Nonlinear vibrational microscopy applied to lipid biology

Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology