Burgoyne Thomas, Lane Amelia, Laughlin William E, Cheetham Michael E, Futter Clare E
Institute of Ophthalmology, University College London, London, United Kingdom.
Primary Ciliary Dyskinesia Service, Electron Microscopy Unit, Department of Paediatrics, Royal Brompton Hospital, Sydney Street, London, United Kingdom.
PLoS One. 2018 Jan 9;13(1):e0191048. doi: 10.1371/journal.pone.0191048. eCollection 2018.
Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM.
correlative light-electron microscopy (CLEM)是一种强大的技术，可将荧光显微镜（FM）图像中特定大分子的定位映射到相应的高分辨率电子显微镜（EM）图像上。现有方法适用于有限的样本类型，且技术上具有挑战性。在此，我们描述了利用常用的FM包埋剂——最佳切割温度（OCT）复合物，在冷冻切片上进行CLEM和免疫电子显微镜（iEM）的新方法。利用这些方法，我们（i）在视网膜组织的视网膜色素上皮（RPE）中通过FM和EM鉴定出相同的吞噬体；（ii）在诱导多能干细胞衍生的视杯中，显示了视紫红质在光感受器外段盘状结构上的正确定位；（iii）在RPE中鉴定出过氧化物酶体与黑素小体以及吞噬体之间的新型相互作用。这些数据表明，冷冻切片能够轻松表征组织样本中靶大分子的定位，从而比许多局限于培养细胞的传统方法有了实质性改进。由于OCT包埋常用于FM，这为通过高分辨率EM进一步分析现有样本提供了一种易于获取且可靠的方法。
