Duindam H J, Vrensen G F, Otto C, Puppels G J, Greve J
The Netherlands Ophthalmic Research Institute, Amsterdam.
J Lipid Res. 1995 May;36(5):1139-46.
Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytical tool for the study of some aspects of the molecular organization of cells and tissues with high spatial resolution. Filipin, a polyene antibiotic, specifically binds to cholesterol, and its molecular structure predicts it to be Raman-active. The aim of the present study was to assess the potentialities of a combined CRM-filipin approach to study the distribution of cholesterol in the human eye lens. Paraformaldehyde-fixed human lenses were sliced (0.7 mm), incubated with filipin, and analyzed by CRM. Filipin proved to give a specific Raman signal at 1586 cm-1, hardly interfering with signals from lens proteins. The CRM-filipin approach proved to be extremely sensitive, allowing detection of cholesterol in the femtogram range. It has an excellent spatial resolution (0.2-0.5 micron 3) when using point measurements. Due to the intrinsic anisotropy of membranes in the eye lens and therefore of the cholesterol distribution, a line-scan approach has to be adopted when fiber-to-fiber changes in cholesterol are of interest. The distribution of filipin along the optical axis of four human eye lenses was compared with data from the literature. The combined CRM-filipin approach is a highly specific and sensitive method for the study of cholesterol within cells and tissues. The spatial resolution is high and can be adapted to the desired discriminative power. The gross distribution of filipin along the optical axis obtained in this study is similar to that found in biochemical studies.
共聚焦拉曼显微光谱（CRM）是一种非侵入性、非破坏性且灵敏的分析工具，用于在高空间分辨率下研究细胞和组织分子组织的某些方面。制霉菌素是一种多烯抗生素，它能特异性结合胆固醇，其分子结构表明它具有拉曼活性。本研究的目的是评估CRM与制霉菌素联合方法在研究人眼晶状体中胆固醇分布方面的潜力。将多聚甲醛固定的人晶状体切片（0.7毫米），与制霉菌素孵育，然后用CRM进行分析。结果表明，制霉菌素在1586厘米-1处产生特定的拉曼信号，几乎不干扰晶状体蛋白的信号。CRM与制霉菌素联合方法被证明极其灵敏，能够检测飞克级的胆固醇。使用点测量时，它具有出色的空间分辨率（0.2-0.5微米3）。由于眼晶状体中膜的固有各向异性以及因此胆固醇分布的各向异性，当关注纤维间胆固醇变化时，必须采用线扫描方法。将四种人眼晶状体沿光轴的制霉菌素分布与文献数据进行了比较。CRM与制霉菌素联合方法是研究细胞和组织内胆固醇的一种高度特异性和灵敏的方法。空间分辨率高，并且可以根据所需的辨别能力进行调整。本研究中获得的制霉菌素沿光轴的总体分布与生化研究中发现的分布相似。
