Chemically‐specific dual/differential CARS micro‐spectroscopy of saturated and unsaturated lipid droplets

Abstract

Journal of BIOPHOTONICSWe have investigated the ability of dual-frequency Coherent Antistokes Raman Scattering (D-CARS) microspectroscopy, based on femtosecond pulses (100 fs or 5 fs) spectrally focussed by glass dispersion, to distinguish the chemical composition of micron-sized lipid droplets consisting of different triglycerides types (polyunsaturated glyceryl trilinolenate, mono-unsaturated glyceryl trioleate and saturated glyceryl tricaprylate and glyceryl tristearate) in a rapid and label-free way. A systematic comparison of Raman spectra with CARS and D-CARS spectra was used to identify D-CARS spectral signatures which distinguish the disordered poly-unsaturated lipids from the more ordered saturated ones both in the CH-stretch vibration region and in the fingerprint region, without the need for lengthy CARS multiplex acquisition and analysis. D-CARS images of the lipid droplets at few selected wavenumbers clearly resolved the lipid composition differences, and exemplify the potential of this technique for label-free chemically selective rapid imaging of cytosolic lipid droplets in living cells.

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

“…Importantly, qDIC is intrinsically label-free, overcoming the inherent drawbacks of fluorescent labeling such as sample modification and photobleaching. Particularly in the context of novel microscopy techniques that have recently emerged to image lipids in a label-free manner, such as coherent anti-Stokes Raman scattering (14,15), the method offers a platform for characterization of vesicle lamellarity compatible with subsequent label-free studies of GUVs. The method is capable to determine the lamellarity from a single DIC image, avoiding motion artifacts and opening up the study of vesicle dynamics.…”

Modern trends in biophotonics for clinical diagnosis and therapy to solve unmet clinical needs

“…This combined multimodal and spectral analysis elucidated the layered structure of rat cornea with molecular and structural information. Dual frequency CARS (D‐CARS) microspectroscopy was able to distinguish the chemical composition of micro‐sized lipid droplets in a rapid and label‐free way . The distinction was achieved by collecting D‐CARS images at few selected wavenumbers without the need for lengthy CARS multiplex acquisition and analysis.…”

Perilipin 5 mediated lipid droplet remodelling revealed by coherent Raman imaging

“…Thus quantifying ester bonds in the LD should yield an indicative measure of the number of esterified acyl chains within the LD and allow us to determine if increased acyl chain packing contributes to the 200-330% increase in methylene groups in LDs of PLIN5-overexpressing muscle. The Raman mode at 1740 cm À1 is attributed to CQO stretching vibration of ester bonds 23,30 (Fig. 1B), so we created vibrational images of CQO groups to investigate the ester content in LDs.…”

“…21,22 CARS has recently been shown to quantitatively agree with GC-MS results for lipid profiling. [22][23][24] Here we use hyperspectral CARS microscopy with in vitro myotubes and ex vivo myofibers expressing endogenous and elevated levels of PLIN5 to investigate whether PLIN5 overexpression modulates composition of individual LDs harboring IMCL in skeletal muscle. We directly measure changes in individual myocellular LD chemistry by quantification of different chemical species at sub-micron resolution.…”