Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA, USA.
Methods Mol Biol. 2021;2246:111-128. doi: 10.1007/978-1-0716-1115-9_8.
High-resolution, spatial characterization of microbial communities is critical for the accurate understanding of microbe-microbe and microbe-plant interactions in leaf surfaces (phyllosphere). However, leaves are specially challenging surfaces for imaging methods due to their high autofluorescence. In this chapter we describe the Leaf-FISH method. Leaf-FISH is a fluorescence in situ hybridization (FISH) method specially adapted to the requirements of plant tissues. Leaf-FISH uses a combination of leaf pretreatments coupled with spectral imaging confocal microscopy and image post-processing to visualize bacterial taxa on a structural-informed context recreated from the residual background autofluorescence of the tissues. Leaf-FISH is suitable for simultaneous identification of multiple bacterial taxa using multiple taxon-specific fluorescently labeled oligonucleotide probes (combinatorial labeling).
高分辨率、空间解析微生物群落对于准确理解叶片表面（叶面）中微生物-微生物和微生物-植物相互作用至关重要。然而，由于叶片具有很强的自发荧光，因此成像方法特别具有挑战性。在本章中，我们描述了 Leaf-FISH 方法。Leaf-FISH 是一种特别适用于植物组织要求的荧光原位杂交（FISH）方法。Leaf-FISH 将叶片预处理与光谱成像共聚焦显微镜以及图像后处理相结合，利用组织残留背景自发荧光重建的结构信息背景，对细菌分类群进行可视化。Leaf-FISH 适用于使用多个分类群特异性荧光标记寡核苷酸探针（组合标记）同时鉴定多种细菌分类群。
