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

Masaaki Uematsu,Takao Shimizu

doi:10.1038/s42003-021-02679-w

Abstract

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, since 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 physical properties of intracellular 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 perturbations attempting to alter the lipid composition. This was likely due to LDs functioning as a buffer against excess gauche/trans ratio, beyond its traditional role as an energy storage organelle. Our novel approach enables the observation of the physical properties of organelle lipids, which is difficult to perform with conventional probes, and is useful for quantitative assessment of the subcellular lipid environment.

Highlights

Background subtraction of in vivo spectraTo simplify the comparison of the spectra, background spectra were subtracted with weighting from representative spectra of lipid droplets (LDs) and non-LD regions
We investigated whether drastic changes in the physical state of lipids, namely, changes in the liquid-solid phase of lipids, are detectable using the Raman spectra of C–D stretch from deuterium-labeled fatty acids
Saturated (Fig. 1a) and unsaturated (Fig. 1b) fatty acids used in this experiment are in solid and liquid states, respectively, at room temperature, while fatty acids incorporated into cells should be closer to the liquid state, as they are mixed with other endogenous lipids

Summary

Introduction

To simplify the comparison of the spectra, background spectra were subtracted with weighting from representative spectra of LD and non-LD regions. The weighting values βLD, βnonLD, γLD, γnonLD, δLD, and δnonLD were calculated to minimize the following RSS values: RSS3 1⁄4 km0LD À ðβLD Á m0BG þ γLD Á sð280Þ þ δLD Á ið280ÞÞk22; RSS4 1⁄4 km0nonLD À ðβnonLD Á m0BG þ γnonLD Á sð280Þ þ δnonLD Á ið280ÞÞk22: ð11Þ. M0LD, m0nonLD, and m0BG represent the concatenated two sub-vectors of the spectra measured in representative LD, non-LD, and background pixels, respectively. The size of each sub-vector was 140, and the region from which subvectors were extracted was 1800–2000 and 2300–2500 cm−1. The procedure for this background subtraction was used only for the visualization of spectra, and not for other analyses. Model membrane systems were created using CHARMMGUI36,37 with the following PC18:1(d0)/PC16:0(d62) ratio (numbers in brackets followed by the ratio represent the numbers of each phospholipid molecule): 0.0 [0/

Methods

Results

Conclusion