In vivo lipidomics using single-cell Raman spectroscopy

Abstract

We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.lipid analysis | bioenergy T he global concerns surrounding unabated fossil fuel consumption and the risk of significant environmental impact caused by the associated greenhouse gas emissions, compounded by potential challenges associated with land-based biofuels, have renewed significant interest in microalgae as an alternative feedstock for the production of biodiesel and other biofuels (1). Microalgae hold considerable promise because of their ability to synthesize and store lipids, such as fatty acids and triacylglycerols (TAGs), which can be readily converted into biodiesel (fatty acid methyl or ethyl esters) through relatively simple chemical reactions (2). Small yet efficient, microalgae are attractive for many reasons, including their rapid, cost-effective, and resource-efficient production on nonarable land or photobioreactors (3), with impaired water, and for especially significant lipid production-up to 20-50% of their total dry weight, with examples of up to 80% under certain conditions reported (4). It has been estimated that lipid production of microalgae could be 30 times more efficient in terms of relative production of lipids per acre per year than any other terrestrial plant oil feedstock (2, 5).Under optimal growth conditions, microalgae synthesize fatty acids in the form of various glycerol-based membrane lipids primarily for structural and functional roles (6). In contrast, adverse environmental or metabolic stress conditions such as nutrient limitation, commonly referred to as "lipid trigger" conditions, result in an increase in carbon partitioning and accumulation of substantial proportions of neutral lipids (20-50% of dry weight), primarily in the form of TAGs. The TAGs are a form of...

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