Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.
Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.
复制标题
DOI：
10.1242/dev.129908
复制
发表时间：
2016-06-15
期刊：
影响因子：
--
通讯作者：
Borri P
中科院分区：
文献类型：
--
作者：
Bradley J;Pope I;Masia F;Sanusi R;Langbein W;Swann K;Borri P
哺乳动物卵母细胞含有储存脂肪酸的脂滴，其新陈代谢在着床前的发育中起着重要作用。荧光染色以前被用来对哺乳动物卵母细胞和胚胎中的脂滴进行成像，但这种方法不是定量的，而且往往与活细胞成像和后续发育不兼容。在这里，我们将化学特异性的、无标记的相干反斯托克斯拉曼散射(CARS)显微镜应用于小鼠卵母细胞和植入前胚胎。我们表明，CARS成像可以定量的大小，数量和空间分布的脂滴在活的小鼠卵母细胞和胚胎，直到囊胚阶段。值得注意的是，它可以在不影响卵母细胞成熟或胚胎发育的情况下使用。我们还将CARS与同时使用荧光脂质探针在固定样本上获得的双光子荧光显微镜进行了关联，发现只有部分相关性，这取决于脂质探针，这清楚地例证了脂质标记的局限性。此外，我们还发现，在添加了不同饱和和不饱和脂肪酸的培养液中成熟的活卵母细胞中，脂滴的化学成分可以用CARS高光谱成像进行检测。这些结果表明，CARS显微镜提供了一种新的非侵入性方法，以亚微米级的分辨率量化活的哺乳动物卵母细胞和胚胎中的脂肪含量、类型和空间分布。摘要：CARS显微镜提供了一种新的、非侵入性的方法来定量活的小鼠卵母细胞和植入前胚胎中的脂肪含量、类型和空间分布。
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. Summary: CARS microscopy provides a novel and non-invasive method of quantifying lipid content, type and spatial distribution in living mouse oocytes and pre-implantation embryos.