Lipid imaging using bifunctional lipid probes

Mechanistic lipid cell biology requires techniques to faithfully image cellular lipid localization and transport in a species-specific manner. This is only partially possible by employing fluorescent lipid analogues and lipid biosensors, creating a need for more precise techniques. Here we outline techniques for using bifunctional (clickable and crosslinkable) lipid probes to visualize individual lipid species in cells and quantify their inter-organelle dynamics. We provide a detailed overview with regard to the nature of the chemical reporter groups, their effects on biophysical lipid properties, provide experimental details for sample preparation, fluorescence imaging, image analysis strategies and data interpretation. We outline pitfalls, limitations and best practices for control experiments. The lipid imaging strategy is described here for immortalized cell lines, but can easily be adapted for other tissue culture systems.