Center for Biophotonics, Science and Technology (CBST), University of California, Davis, CA 95817, USA.
Analyst. 2013 Nov 7;138(21):6662-70. doi: 10.1039/c3an00970j. Epub 2013 Sep 2.
Cellular lipid droplets are the least studied and least understood cellular organelles in eukaryotic and prokaryotic cells. Despite a significant body of research studying the physiology of lipid droplets it has not yet been possible to fully determine the composition of individual cellular lipid droplets. In this paper we use Raman spectroscopy on single cellular lipid droplets and least-squares fitting of pure fatty acid spectra to determine the composition of individual lipid droplets in cells after treatment with different ratios of oleic and palmitic acid. We validate the results of the Raman spectroscopy-based single lipid droplet analysis with results obtained by gas chromatography analysis of millions of cells, and find that our approach can accurately predict the relative amount of a specific fatty acid in the lipid droplet. Based on these results we show that the fatty acid composition in individual lipid droplets is on average similar to that of all lipid droplets found in the sample. Furthermore, we expand this approach to the investigation of the lipid composition in single cellular peroxisomes. We determine the location of cellular peroxisomes based on two-photon excitation fluorescence (TPEF) imaging of peroxisomes labeled with the green fluorescent protein, and successive Raman spectroscopy of peroxisomes. We find that in some cases peroxisomes can produce a detectable CARS signal, and that the peroxisomal Raman spectra exhibit an oleic acid-like signature.
细胞脂质滴是真核细胞和原核细胞中研究最少、了解最少的细胞器。尽管有大量研究脂质滴生理学的文献，但仍未能完全确定单个细胞脂质滴的组成。在本文中，我们使用拉曼光谱对单个细胞脂质滴进行研究，并对纯脂肪酸光谱进行最小二乘拟合，以确定在用不同比例油酸和棕榈酸处理后细胞中单个脂质滴的组成。我们通过对数百万个细胞进行气相色谱分析来验证基于拉曼光谱的单个脂质滴分析的结果，并发现我们的方法可以准确预测脂质滴中特定脂肪酸的相对含量。基于这些结果，我们表明单个脂质滴中的脂肪酸组成平均与在样本中发现的所有脂质滴中的脂肪酸组成相似。此外，我们将这种方法扩展到对单个细胞过氧化物酶体中脂质组成的研究。我们基于过氧化物酶体用绿色荧光蛋白标记的双光子激发荧光（TPEF）成像来确定细胞过氧化物酶体的位置，并对过氧化物酶体进行连续的拉曼光谱分析。我们发现，在某些情况下，过氧化物酶体可以产生可检测的 CARS 信号，并且过氧化物酶体的拉曼光谱表现出类似于油酸的特征。
