Imaging lipids in live microalgae

Abstract

Microalgae are capable of producing lipids from CO2 and sunlight and as such the primary producers of n-3 fatty acids. Intense research is underway to understand the conditions under which optimal lipid accumulation occurs, not only for neutraceutical applications, but also for biodiesel production. To aid this research we propose the application of a powerful microscopic technique that allows monitoring of lipids with chemical specificity at intra-cellular level in living cells: Coherent anti-Stokes Raman spectroscopy (CARS). CARS is a non-linear microscopy technique that can be used to probe C-H bonds especially abundant in lipids by a process involving four-wave mixing: two or three coherent beams of different near-infra red (NIR) wavelengths are tuned to induce a resonant vibration in the C-H bonds, and generate a blue-shifted CARS signal. The NIR light used to probe the sample allows good penetration which in turn makes optical sectioning possible. By taking many optical sections of the sample, a 3D image can be constructed, from which the volume of lipids in the cell can be calculated, allowing quantitative studies of lipid accumulation in single microalgae. In our study, we used Phaeodactylum tricornutum grown under normal, light-starved and nitrogen-starved conditions. CARS microscopy detected statistically significant differences in lipid droplet number and their volumes when comparing growth conditions at single cell level. The whole population was then subjected to traditional lipid extraction and chromatographic separation of fatty acids. Average lipid volumes, as calculated from CARS microscopy, correlated well with traditional chemical analysis. As a conclusion, CARS could be applied to the in vivo study of culture condition effect on lipid accumulation in microalgae