CARS microscopy of lipid stores in yeast: the impact of nutritional state and genetic background

We have developed a protocol for sub-micrometer resolved and chemically specific imaging of lipid storage in vivo employing coherent anti-Stokes Raman scattering (CARS) microscopy of one of the most important model organisms Saccharomyces cerevisiae - the yeast cell. By probing the carbon-hydrogen vibration using the nonlinear process of CARS, lipid droplets in the yeast cells clearly appear, as confirmed by comparative studies on relevant labeled organelles using two-photon fluorescence microscopy. From the images, unique quantitative data can be deduced with high three-dimensional resolution, such as the volume, shape, number, and intracellular location of the neutral lipid stores. We exemplify the strength and usability of the method for two cases: the impact on lipid storage of the nutritional condition (starvation and type of carbon source available) as well as of genetic modification of two fundamental metabolic regulation pathways involving carbohydrate and lipid storage (BCY1 and DGA1, LRO1, ARE1/2 deletions), respectively. While the impact of carbon source on the total cellular lipid volume was minimal, long-term starvation induces a significant accumulation of lipid droplets. We also confirm that the lipid-storage-deficient mutant is indeed unable to synthesize lipid droplets, and that the inability of the bcy1-mutant to store carbohydrates is compensated by a two-fold increase in stored neutral lipids. We note that there is a significant cell-to-cell variability in neutral lipid storage in general, i.e. that there is a correspondence to the noise found for gene expression also in lipidomics. Copyright (C) 2009 John Wiley & Sons, Ltd.