Raman microscopy-based quantification of the physical properties of intracellular lipids

The physical properties of lipids, such as viscosity, are homeostatically maintained in cells, and are intimately involved in physiological roles. Measurement of the physical properties of plasma membranes has been achieved primarily through chemical or genetically encoded fluorescent probes, however, the effect of the probes themselves on these physical properties hampered accurate measurements. In addition, as most probes target plasma membranes, physical properties of lipids in intracellular organelles, including lipid droplets (LDs) are yet to be analyzed. Here, we present a novel Raman microscopy-based approach for quantifying the intracellular physical properties of lipids under deuterium-labeled fatty acid treatment conditions. Focusing on the fact that Raman spectra of carbon-deuterium vibration are altered depending on the surrounding lipid species, we quantitatively represented the physical properties of lipids as the gauche/trans conformational ratio of the introduced labeled fatty acids, which can be used as an indicator of viscosity. Intracellular Raman imaging revealed that the gauche/trans ratio of cytosolic regions was robustly preserved against stimuli attempting to alter the lipid composition. This was likely due to LDs functioning as a buffer against excess gauche/trans ratio. The novel approach, that is, using the target lipid itself as a probe, overcomes the issues presented by conventional probes, making it useful for the quantitative evaluation of biological functions and regulatory mechanisms associated with the physical state of intracellular lipid environments.