title: Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.
abstract: Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos.
authors.affiliation: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK.
authors.forename: Josephine
authors.initials: J
authors.lastname: Bradley
authors.affiliation: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK.
authors.forename: Iestyn
authors.initials: I
authors.lastname: Pope
authors.affiliation: Cardiff School of Physics and Astronomy, The Parade, Cardiff CF24 3AA, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: Masia
authors.affiliation: Cardiff University School of Medicine, Sir Geraint Evans Building, Heath Park, Cardiff CF14 4XN, UK.
authors.forename: Randa
authors.initials: R
authors.lastname: Sanusi
authors.affiliation: Cardiff School of Physics and Astronomy, The Parade, Cardiff CF24 3AA, UK.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Langbein
authors.affiliation: Cardiff University School of Medicine, Sir Geraint Evans Building, Heath Park, Cardiff CF14 4XN, UK SwannK1@cardiff.ac.uk BorriP@cardiff.ac.uk.
authors.forename: Karl
authors.identifier: 0000-0002-4355-1449
authors.initials: K
authors.lastname: Swann
authors.affiliation: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK SwannK1@cardiff.ac.uk BorriP@cardiff.ac.uk.
authors.forename: Paola
authors.initials: P
authors.lastname: Borri
mesh_terms: D000818:Animals; D002470:Cell Survival; D016000:Cluster Analysis; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D047108:Embryonic Development; D005260:Female; D021621:Imaging, Three-Dimensional; D066292:Lipid Droplets / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D013059:Spectrum Analysis, Raman
chemical_list: D008055:Lipids
keywords: Egg; Embryo; Lipid; Microscopy; Oocyte