Imaging lipids in live microalgae

Conference poster, 2012

Microalgae are the primary producers of n-3 fatty acids in the aquatic food web, capable of producing lipids from CO2 and sunlight. Intense research is underway to understand the conditions under which optimal lipid accumulation occurs. 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. While fluorescence microscopy with an appropriate dye can be highly specific, cells must be fixed and permeabilized, excluding live-cell studies; other techniques, such as phase contrast microscopy can be applied to living cells, but they lack chemical specificity. Coherent anti-Stokes Raman spectroscopy (CARS) is a non-linear microscopy technique that probes 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 in the visible region. Multi-photon autofluorescence from e.g. pigments in the sample can be detected simultaneously. The NIR light used to illuminate 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. We herein present a proof-of-concept in the comparison of Phaeodactylum tricornutum grown under normal light-starved and nitrogen-starved conditions.