Leaf-FISH: In Situ Hybridization Method for Visualizing Bacterial Taxa on Plant Surfaces.

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).