Imaging Cellular Metabolism with Timelapse Stimulated Raman Scattering Microscopy

Abstract

Cellular metabolism depends on the intricate coordination of many biochemical processes across space and time. While many of these processes are well understood on a chemical level, it has been much more challenging to see them play out in single cells. Stimulated Raman Scattering (SRS) microscopy is an imaging technique that has great potential for metabolic imaging due to its sensitivity to the vibrational energy levels of molecules. Previous advancements in SRS imaging have increased spatial resolution and focused on large samples such as tissues or organisms. Here, I describe experiments that push the boundaries of SRS microscopy to enable dynamic metabolic imaging of growing microbes. My work demonstrates that SRS microscopy can image hundreds of small cells over long periods of time while collecting many images per cell division, opening up an exciting new regime of potential applications for SRS microscopy in biological imaging. I developed a novel computational approach that corrects for non-uniform backgrounds in SRS images and enables the extraction of small SRS signals. With this approach, and taking advantange of the unique capability of SRS to detect isotopically-labeled compounds, I report the first timelapse SRS measurements of glucose consumption in single budding yeast cells. Combining SRS imaging with bright field and fluorescence modalities, I investigate the production of biomass in different subpopulations of cells and directly observe that mother cells preferentially retain older biomass though cell division. Furthermore, I show how net glucose uptake dynamics in cells expressing a single glucose transporter are different from those of wild type cells, suggesting that SRS may offer insights into the transport rates of individual proteins in the cellular context. Together, these results establish timelapse SRS microscopy as a powerful tool for measuring cellular metabolism.