Single-Cell Characterization of Microalgal Lipid Contents with Confocal Raman Microscopy

The environmental impacts from consumption of fossil fuels have raised interest in finding renewable energy resources throughout the globe. Much focus has been placed on optimizing microalgae to efficiently produce compounds that can substitute for fossil fuels. However, the path to achieving economical feasibility of this substitution is likely to require strain optimization through mutagenesis screens as well as other available approaches and tools. Rapid characterization of the type of fatty acid expressed at a single-cell level can help identify screened cells with the desired lipid characteristics such as chain length and saturation status. Confocal Raman microscopy is a powerful tool for physicochemical characterization of biological samples. It enables single-cell, in vivo monitoring of various cellular components in a rapid, quantitative, label-free, and nondestructive manner. In this chapter, we describe recent advances in this method, which have resulted in remarkable enhancements in the sensitivity, specificity, and spatiotemporal resolution of the technique. We utilize this technique for analyzing lipid content of algal isolates obtained through a mutagenesis screen of the green alga, Chlamydomonas reinhardtii, for increased lipid production at the single-cell level. Our results demonstrate cell-to-cell variation in structural features of expressed lipids among the screened C. reinhardtii mutants, while clonal isolates show little to no variability in expressed lipids. The lack of stochasticity in expression of lipids in clonal populations of C. reinhardtii is a desired feature when accompanied by expression of fatty acids suitable for use as biofuel feedstock.