tools to measure cell composition have significant limitations. Information-rich methods, such as mass spectrometry, are lethal, while fluorescence methods provide good time resolution and work on live cells, but are limited in what they can measure. Prior work has demonstrated that spontaneous Raman spectroscopy can be a powerful tool to measure cellular composition. However, low throughput has limited its potential for discovery. Here, I describe my contributions to correlated epifluorescence and laser scanning Raman microscopy to enable the collection of single-cell spectra from many single cells in long-term time-lapse experiments, with imaging every 15 minutes. I then establish that this single-cell Raman imaging (scRaman) system can be used without substantial phototoxic effects to image the yeast Saccharomyces cerevisiae. To benchmark the ability to track biologically important changes in cellular composition, I study S. cerevisiae under conditions of nitrogen starvation and repletion. I demonstrate the ability of this system to resolve the dynamics of compositional changes at a single-cell level, mapping the observed dynamics to known biological mechanisms. I describe the analysis pipeline required for this work and the advances in combined Raman and microscope control software to achieve these experiments. Finally, I describe a separate project in which I developed an analytical framework for interpreting time-resolved Stark effect spectroscopy data obtained from single protein crystals.